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CA 02850095 2014-03-25
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BIOLOGICAL METHODS FOR PREPARING ADIPIC ACID
Related Patent Applications
This patent application claims the benefit of U.S. patent application no.
13/245,780 filed on
September 26, 2011, entitled BIOLOGICAL METHODS FOR PREPARING ADIPIC ACID,
naming
Stephen Picataggio and Tom Beardslee as inventors, and designated by Attorney
Docket No.
VRD-1001-UT. This patent application also claims the benefit of U.S. patent
application no.
13/245,782 filed on September 26, 2011, entitled BIOLOGICAL METHODS FOR
PREPARING
ADIPIC ACID, naming Stephen Picataggio and Tom Beardslee as inventors, and
designated by
Attorney Docket No. VRD-1001-UT2.
This patent application is related to U.S. provisional patent application no.
61/222,902 filed on July
2, 2009, entitled BIOLOGICAL METHODS FOR PREPARING ADIPIC ACID, naming Stephen
Picataggio as inventor, and designated by Attorney Docket No. VRD-1001-PV.
This patent
application also is related to International patent application no.
PCT/U52010/040837 filed on July
1, 2010, entitled BIOLOGICAL METHODS FOR PREPARING ADIPIC ACID, naming Stephen
Picataggio and Tom Beardslee as inventors, and designated by Attorney Docket
No. VRD-1001-
PC. This patent application is related to U.S. provisional patent application
no. 61/430,097 filed on
January 5, 2011, entitled BIOLOGICAL METHODS FOR PREPARING ADIPIC ACID, naming
Stephen Picataggio and Tom Beardslee as inventors, and designated by Attorney
Docket No.
VRD-1001-PV2. This patent application also is related to U.S. provisional
patent application no.
61/482,160 filed on May 3, 2011, entitled BIOLOGICAL METHODS FOR PREPARING
ADIPIC
ACID, naming Stephen Picataggio and Tom Beardslee as inventors, and designated
by Attorney
Docket No. VRD-1001-PV3. This patent application also is related to U.S.
provisional patent
application no. 13/245,777 filed on September 26, 2011, entitled BIOLOGICAL
METHODS FOR
PREPARING ADIPIC ACID, naming Stephen Picataggio and Tom Beardslee as
inventors, and
designated by Attorney Docket No. VRD-1001-CT. This patent application also is
related to
International patent application no. PCT/U52012/020230 filed on January 4,
2012, entitled
BIOLOGICAL METHODS FOR PREPARING ADIPIC ACID, naming Stephen Picataggio and
Tom
Beardslee as inventors, and designated by Attorney Docket No. VRD-1001-PC2.
The entire content of each of the foregoing patent applications is
incorporated herein by reference,
including, without limitation, all text, tables and drawings.
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Field
The technology relates in part to biological methods for producing adipic acid
and engineered
microorganisms capable of such production.
Background
Microorganisms employ various enzyme-driven biological pathways to support
their own
metabolism and growth. A cell synthesizes native proteins, including enzymes,
in vivo from
deoxyribonucleic acid (DNA). DNA first is transcribed into a complementary
ribonucleic acid (RNA)
that comprises a ribonucleotide sequence encoding the protein. RNA then
directs translation of
the encoded protein by interaction with various cellular components, such as
ribosomes. The
resulting enzymes participate as biological catalysts in pathways involved in
production of
molecules by the organism.
These pathways can be exploited for the harvesting of the naturally produced
products. The
pathways also can be altered to increase production or to produce different
products that may be
commercially valuable. Advances in recombinant molecular biology methodology
allow
researchers to isolate DNA from one organism and insert it into another
organism, thus altering the
cellular synthesis of enzymes or other proteins. Such genetic engineering can
change the
biological pathways within the host organism, causing it to produce a desired
product.
Microorganic industrial production can minimize the use of caustic chemicals
and the production of
toxic byproducts, thus providing a "clean" source for certain compounds.
Summary
Provided herein are engineered microorganisms that produce six-carbon organic
molecules such
as adipic acid, methods for manufacturing such microorganisms and methods for
using them to
produce adipic acid and other six-carbon organic molecules. Also provided
herein are engineered
microorganisms including genetic alterations that direct carbon flux towards
adipic acid through
increased fatty acid production in conjunction with increased omega and beta
oxidation activities,
methods for manufacturing such organisms and methods for using them to produce
adipic acid and
other six-carbon organic molecules.
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Thus, provided herein in some embodiments are engineered microorganisms
capable of producing
adipic acid, which microorganisms comprise one or more altered activities
selected from the group
consisting of aldehyde dehydrogenase activity (e.g., 6-oxohexanoic acid
dehydrogenase activity,
omega oxo fatty acid dehydrogenase activity), fatty alcohol oxidase activity
(e.g., 6-
hydroxyhexanoic acid dehydrogenase activity, omega hydroxyl fatty acid
dehydrogenase activity),
glucose-6-phosphate dehydrogenase activity, hexanoate synthase activity,
lipase activity, fatty acid
synthase activity, acetyl CoA carboxylase activity, acyl-CoA hydrolase (e.g.,
ACH; thioesterase)
activity, acyl-CoA thioesterase (e.g., TESA) activity, acyl-CoA synthetase
(e.g., ACS1) activity, long
chain acyl-CoA synthetase (e.g., FAT1) activity, acyl-CoA sterol acyl
transferase (e.g., ARE1,
ARE2, or ARE1 and ARE2) activity, acyltransferase activity (e.g., diacyl-
glycerol acyl transferase,
DGA1, LR01, or DGA1 and LR01) activity and monooxygenase activity.
In certain embodiments, the microorganism comprises a genetic modification
that adds or
increases the aldehyde dehydrogenase activity (e.g., 6-oxohexanoic acid
dehydrogenase activity,
omega oxo fatty acid dehydrogenase activity), fatty alcohol oxidase activity
(e.g., 6-
hydroxyhexanoic acid dehydrogenase activity, omega hydroxyl fatty acid
dehydrogenase activity),
glucose-6-phosphate dehydrogenase activity, hexanoate synthase activity,
lipase activity, fatty acid
synthase activity, acetyl CoA carboxylase activity, monooxygenase activity,
monooxygenase
reductase activity, fatty alcohol oxidase activity, acyl-CoA ligase activity,
acyl-CoA oxidase activity,
enoyl-CoA hydratase activity, 3-hydroxyacyl-CoA dehydrogenase activity, acyl-
CoA hydrolase
activity, acyl-CoA thioesterase activity and/or acetyl-CoA C-acyltransferase
activity. Also provided
in some embodiments, are engineered microorganisms that produce adipic acid,
which
microorganisms comprise an altered monooxygenase activity. Provided also
herein in some
embodiments are engineered microorganisms that include a genetic modification
that reduces the
acyl-CoA oxidase activity, acyl-CoA synthetase activity, long chain acyl-CoA
synthetase activity,
acyl-CoA sterol acyl transferase activity, and/or acyltransferase activity
(e.g., diacyl-glycerol
acyltransferase).
In some embodiments, an engineered microorganism includes a genetic
modification that includes
multiple copies of a polynucleotide that encodes a polypeptide having aldehyde
dehydrogenase
activity (e.g., 6-oxohexanoic acid dehydrogenase activity, omega oxo fatty
acid dehydrogenase
activity), fatty alcohol oxidase activity (e.g., 6-hydroxyhexanoic acid
dehydrogenase activity, omega
hydroxyl fatty acid dehydrogenase activity), glucose-6-phosphate dehydrogenase
activity,
hexanoate synthase activity, lipase activity, fatty acid synthase activity,
acetyl CoA carboxylase
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activity, monooxygenase activity, monooxygenase reductase activity, fatty
alcohol oxidase activity,
acyl-CoA ligase activity, acyl-CoA oxidase activity, enoyl-CoA hydratase
activity, 3-hydroxyacyl-
CoA dehydrogenase activity, acyl-CoA hydrolase activity, acyl-CoA thioesterase
activity and/or
acetyl-CoA C-acyltransferase activity. In some embodiments, 2, 3, 4, 5, 6, 7,
8, 9, 10, 15, 20, 25,
30, 35, 40, 45, 50 or more copies of the particular polynucleotide are present
in the microbe. In
certain embodiments, an engineered microorganism includes a heterologous
promoter (and/or
5'UTR) in functional connection with a polynucleotide that encodes a
polypeptide having aldehyde
dehydrogenase activity (e.g., 6-oxohexanoic acid dehydrogenase activity, omega
oxo fatty acid
dehydrogenase activity), fatty alcohol oxidase activity (e.g., 6-
hydroxyhexanoic acid
dehydrogenase activity, omega hydroxyl fatty acid dehydrogenase activity),
glucose-6-phosphate
dehydrogenase activity, hexanoate synthase activity, lipase activity, fatty
acid synthase activity,
acetyl CoA carboxylase activity, monooxygenase activity, monooxygenase
reductase activity, fatty
alcohol oxidase activity, acyl-CoA ligase activity, acyl-CoA oxidase activity,
enoyl-CoA hydratase
activity, 3-hydroxyacyl-CoA dehydrogenase activity, acyl-CoA hydrolase
activity, acyl-CoA
thioesterase activity and/or acetyl-CoA C-acyltransferase activity. In some
embodiments, the
promoter is a PDX4 or P0X5 promoter or monooxygenase promoter from a yeast
(e.g., Candida
yeast strain (e.g., C. tropicalis, C. viswanithii)), or other promoter.
Examples of promoters that can
be utilized are described herein. The promoter sometimes is exogenous or
endogenous with
respect to the microbe.
Also provided herein is an engineered microorganism that produces adipic acid,
where the
microorganism includes an altered monooxygenase activity. In certain
embodiments, an
engineered microorganism comprises a genetic modification that alters a
monooxygenase activity.
In some embodiments, an engineered microorganism includes a genetic
modification that alters a
monooxygenase activity selected from the group consisting of CYP52Al2
activity, CYP52A13
activity, CYP52A14 activity, CYP52A15 activity, CYP52A16 activity, CYP52A17
activity,
CYP52A18 activity, CYP52A19 activity, CYP52A20 activity, CYP52D2 activity,
and/or BM3 activity
(e.g., from B. megaterium). In certain embodiments, an engineered
microorganism includes one or
more genetically modified monooxygenase activities selected from the group
consisting of
CYP52Al2 activity, CYP52A13 activity, CYP52A14 activity, CYP52A15 activity,
CYP52A16
activity, CYP52A17 activity, CYP52A18 activity, CYP52A19 activity, CYP52A20
activity, CYP52D2
activity, and/or BM3 activity. In some embodiments, the monooxygenase activity
is encoded by a
CYP52Al2 polynucleotide, a CYP52A13 polynucleotide, a CYP52A14 polynucleotide,
a
CYP52A15 polynucleotide, a CYP52A16 polynucleotide, a CYP52A17 polynucleotide,
a
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CYP52A18 polynucleotide, a CYP52A19 polynucleotide, a CYP52A20 polynucleotide,
a CYP52D2
polynucleotide, and/or a BM3 polynucleotide. In certain embodiments, an
engineered
microorganism includes one or more monooxygenase activities encoded by
polynucleotides
selected from the group consisting of a CYP52Al2 polynucleotide, a CYP52A13
polynucleotide, a
CYP52A14 polynucleotide, a CYP52A15 polynucleotide, a CYP52A16 polynucleotide,
a
CYP52A17 polynucleotide, a CYP52A18 polynucleotide, a CYP52A19 polynucleotide,
a
CYP52A20 polynucleotide, a CYP52D2 polynucleotide, and/or a BM3
polynucleotide. In some
embodiments, the genetic modification increases monooxygenase activity. In
certain
embodiments, the genetic modification increases the copy number of an
endogenous
polynucleotide that encodes a polypeptide having the monooxygenase activity
(e.g., 2, 3, 4, 5, 6, 7,
8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more copies of the
polynucleotide). In certain
embodiments, an engineered microorganism comprises one or more polynucleotides
that includes
a promoter (e.g., promoter and/or 5'UTR) and encodes a polypeptide having a
monooxygenase
activity. The promoter may be exogenous or endogenous with respect to the
microbe. An
engineered microorganism in certain embodiments comprises one or more
heterologous
polynucleotides encoding a polypeptide having monooxygenase activity. In
related embodiments,
the heterologous polynucleotide is from a yeast, such as a Candida yeast in
certain embodiments
(e.g., C. tropicalis, C. viswanithii), or from a bacteria, such as Bacillus
bacteria in some
embodiments (e.g., B. megaterium).
In certain embodiments, an engineered microorganism comprises a genetic
modification that alters
monooxygenase reductase activity. In some embodiments, an engineered
microorganism includes
a genetic modification that alters a monooxygenase reductase activity selected
from the group
consisting of NADPH cytochrome P450 reductase (e.g., CPR, from C. tropicalis
strain ATCC750),
NADPH cytochrome P450 reductase A (e.g., CPRA, from C. tropicalis strain
ATCC20336), NADPH
cytochrome P450 reductase B (e.g., CPRB, from C. tropicalis strain ATCC20336)
and/or
cytochrome P450:NADPH P450 reductase (e.g., B. megaterium). In certain
embodiments, an
engineered microorganism includes one or more genetically modified
monooxygenase reductase
activities selected from the group consisting of NADPH cytochrome P450
reductase (e.g., CPR),
NADPH cytochrome P450 reductase A (e.g., CPRA), NADPH cytochrome P450
reductase B (e.g.,
CPRB) and/or cytochrome P450:NADPH P450 reductase. In some embodiments, the
genetic
modification increases the copy number of an endogenous polynucleotide that
encodes a
polypeptide having monooxygenase reductase activity (e.g., 2, 3, 4, 5, 6, 7,
8, 9, 10, 15, 20, 25, 30,
35, 40, 45, 50 or more copies of the polynucleotide). In certain embodiments,
an engineered
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microorganism comprises one or more polynucleotides that includes a promoter
(e.g., promoter
and/or 5'UTR) and encodes a polypeptide having a monooxygenase reductase
activity. The
promoter may be exogenous or endogenous with respect to the microbe. In some
embodiments,
the polynucleotide is from a yeast, and in certain embodiments the yeast is a
Candida yeast (e.g.,
C. tropicalis, C. viswanithii). In some embodiments, the polynucleotide is
from a bacteria, and in
certain embodiments the bacteria is a Bacillus bacteria (e.g., B. megaterium).
An engineered microorganism in some embodiments comprises an altered
thioesterase activity. In
some embodiments, an engineered microorganism comprises a genetic modification
that alters the
thioesterase activity, and in certain embodiments, the engineered
microorganism comprises a
genetic alteration that adds or increases a thioesterase activity. In some
embodiments, the
engineered microorganism comprises a heterologous polynucleotide encoding a
polypeptide
having thioesterase activity. In certain embodiments, an engineered
microorganism includes a
genetic modification that alters a thioesterase activity selected from the
group consisting of acyl-
CoA hydrolase activity (e.g., ACHA, ACHB, ACHA and ACHB, from C. tropicalis),
acyl-CoA
thioesterase activity (e.g., TESA, from E. coli), and/or acyl-CoA hydrolase
and acyl-CoA
thioesterase activity. In some embodiments, the genetic modification increases
the copy number
of an endogenous polynucleotide that encodes a polypeptide having thioesterase
activity (e.g., 2,
3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more copies of the
polynucleotide). In
certain embodiments, an engineered microorganism comprises one or more
polynucleotides that
includes a promoter (e.g., promoter and/or 5'UTR) and encodes a polypeptide
having a
thioesterase activity. The promoter may be exogenous or endogenous with
respect to the microbe.
In some embodiments, the polynucleotide is from a yeast, and in certain
embodiments the yeast is
a Candida yeast (e.g., C. tropicalis, C. viswanithii). In some embodiments,
the polynucleotide is
from a bacteria, and in certain embodiments the bacteria is an Enteric
bacteria (e.g., Eschericia
coli). Examples of polynucleotide sequences that encode peptides with
thioesterase activity, and
polypeptide sequences with thioesterase activity are provided herein (e.g.,
SEQ ID NOs: 42-47)
An engineered microorganism in some embodiments comprises an altered fatty
alcohol oxidase
activity. In some embodiments, an engineered microorganism comprises a genetic
modification
that alters the fatty alcohol oxidase activity, and in certain embodiments,
the engineered
microorganism comprises a genetic alteration that adds or increases a fatty
alcohol oxidase
activity. In some embodiments, the genetic modification increases the copy
number of an
endogenous polynucleotide that encodes a polypeptide having fatty alcohol
oxidase activity (e.g.,
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2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more copies of
the polynucleotide). An
engineered microorganism in certain embodiments comprises a heterologous
promoter (e.g.,
endogenous or exogenous promoter with respect to the microbe) in functional
connection with a
polynucleotide that encodes a polypeptide having fatty alcohol oxidase
activity. In some
embodiments, the engineered microorganism comprises a heterologous
polynucleotide encoding a
polypeptide having fatty alcohol oxidase activity. In some embodiments, the
polynucleotide is from
a yeast, and in certain embodiments the yeast is Candida (e.g., a C.
tropicalis strain).
An engineered microorganism in some embodiments comprises an altered 6-
oxohexanoic acid
dehydrogenase activity or an altered omega oxo fatty acid dehydrogenase
activity. In some
embodiments, an engineered microorganism comprises a genetic modification that
adds or
increases 6-oxohexanoic acid dehydrogenase activity or omega oxo fatty acid
dehydrogenase
activity, and in certain embodiments, an engineered microorganism comprises a
heterologous
polynucleotide encoding a polypeptide having 6-oxohexanoic acid dehydrogenase
activity or
omega oxo fatty acid dehydrogenase activity. In related embodiments, the
heterologous
polynucleotide sometimes is from a bacterium, such as an Acinetobacter,
Nocardia, Pseudomonas
or Xanthobacter bacterium in some embodiments.
An engineered microorganism in some embodiments comprises an altered 6-
hydroxyhexanoic acid
dehydrogenase activity or an altered omega hydroxyl fatty acid dehydrogenase
activity. In some
embodiments, an engineered microorganism comprises a genetic modification that
adds or
increases the 6-hydroxyhexanoic acid dehydrogenase activity or omega hydroxyl
fatty acid
dehydrogenase activity, and in certain embodiments, an engineered
microorganism comprises a
heterologous polynucleotide encoding a polypeptide having 6-hydroxyhexanoic
acid
dehydrogenase activity or omega hydroxyl fatty acid dehydrogenase activity. In
related
embodiments, the heterologous polynucleotide is from a bacterium, such as an
Acinetobacter,
Nocardia, Pseudomonas or Xanthobacter bacterium in some embodiments.
An engineered microorganism in some embodiments comprises an altered fatty
acid synthase
activity. In some embodiments, an engineered microorganism comprises a genetic
modification
that alters fatty acid synthase activity. In certain embodiments, an
engineered microorganism
includes a genetic alteration that adds or increases fatty acid synthase
activity. In some
embodiments, the genetic modification increases the copy number of endogenous
polynucleotides
that encode polypeptides having fatty acid synthase activity (e.g., 2, 3, 4,
5, 6, 7, 8, 9, 10, 15, 20,
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25, 30, 35, 40, 45, 50 or more copies of a polynucleotide). An engineered
microorganism in certain
embodiments comprises a heterologous promoter (e.g., endogenous or exogenous
promoter with
respect to the microbe) in functional connection with a polynucleotide that
encodes a polypeptide
having fatty acid synthase activity. In some embodiments, the engineered
microorganism
comprises a heterologous polynucleotide encoding a polypeptide having fatty
acid synthase
activity. In certain embodiments, the fatty acid synthase activity is provided
by one or more
polypeptides having fatty acid synthase activity (e.g., a single subunit
protein or multi subunit
protein). In certain embodiments, the fatty acid synthase activity is provided
by a polypeptide
having fatty acid synthase subunit alpha (e.g., FAS2) activity, fatty acid
synthase subunit beta
(e.g., FAS1) activity, or fatty acid synthase subunit alpha activity and fatty
acid synthase subunit
beta activity. In some embodiments a fatty acid synthase activity comprises a
hexanoate synthase
activity. In certain embodiments, the polynucleotide is from a yeast, and in
certain embodiments
the yeast is a Candida yeast (e.g., C. tropicalis, C. viswanithii). Examples
of polynucleotides that
encode fatty acid synthase molecules (e.g., FAS1, FAS2) are provided herein
(e.g., SEQ ID NOs:
31 and 32).
An engineered microorganism in some embodiments comprises an altered hexanoate
synthase
activity. In some embodiments, an engineered microorganism comprises a genetic
modification
that alters hexanoate synthase activity. In certain embodiments, an engineered
microorganism
includes a genetic alteration that adds or increases hexanoate synthase
activity. In some
embodiments, an engineered microorganism comprises a heterologous
polynucleotide encoding a
polypeptide having hexanoate synthase activity. In certain embodiments, the
hexanoate synthase
activity is provided by a polypeptide having hexanoate synthase activity. In
certain embodiments,
the hexanoate synthase activity is provided by a polypeptide having hexanoate
synthase subunit A
activity, hexanoate synthase subunit B activity, or hexanoate synthase subunit
A activity and
hexanoate synthase subunit B activity. In some embodiments, the heterologous
polynucleotide is
from a fungus, such as an Aspergillus fungus in certain embodiments (e.g., A.
parasiticus, A.
nidulans).
In certain embodiments, an engineered microorganism comprises a genetic
modification that
results in substantial (e.g., primary) hexanoate usage by monooxygenase
activity. In related
embodiments, the genetic modification reduces a polyketide synthase activity.
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An engineered microorganism in some embodiments comprises an altered lipase
activity. In
certain embodiments, an engineered microorganism includes a genetic alteration
that adds or
increases a lipase activity. In some embodiments, the genetic modification
increases the copy
number of endogenous polynucleotides that encode polypeptides having lipase
activity (e.g., 2, 3,
4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more copies of a
polynucleotide). An
engineered microorganism in certain embodiments comprises a heterologous
promoter (e.g.,
endogenous or exogenous promoter with respect to the microbe) in functional
connection with a
polynucleotide that encodes a polypeptide having lipase activity. In some
embodiments, the
engineered microorganism comprises a heterologous polynucleotide encoding a
polypeptide
having lipase activity. In certain embodiments, the lipase activity is
provided by one or more
polypeptides having lipase activity (e.g., a single subunit protein or multi
subunit protein). In
certain embodiments, the lipase activity is provided by a polypeptide
comprising all or part of the
amino acid sequence of SEQ ID NO: 28 or 29, and sometimes the polypeptide is
encoded by a
polynucleotide of SEQ ID NO: 27. In certain embodiments, the polynucleotide is
from a yeast, and
in certain embodiments the yeast is a Candida yeast (e.g., C. tropicalis, C.
viswanithii).
An engineered microorganism in some embodiments comprises an altered acetyl-
CoA carboxylase
activity. In certain embodiments, an engineered microorganism includes a
genetic alteration that
adds or increases a acetyl-CoA carboxylase activity. In some embodiments, the
genetic
modification increases the copy number of endogenous polynucleotides that
encode polypeptides
having acetyl-CoA carboxylase activity (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,
20, 25, 30, 35, 40, 45, 50
or more copies of a polynucleotide). An engineered microorganism in certain
embodiments
comprises a heterologous promoter (e.g., endogenous or exogenous promoter with
respect to the
microbe) in functional connection with a polynucleotide that encodes a
polypeptide having acetyl-
CoA carboxylase activity. In some embodiments, the engineered microorganism
comprises a
heterologous polynucleotide encoding a polypeptide having acetyl-CoA
carboxylase activity. In
some embodiments, the engineered microorganism comprises a heterologous
polynucleotide
encoding a polypeptide having acetyl-CoA carboxylase activity. In some
embodiments, the
polynucleotide is from a yeast, and in certain embodiments the yeast is
Candida (e.g., a C.
tropicalis strain). In some embodiments an acetyl-CoA carboxylase polypeptide
is encoded by a
polynucleotide comprising the sequence of SEQ ID NO 30.
An engineered microorganism in some embodiments is a non-prokaryotic organism,
and
sometimes is a eukaryote. A eukaryote can be a yeast in some embodiments, such
as a Candida
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yeast (e.g., C. tropicalis, C. viswanithii), or a Yarrowia yeast (e.g., Y.
lipolytica), for example. In
certain embodiments a eukaryote is a fungus such as an Aspergillus fungus
(e.g., A. parasiticus or
A. nidulans), for example.
In some embodiments, an engineered microorganism comprises a genetic
modification that
reduces 6-hydroxyhexanoic acid conversion. In related embodiments, the genetic
modification
reduces 6-hydroxyhexanoic acid dehydrogenase activity or omega hydroxyl fatty
acid
dehydrogenase activity.
In certain embodiments, an engineered microorganism comprises a genetic
modification that
reduces beta-oxidation activity, and in some embodiments, the genetic
modification renders beta-
oxidation activity undetectable (e.g., completely blocked beta-oxidation
activity). In certain
embodiments, the genetic modification partially reduces beta-oxidation
activity.
A fatty acid-CoA derivative, or dicarboxylic acid-CoA derivative, can be
converted to a trans-2,3-
dehydroacyl-CoA derivative by the activity of acyl-CoA oxidase (e.g., also
known as or referred to
as acyl-CoA oxidoreductase and fatty acyl-coenzyme A oxidase), in many
organisms. In some
embodiments, an engineered microorganism comprises a genetic modification that
alters the
specificity of and/or reduces the activity of an acyl-CoA oxidase activity. In
certain embodiments,
the genetic modification disrupts an acyl-CoA oxidase activity. In some
embodiments, the genetic
modification includes disrupting a polynucleotide that encodes a polypeptide
having an acyl-CoA
oxidase activity. In certain embodiments, the genetic modification includes
disrupting a promoter
and/or 5'UTR in functional connection with a polynucleotide that encodes a
polypeptide having the
acyl-CoA oxidase activity. In some embodiments, the polypeptide having acyl-
CoA activity is a
PDX polypeptide. In certain embodiments, the PDX polypeptide is a PDX4
polypeptide, a P0X5
polypeptide, or a PDX4 polypeptide and PDX5 polypeptide. In certain
embodiments, the genetic
modification disrupts an acyl-CoA activity by disrupting a PDX4 nucleotide
sequence, a PDX5
nucleotide sequence, or a PDX4 and PDX5 nucleotide sequence.
In some embodiments, an engineered microorganism comprises a genetic
modification that
increases beta-oxidation activity. In certain embodiments, the beta-oxidation
increase in beta-
oxidation activity is the result of an increase in activity in one or more
activities involved in beta-
oxidation. In some embodiments, the genetic modification increases the copy
number of an
endogenous polynucleotide that encodes a polypeptide having an activity
involved in beta-
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oxidation (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or
more copies of the
polynucleotide). An engineered microorganism in certain embodiments comprises
a heterologous
promoter (e.g., endogenous or exogenous promoter with respect to the microbe)
in functional
connection with a polynucleotide that encodes a polynucleotide that encodes a
polypeptide having
an activity involved in beta-oxidation. In some embodiments, the engineered
microorganism
comprises a heterologous polynucleotide encoding a polynucleotide that encodes
a polypeptide
having an activity involved in beta-oxidation. In certain embodiments, beta
oxidation activity that is
increased is an acyl-CoA oxidase activity, and in some embodiments the acyl-
CoA oxidase activity
is an activity encoded by the PDX5 gene. In some embodiments, the altered
activity (e.g.,
increased activity) is provided by a polypeptide encoded by a polynucleotide
native to the host
organism (e.g., multiple copies of the native polynucleotide, promoter
inserted in functional
connection with the with the native polynucleotide). In certain embodiments,
the polynucleotide is
from a yeast, and in certain embodiments the yeast is Candida (e.g., a C.
tropicalis strain, C.
viswanithii strain).
In some embodiments, an engineered microorganism comprises a genetic
modification that
increases omega-oxidation activity. In some embodiments, an engineered
microorganism
comprises one or more genetic modifications that alter a reverse activity in a
beta oxidation
pathway, an omega oxidation pathway, or a beta oxidation and omega oxidation
pathway, thereby
increasing carbon flux through the respective pathways, due to the reduction
in one or more
reverse enzymatic activities.
An engineered microorganism can include a heterologous polynucleotide that
encodes a
polypeptide providing an activity described above, and the heterologous
polynucleotide can be
from any suitable microorganism. Examples of microorganisms are described
herein (e.g.,
Candida yeast, Saccharomyces yeast, Yarrowia yeast, Pseudomonas bacteria,
Bacillus bacteria,
Clostridium bacteria, Eubacterium bacteria and others include Megasphaera
bacteria.
Also provided herein are engineered microorganisms including genetic
alterations that direct
carbon flux (e.g., carbon metabolism) towards the production of adipic acid by
increasing
production and/or accumulation of fatty acids and increasing omega oxidation
and beta oxidation
activities. In certain embodiments, the genetic alterations are selected to
maximize production of
adipic acid from certain feedstocks (e.g., sugars, cellulose,
triacylglycerides, fatty acids, the like
and combinations thereof). In some embodiments, an engineered microorganism
comprises a
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genetic modification that reduces activities associated with generation of
biomass and/or carbon
storage molecules (e.g., various storage triglycerides), or with utilization
of fatty acids for energy
via beta oxidation and in certain embodiments, the genetic modification
renders the activities
associated with generation of biomass and/or carbon storage molecules or
utilization of fatty acids
for energy undetectable. In some embodiments, the activity associated with
generation of biomass
and/or carbon storage molecules is an activity that generates phospholipids,
triacylglycerides,
and/or steryl esters. In certain embodiments, the activity associated with
generation of biomass
and/or carbon storage or utilization of fatty acids for energy is selected
from acyl-CoA synthetase
(e.g., ACS1) activity, long chain acyl-CoA synthetase (e.g., FAT1) activity,
acyl-CoA sterol acyl
transferase (e.g., ARE1, ARE2, or ARE1 and ARE2) activity, and/or diacyl-
glycerol acyl transferase
(e.g., DGA1, LR01, or DGA1 and LR01) activity.
In some embodiments, an engineered microorganism comprises a genetic
modification that alters
the specificity of and/or reduces the activity of an acyl-CoA synthetase
activity. In certain
embodiments, the genetic modification disrupts an acyl-CoA synthetase
activity. In some
embodiments, the genetic modification includes disrupting a polynucleotide
that encodes a
polypeptide having an acyl-CoA synthetase activity. In certain embodiments,
the genetic
modification includes disrupting a promoter and/or 5'UTR in functional
connection with a
polynucleotide that encodes a polypeptide having the acyl-CoA synthetase
activity, and in some
embodiments, the genetic modification includes disrupting a portion or all of
the nucleotide
sequence which encodes the polypeptide having acyl-CoA synthetase activity. In
some
embodiments, the polypeptide having acyl-CoA synthetase activity is an ACS
polypeptide. In
certain embodiments, the ACS polypeptide is an ACS1 polypeptide, an ACS2
polypeptide, or an
ACS1 polypeptide and ACS2 polypeptide. In certain embodiments, the genetic
modification
disrupts an acyl-CoA synthetase activity by disrupting an ACS1 nucleotide
sequence, an ACS2
nucleotide sequence, or an ACS1 and ACS2 nucleotide sequence. In some
embodiments, an
acyl-CoA synthetase activity is disrupted by disrupting an ACS1 nucleotide
sequence substantially
similar to the nucleotide sequence of SEQ ID No: 48. In certain embodiments
involving disruption
of an acyl-CoA synthetase activity, a polypeptide corresponding to SEQ ID NO:
49 is below the
limits of detection using currently available detection methods (e.g.,
immunodetection, enzymatic
assay, the like and combinations thereof), in a host organism.
In some embodiments, an engineered microorganism comprises a genetic
modification that alters
the specificity of and/or reduces the activity of a long chain acyl-CoA
synthetase activity. In certain
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embodiments, the genetic modification disrupts a long chain acyl-CoA
synthetase activity. In some
embodiments, the genetic modification includes disrupting a polynucleotide
that encodes a
polypeptide having a long chain acyl-CoA synthetase activity. In certain
embodiments, the genetic
modification includes disrupting a promoter and/or 5'UTR in functional
connection with a
polynucleotide that encodes a polypeptide having the long chain acyl-CoA
synthetase activity, and
in some embodiments, the genetic modification includes disrupting a portion or
all of the nucleotide
sequence which encodes the polypeptide having long chain acyl-CoA synthetase
activity. In some
embodiments, the polypeptide having long chain acyl-CoA synthetase activity is
a FAT1
polypeptide. In certain embodiments, the genetic modification disrupts a long
chain acyl-CoA
synthetase activity by disrupting a FAT1 nucleotide sequence. In some
embodiments, a long chain
acyl-CoA synthetase activity is disrupted by disrupting a FAT1 nucleotide
sequence substantially
similar to the nucleotide sequence of SEQ ID No: 50. In certain embodiments
involving disruption
of a long chain acyl-CoA synthetase activity, a polypeptide corresponding to
SEQ ID NO: 51 is
below the limits of detection using currently available detection methods
(e.g., immunodetection,
enzymatic assay, the like and combinations thereof), in a host organism.
In some embodiments, an engineered microorganism comprises a genetic
modification that alters
the specificity of and/or reduces the activity of an acyl-CoA sterol
acyltransferase activity. In
certain embodiments, the genetic modification disrupts an acyl-CoA sterol
acyltransferase activity.
In some embodiments, the genetic modification includes disrupting a
polynucleotide that encodes a
polypeptide having an acyl-CoA sterol acyltransferase activity. In certain
embodiments, the
genetic modification includes disrupting a promoter and/or 5'UTR in functional
connection with a
polynucleotide that encodes a polypeptide having the acyl-CoA sterol
acyltransferase activity, and
in some embodiments, the genetic modification includes disrupting a portion or
all of the nucleotide
sequence which encodes the polypeptide having acyl-CoA sterol acyltransferase
activity. In some
embodiments, the polypeptide having acyl-CoA sterol acyltransferase activity
is an ARE
polypeptide. In certain embodiments, the ARE polypeptide is an ARE1
polypeptide, an ARE2
polypeptide, or an ARE1 polypeptide and ARE2 polypeptide. In certain
embodiments, the genetic
modification disrupts an acyl-CoA sterol acyltransferase activity by
disrupting an ARE1 nucleotide
sequence, an ARE2 nucleotide sequence, or an ARE1 and ARE2 nucleotide
sequence. In some
embodiments, an acyl-CoA sterol acyltransferase activity is disrupted by
disrupting an ARE
nucleotide sequence substantially similar to a nucleotide sequence
corresponding to SEQ ID NOs:
52 and/or 54. In certain embodiments involving disruption of an acyl-CoA
sterol acyltransferase
activity, a polypeptide substantially similar to an amino acid sequence
corresponding to SEQ ID
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NOs: 53 and/or 55 is below the limits of detection using currently available
detection methods (e.g.,
immunodetection, enzymatic assay, the like and combinations thereof), in a
host organism.
In some embodiments, an engineered microorganism comprises a genetic
modification that alters
the specificity of and/or reduces the activity of a diacylglycerol
acyltransferase activity. In certain
embodiments, the genetic modification disrupts a diacylglycerol
acyltransferase activity. In some
embodiments, the genetic modification includes disrupting a polynucleotide
that encodes a
polypeptide having a diacylglycerol acyltransferase activity. In certain
embodiments, the genetic
modification includes disrupting a promoter and/or 5'UTR in functional
connection with a
polynucleotide that encodes a polypeptide having the diacylglycerol
acyltransferase activity, and in
some embodiments, the genetic modification includes disrupting a portion or
all of the nucleotide
sequence which encodes the polypeptide having a diacylglycerol acyltransferase
activity. In some
embodiments, the polypeptide having diacylglycerol acyltransferase activity is
a DGA1 polypeptide.
In certain embodiments, the genetic modification disrupts a diacylglycerol
acyltransferase activity
by disrupting a DGA1 nucleotide sequence. In some embodiments, a
diacylglycerol
acyltransferase activity is disrupted by disrupting a DGA1 nucleotide sequence
substantially similar
to the nucleotide sequence of SEQ ID No: 56. In certain embodiments involving
disruption of a
diacylglycerol acyltransferase activity, a polypeptide corresponding to SEQ ID
NO: 57 is below the
limits of detection using currently available detection methods (e.g.,
immunodetection, enzymatic
assay, the like and combinations thereof), in a host organism.
In some embodiments, an engineered microorganism comprises a genetic
modification that alters
the specificity of and/or reduces the activity of an acyltransferase activity
(e.g., LRO1). In certain
embodiments, the genetic modification disrupts an acyltransferase activity. In
some embodiments,
the genetic modification includes disrupting a polynucleotide that encodes a
polypeptide having an
acyltransferase activity. In certain embodiments, the genetic modification
includes disrupting a
promoter and/or 5'UTR in functional connection with a polynucleotide that
encodes a polypeptide
having an acyltransferase activity, and in some embodiments, the genetic
modification includes
disrupting a portion or all of the nucleotide sequence which encodes the
polypeptide having an
acyltransferase activity. In some embodiments, the polypeptide having
acyltransferase activity is a
LRO1 polypeptide. In certain embodiments, the genetic modification disrupts an
acyltransferase
activity by disrupting a LRO1 nucleotide sequence. In some embodiments, an
acyltransferase
activity is disrupted by disrupting a LRO1 nucleotide sequence substantially
similar to the
nucleotide sequence of SEQ ID No: 58. In certain embodiments involving
disruption of an
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acyltransferase activity, a polypeptide corresponding to SEQ ID NO: 59 is
below the limits of
detection using currently available detection methods (e.g., immunodetection,
enzymatic assay,
the like and combinations thereof), in a host organism.
Also provided in some embodiments are methods for manufacturing adipic acid,
which comprise
culturing an engineered microorganism described herein under culture
conditions in which the
cultured microorganism produces adipic acid. In some embodiments, the host
microorganism from
which the engineered microorganism is generated does not produce a detectable
amount of adipic
acid. In certain embodiments, the culture conditions comprise fermentation
conditions, introduction
of biomass, introduction of glucose, introduction of a paraffin (e.g., plant
or petroleum based, such
as hexane or coconut oil, for example) and/or combinations thereof. In some
embodiments, the
adipic acid is produced with a yield of greater than about 0.3 grams per gram
of glucose added. In
related embodiments, a method comprises purifying the adipic acid from the
cultured
microorganisms and or modifying the adipic acid, thereby producing modified
adipic acid. In
certain embodiments, a method comprises placing the cultured microorganisms,
the adipic acid or
the modified adipic acid in a container, and optionally, shipping the
container.
Provided also in certain embodiments are methods for manufacturing 6-
hydroxyhexanoic acid,
which comprise culturing an engineered microorganism described herein under
culture conditions
in which the cultured microorganism produces 6-hydroxyhexanoic acid. In some
embodiments, the
host microorganism from which the engineered microorganism is generated does
not produce a
detectable amount of 6-hydroxyhexanoic acid. In certain embodiments, the
culture conditions
comprise fermentation conditions, introduction of biomass, introduction of
glucose, and/or
introduction of hexane. In some embodiments, the 6-hydroxyhexanoic acid is
produced with a
yield of greater than about 0.3 grams per gram of glucose added. In related
embodiments, a
method comprises purifying the 6-hydroxyhexanoic acid from the cultured
microorganisms and or
modifying the 6-hydroxyhexanoic acid, thereby producing modified 6-
hydroxyhexanoic acid. In
certain embodiments, a method comprises placing the cultured microorganisms,
the 6-
hydroxyhexanoic acid or the modified 6-hydroxyhexanoic acid in a container,
and optionally,
shipping the container.
Also provided in some embodiments are methods for preparing an engineered
microorganism that
produces adipic acid, which comprise: (a) introducing a genetic modification
to a host organism
that adds or increases monooxygenase activity, thereby producing engineered
microorganisms
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having detectable and/or increased monooxygenase activity; and (b) selecting
for engineered
microorganisms that produce adipic acid. Provided also herein in some
embodiments are methods
for preparing an engineered microorganism that produces adipic acid, which
comprise: (a)
culturing a host organism with hexane as a nutrient source, thereby producing
engineered
microorganisms having detectable monooxygenase activity; and (b) selecting for
engineered
microorganisms that produce adipic acid. In some embodiments the monooxygenase
activity is
incorporation of a hydroxyl moiety into a six-carbon molecule, and in certain
embodiments, the six-
carbon molecule is hexanoate. In related embodiments, a method comprises
selecting the
engineered microorganisms that have a detectable amount of the monooxygenase
activity. In
some embodiments, a method comprises introducing a genetic modification that
adds or increases
a hexanoate synthase activity, thereby producing engineered microorganisms,
and selecting for
engineered microorganisms having detectable and/or increased hexanoate
synthase activity. In
related embodiments, the genetic modification encodes a polypeptide having a
hexanoate
synthase subunit A activity, a hexanoate synthase subunit B activity, or a
hexanoate synthase
subunit A activity and a hexanoate synthase subunit B activity.
In some embodiments, a method comprises introducing a genetic modification
that adds or
increases an aldehyde dehydrogenase activity (e.g., 6-oxohexanoic acid
dehydrogenase activity,
omega oxo fatty acid dehydrogenase), thereby producing engineered
microorganisms, and
selecting for engineered microorganisms having detectable and/or increased 6-
oxohexanoic acid
dehydrogenase activity or omega oxo fatty acid dehydrogenase relative to the
host microorgansim.
In certain embodiments, a method for preparing microorganisms that produce
adipic acid includes
selecting for engineered microorganisms having one or more detectable and/or
increased activities
selected from the group consisting of an aldehyde dehydrogenase activity
(e.g., 6-oxohexanoic
acid dehydrogenase activity, omega oxo fatty acid dehydrogenase), fatty
alcohol oxidase activity
(e.g., 6-hydroxyhexanoic acid dehydrogenase activity, omega hydroxyl-fatty
acid dehydrogenase),
glucose-6-phosphate dehydrogenase activity, hexanoate synthase activity,
lipase activity, fatty acid
synthase activity, acetyl CoA carboxylase activity, monooxygenase activity,
monooxygenase
reductase activity, fatty alcohol oxidase activity, acyl-CoA ligase activity,
acyl-CoA oxidase activity,
acyl-CoA hydrolase, acyl-CoA thioesterase enoyl-CoA hydratase activity, 3-
hydroxyacyl-CoA
dehydrogenase activity, and/or acetyl-CoA C-acyltransferase activity.
In certain embodiments, a method comprises introducing a genetic modification
that adds or
increases a fatty alcohol oxidase activity (e.g., 6-hydroxyhexanoic acid
dehydrogenase activity,
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omega hydroxyl fatty acid dehydrogenase activity) thereby producing engineered
microorganisms,
and selecting for engineered microorganisms having a detectable and/or
increased 6-
hydroxyhexanoic acid dehydrogenase activity or omega hydroxyl fatty acid
dehydrogenase activity
relative to the host microorganism. In some embodiments, a method comprises
introducing a
genetic modification that adds or increases a thioesterase activity, thereby
producing engineered
microorganisms, and selecting for engineered microorganisms having a
detectable and/or
increased thioesterase activity relative to the host microorganism.
In certain embodiments, a method comprises introducing a genetic modification
that reduces 6-
hydroxyhexanoic acid conversion, thereby producing engineered microorganisms,
and selecting for
engineered microorganisms having reduced 6-hydroxyhexanoic acid conversion
relative to the host
microorganism. In some embodiments, a method comprises introducing a genetic
modification that
reduces beta-oxidation activity, thereby producing engineered microorganisms,
and selecting for
engineered microorganisms having reduced beta-oxidation activity relative to
the host
microorganism. In certain embodiments, a method comprises introducing a
genetic modification
that reduces activities associated with generation of biomass and/or carbon
storage molecules
and/or utilization of fatty acids for energy, thereby producing engineered
microorganisms, and
selecting for engineered microorganisms having reduced activities associated
with generation of
biomass and/or carbon storage molecules and/or utilization of fatty acids for
energy relative to the
host microorganism. In certain embodiments, a method comprises introducing a
genetic
modification that results in substantial hexanoate usage by the monooxygenase
activity, thereby
producing engineered microorganisms, and selecting for engineered
microorganisms in which
substantial hexanoate usage is by the monooxygenase activity relative to the
host microorganism.
In some embodiments, a method comprises introducing a genetic modification
that increases
omega- oxidation activity, thereby producing engineered microorganisms, and
selecting for
engineered microorganisms having increased omega-oxidation activity relative
to the host
microorganism.
Provided also herein in certain embodiments are methods for preparing a
microorganism that
produces adipic acid, which comprise: (a) introducing one or more genetic
modifications to a host
organism that add or increase one or more activities selected from the group
consisting of 6-
oxohexanoic acid dehydrogenase activity, omega oxo fatty acid dehydrogenase
activity, 6-
hydroxyhexanoic acid dehydrogenase activity, omega hydroxyl fatty acid
dehydrogenase activity,
glucose-6-phosphate dehydrogenase activity, hexanoate synthase activity,
lipase activity, fatty acid
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synthase activity, acetyl CoA carboxylase activity, monooxygenase activity,
monooxygenase
reductase activity, fatty alcohol oxidase activity, acyl-CoA ligase activity,
acyl-CoA oxidase activity,
acyl-CoA hydrolase activity, acyl-CoA thioesterase activity, enoyl-CoA
hydratase activity, 3-
hydroxyacyl-CoA dehydrogenase activity, and/or acetyl-CoA C-acyltransferase
activity, thereby
producing engineered microorganisms, and (b) selecting for engineered
microorganisms that
produce adipic acid. In some embodiments, a method comprises selecting for
engineered
microorganisms having one or more detectable and/or increased activities
selected from the group
consisting of 6-oxohexanoic acid dehydrogenase activity, 6-hydroxyhexanoic
acid dehydrogenase
activity, glucose-6-phosphate dehydrogenase activity, hexanoate synthase
activity, lipase activity,
fatty acid synthase activity, acetyl CoA carboxylase activity, monooxygenase
activity,
monooxygenase reductase activity, fatty alcohol oxidase activity, acyl-CoA
ligase activity, acyl-CoA
oxidase activity, acyl-CoA hydrolase activity, acyl-CoA thioesterase activity,
enoyl-CoA hydratase
activity, 3-hydroxyacyl-CoA dehydrogenase activity, and/or acetyl-CoA C-
acyltransferase activity,
relative to the host microorganism.
In certain embodiments, a method comprises introducing a genetic modification
that reduces 6-
hydroxyhexanoic acid conversion, thereby producing engineered microorganisms,
and selecting for
engineered microorganisms having reduced 6-hydroxyhexanoic acid conversion
relative to the host
microorganism. In some embodiments, a method comprises introducing a genetic
modification that
reduces beta-oxidation activity, thereby producing engineered microorganisms,
and selecting for
engineered microorganisms having reduced beta-oxidation activity relative to
the host
microorganism. In certain embodiments, a method comprises introducing a
genetic modification
that reduces activities associated with generation of biomass and/or carbon
storage molecules
and/or utilization of fatty acids for energy, thereby producing engineered
microorganisms, and
selecting for engineered microorganisms having reduced activities associated
with generation of
biomass and/or carbon storage molecules and/or utilization of fatty acids for
energy relative to the
host microorganism. In certain embodiments, a method comprises introducing a
genetic
modification that results in substantial hexanoate usage by the monooxygenase
activity, thereby
producing engineered microorganisms, and selecting for engineered
microorganisms in which
substantial hexanoate usage is by the monooxygenase activity relative to the
host microorganism.
Also provided in some embodiments are methods for preparing a microorganism
that produces 6-
hydroxyhexanoic acid, which comprise: (a) introducing one or more genetic
modifications to a host
organism that add or increase one or more activities selected from the group
consisting of 6-
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oxohexanoic acid dehydrogenase activity, glucose-6-phosphate dehydrogenase
activity,
hexanoate synthase activity, lipase activity, fatty acid synthase activity,
acetyl CoA carboxylase
activity, monooxygenase activity, monooxygenase reductase activity, fatty
alcohol oxidase activity,
acyl-CoA ligase activity, acyl-CoA oxidase activity, acyl-CoA hydrolase, acyl-
CoA thioesterase
enoyl-CoA hydratase activity, 3-hydroxyacyl-CoA dehydrogenase activity, and/or
acetyl-CoA C-
acyltransferase activity, thereby producing engineered microorganisms, (b)
introducing a genetic
modification to the host organism that reduces 6-hydroxyhexanoic acid
conversion, and (c)
selecting for engineered microorganisms that produce 6-hydroxyhexanoic acid.
In certain
embodiments, a method comprises selecting for engineered microorganisms having
reduced 6-
hydroxyhexanoic acid conversion relative to the host microorganism. In some
embodiments, a
method comprises selecting for engineered microorganisms having one or more
detectable and/or
increased activities selected from the group consisting of aldehyde
dehydrogenase activity (e.g., 6-
oxohexanoic acid dehydrogenase activity, omega oxo fatty acid dehydrogenase
activity), fatty
alcohol oxidase activity (e.g., 6-hydroxyhexanoic acid dehydrogenase activity,
omega hydroxyl
fatty acid dehydrogenase activity), glucose-6-phosphate dehydrogenase
activity, hexanoate
synthase activity, lipase activity, fatty acid synthase activity, acetyl CoA
carboxylase activity,
monooxygenase activity, monooxygenase reductase activity, fatty alcohol
oxidase activity, acyl-
CoA ligase activity, acyl-CoA oxidase activity, acyl-CoA hydrolase, acyl-CoA
thioesterase enoyl-
CoA hydratase activity, 3-hydroxyacyl-CoA dehydrogenase activity, and/or
acetyl-CoA C-
acyltransferase activity, relative to the host microorganism. In certain
embodiments, a method
comprises introducing a genetic modification that reduces beta-oxidation
activity, thereby
producing engineered microorganisms, and selecting for engineered
microorganisms having
reduced beta-oxidation activity relative to the host microorganism. In some
embodiments, a
method comprises introducing a genetic modification that results in
substantial hexanoate usage
by the monooxygenase activity, thereby producing engineered microorganisms,
and selecting for
engineered microorganisms in which substantial hexanoate usage is by the
monooxygenase
activity relative to the host microorganism.
Also provided are methods that include contacting an engineered microorganism
with a feedstock
including one or more polysaccharides, wherein the engineered microorganism
includes: (a) a
genetic alteration that blocks beta oxidation activity, and (b) a genetic
alteration that adds or
increases a monooxygenase activity, a genetic alteration that adds or
increases a fatty acid
synthase activity, and/or a genetic alteration that adds or increases a
hexanoate synthetase
activity, and culturing the engineered microorganism under conditions in which
adipic acid is
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produced. In some embodiments, the engineered microorganism comprises a
genetic alteration
that adds or increases fatty acid synthase activity and/or hexanoate
synthetase activity. In certain
embodiments, the engineered microorganism comprises a heterologous
polynucleotide encoding a
polypeptide having fatty acid synthase subunit alpha activity, and in some
embodiments the
engineered microorganism comprises a heterologous polynucleotide encoding a
polypeptide
having fatty acid synthase subunit beta activity. In certain embodiments, the
engineered
microorganism comprises a heterologous polynucleotide encoding a polypeptide
having hexanoate
synthase subunit A activity, and in some embodiments the engineered
microorganism comprises a
heterologous polynucleotide encoding a polypeptide having hexanoate synthase
subunit B activity.
In certain embodiments, the heterologous polynucleotide independently is
selected from a fungus.
In some embodiments, the fungus is an Aspergillus fungus, and in certain
embodiments the
Aspergillus fungus is A. parasiticus. In some embodiments, the microorganism
is a Candida yeast.
In certain embodiments, the microorganism is a C. tropicalis strain, and in
some embodiments, the
microorganism is a C. viswanithii strain. In certain embodiments, the
microorganism is a Yarrowia
yeast, and in some embodiments, the microorganism is a Yarrowia lipolytica
strain.
Provided also are methods that include contacting an engineered microorganism
with a feedstock
comprising one or more paraffins, wherein the engineered microorganism
comprises a genetic
alteration that partially blocks beta oxidation activity and culturing the
engineered microorganism
under conditions in which adipic acid is produced. In certain embodiments, the
microorganism
comprises a genetic alteration that increases a monooxygenase activity. In
some embodiments,
the microorganism is a Candida yeast. In certain embodiments, the
microorganism is a C.
tropicalis strain, and in some embodiments, the microorganism is a C.
viswanithii strain. In certain
embodiments, the microorganism is a Yarrowia yeast, and in some embodiments,
the
microorganism is a Yarrowia lipolytica strain.
In some embodiments, the genetic alteration that increases monooxygenase
activity comprises a
genetic alteration that increases monooxygenase (e.g., Cytochrome P450)
reductase activity. In
certain embodiments, the genetic alteration increases the number of copies of
a polynucleotide
that encodes a polypeptide having the Cytochrome P450 reductase activity. In
some
embodiments, the genetic alteration places a promoter and/or 5'UTR in
functional connection with
a polynucleotide that encodes a polypeptide having the Cytochrome P450
reductase activity. In
certain embodiments the monooxygenase reductase activity is selected from the
group consisting
of cytochrome P450:NADPH P450 reductase (B. megaterium), NADPH cytochrome P450
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reductase (CPR; C. tropicalis strain ATCC750), NADPH cytochrome P450 reductase
A (e.g.,
CPRA; C. tropicalis strain ATCC20336), and NADPH cytochrome P450 reductase B
(CPRB; C.
tropicalis strain ATCC20336). In some embodiments the monooxygenase reductase
activity is
provided by a polypeptide encoded by a polynucleotide of any one of SEQ ID
NOs: 23-26.
In certain embodiments, the genetic alteration that blocks beta oxidation
activity disrupts acyl-CoA
oxidase activity. In some embodiments, the genetic alteration disrupts PDX4
and/or PDX5 activity.
In certain embodiments, the genetic alteration disrupts a polynucleotide that
encodes a polypeptide
having the acyl-CoA oxidase activity. In some embodiments, the genetic
alteration disrupts a
promoter and/or 5'UTR in functional connection with a polynucleotide that
encodes a polypeptide
having the acyl-CoA oxidase activity.
In some embodiments a genetic alteration that increases beta oxidation
activity increases acyl-CoA
oxidase activity. In certain embodiments, the genetic alteration that
increases beta oxidation
activity adds or increases the number of copies of a polynucleotide encoding
an acyl-CoA oxidase
activity. In some embodiments, the genetic alteration increases the activity
of a promoter and/or
5'UTR in functional connection with a polynucleotide encoding an acyl-CoA
oxidase activity. In
certain embodiments, the genetic alteration adds or increases the number of
copies of a
polynucleotide encoding an acyl-CoA oxidase activity and/or increases the
activity of a promoter
and/or 5'UTR in functional connection with a polynucleotide encoding an acyl-
CoA oxidase activity.
In some embodiments, the feedstock comprises a 6-carbon sugar. In certain
embodiments, the
feedstock comprises a 5-carbon sugar. In some embodiments, the feedstock
comprises a fatty
acid, and in certain embodiments the feedstock comprises a mixture of fatty
acids. In some
embodiments, the feedstock comprises a triacylglyceride. In certain
embodiments, the adipic acid
is produced at a level of about 80% or more of theoretical yield. In some
embodiments, the
amount of adipic acid produced is detected. In certain embodiments, the adipic
acid produced is
isolated (e.g., partially or completely purified). In some embodiments, the
culture conditions
comprise fermenting the engineered microorganism.
Provided also herein are engineered microorganisms in contact with a
feedstock. In some
embodiments, the feedstock includes a saccharide. In certain embodiments, the
saccharide is a
monosaccharide, polysaccharide, or a mixture of a monosaccharide and
polysaccharide. In some
embodiments, the feedstock includes a paraffin. In certain embodiments, the
paraffin is a
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saturated paraffin, unsaturated paraffin, substituted paraffin, branched
paraffin, linear paraffin, or
combination thereof.
In some embodiments, the paraffin includes about 1 to about 60 carbon atoms
(e.g., between
about 1 carbon atom, about 2 carbon atoms, about 3 carbon atoms, about 4
carbon atoms, about 5
carbon atoms, about 6 carbon atoms, about 7 carbon atoms, about 8 carbon
atoms, about 9
carbon atoms, about 10 carbon atoms, about 12 carbon atoms, about 14 carbon
atoms, about 16
carbon atoms, about 18 carbon atoms, about 20 carbon atoms, about 22 carbon
atoms, about 24
carbon atoms, about 26 carbon atoms, about 28 carbon atoms, about 30 carbon
atoms, about 32
carbon atoms, about 34 carbon atoms, about 36 carbon atoms, about 38 carbon
atoms, about 40
carbon atoms, about 42 carbon atoms, about 44 carbon atoms, about 46 carbon
atoms, about 48
carbon atoms, about 50 carbon atoms, about 52 carbon atoms, about 54 carbon
atoms, about 56
carbon atoms, about 58 carbon atoms and about 60 carbon atoms). In certain
embodiments, the
paraffin is in a mixture of paraffins. In some embodiments, the paraffins in
the mixture of paraffins
have a mean number of carbon atoms of about 8 carbon atoms to about 18 carbon
atoms (e.g.,
about 8 carbon atoms, about 9 carbon atoms, about 10 carbon atoms, about 11
carbon atoms,
about 12 carbon atoms, about 13 carbon atoms, about 14 carbon atoms, about 15
carbon atoms,
about 16 carbon atoms, about 17 carbon atoms or about 18 carbon atoms). In
certain
embodiments, the paraffin is in a wax, and in some embodiments, the paraffin
is in an oil. In some
embodiments, the paraffin contains one or more fatty acids. In certain
embodiments, the paraffin is
from a petroleum product, and in some embodiments, the petroleum product is a
petroleum
distillate. In certain embodiments, the paraffin is from a plant or plant
product.
Also provided herein, is an isolated polynucleotide selected from the group
including a
polynucleotide having a nucleotide sequence 96% or more (e.g., 96% or more,
97% or more, 98%
or more, 99% or more, or 100%) identical to the nucleotide sequence of SEQ ID
NO: 1, a
polynucleotide having a nucleotide sequence that encodes a polypeptide of SEQ
ID NO: 8, and a
polynucleotide having a portion of a nucleotide sequence 96% or more identical
to the nucleotide
sequence of SEQ ID NO: 1 and encodes a polypeptide having fatty alcohol
oxidase activity.
Also provided herein, is an isolated polynucleotide selected from the group
including a
polynucleotide having a nucleotide sequence 98% or more (e.g., 98% or more,
99% or more, or
100%) identical to the nucleotide sequence of SEQ ID NO: 2, a polynucleotide
having a nucleotide
sequence that encodes a polypeptide of SEQ ID NO:10, and a polynucleotide
having a portion of a
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nucleotide sequence 98% or more identical to the nucleotide sequence of SEQ ID
NO: 2 and
encodes a polypeptide having fatty alcohol oxidase activity.
Also provided herein, is an isolated polynucleotide selected from the group
including a
polynucleotide having a nucleotide sequence 95% or more (e.g., 95% or more,
96% or more, 97%
or more, 98% or more, 99% or more, or 100%) identical to the nucleotide
sequence of SEQ ID NO:
3, a polynucleotide having a nucleotide sequence that encodes a polypeptide of
SEQ ID NO: 9,
and a polynucleotide having a portion of a nucleotide sequence 95% or more
identical to the
nucleotide sequence of SEQ ID NO: 3 and encodes a polypeptide having fatty
alcohol oxidase
activity.
Also provided herein, is an isolated polynucleotide selected from the group
including a
polynucleotide having a nucleotide sequence 83% or more (e.g., 83% or more,
84% or more, 85%
or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91%
or more, 92%
or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98%
or more, 99%
or more, or 100%) identical to the nucleotide sequence of SEQ ID NO: 4, a
polynucleotide having a
nucleotide sequence that encodes a polypeptide of SEQ ID NO: 11, and a
polynucleotide having a
portion of a nucleotide sequence 83% or more identical to the nucleotide
sequence of SEQ ID NO:
3 and encodes a polypeptide having fatty alcohol oxidase activity.
Also provided herein, is an isolated polynucleotide selected from the group
including a
polynucleotide having a nucleotide sequence 82% or more (e.g., 82% or more,
83% or more, 84%
or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90%
or more, 91%
or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97%
or more, 98%
or more, 99% or more, or 100%) identical to the nucleotide sequence of SEQ ID
NO: 5, a
polynucleotide having a nucleotide sequence that encodes a polypeptide of SEQ
ID NO: 12, and a
polynucleotide having a portion of a nucleotide sequence 82% or more identical
to the nucleotide
sequence of SEQ ID NO: 3 and encodes a polypeptide having fatty alcohol
oxidase activity.
Also provided herein, is an isolated polynucleotide having a polynucleotide
identical to the
polynucleotide of SEQ ID NO: 13, or fragments thereof and encodes a
polypeptide having
monooxygenase activity. Also provided herein, is an isolated polynucleotide
having a
polynucleotide 96% or more identical to the polynucleotide of SEQ ID NO: 14 or
15, or fragments
thereof and encodes a polypeptide having monooxygenase activity. Also provided
herein, is an
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isolated polynucleotide having a polynucleotide 96% or more identical to the
polynucleotide of SEQ
ID NO 16 or 17, or fragments thereof and encodes a polypeptide having
monooxygenase activity.
Also provided herein, is an isolated polynucleotide having a polynucleotide
94% or more identical
to the polynucleotide of SEQ ID NO 18 or 19, or fragments thereof and encodes
a polypeptide
having monooxygenase activity. Also provided herein, is an isolated
polynucleotide having a
polynucleotide 95% or more identical to the polynucleotide of SEQ ID NO 20 or
21, or fragments
thereof and encodes a polypeptide having monooxygenase activity. Also provided
herein, is an
isolated polynucleotide comprising a nucleotide sequence of any one of SEQ ID
NOs: 23 to 26, or
fragment thereof that encodes a polypeptide having monooxygenase reductase
activity.
Also provided herein, is an isolated polynucleotide (i) comprising a
nucleotide sequence identical to
the nucleotide sequence of SEQ ID NO: 27 or fragment thereof, that encodes a
polypeptide, or (ii)
a polynucleotide that encodes a polypeptide of SEQ ID NO: 28, the polypeptide
having lipase
activity. Also provided herein is a polypeptide having an amino acid sequence
identical to the
polypeptide of SEQ ID NO: 29, the polypeptide having lipase activity.
Also provided herein, is an isolated polynucleotide having a nucleotide
sequence identical to the
nucleotide sequence of SEQ ID NO: 30 or fragments thereof and encodes a
polypeptide having
acetyl-CoA carboxylase activity. Also provided herein, is an isolated
polynucleotide selected from
the group including polynucleotides having a nucleotide sequence identical to
the nucleotide
sequence of any one of SEQ ID NOs: 31 and 32, or fragments thereof and encodes
a polypeptide
having fatty acid synthase activity.
Also provided herein, is an isolated polynucleotide having a nucleotide
sequence identical to the
nucleotide sequence of SEQ ID NO: 33 or fragments thereof and encoding a
polypeptide having
glucose-6-phosphate dehydrogenase activity. Also provided herein is a
polypeptide having an
amino acid sequence identical to the polypeptide of SEQ ID NO: 34, the
polypeptide having
glucose-6-phosphate dehydrogenase activity.
Also provided herein, is an isolated polynucleotide selected from the group
including a
polynucleotide having a nucleotide sequence 96% or more (e.g., 96% or more,
97% or more, 98%
or more, 99% or more, or 100%) identical to the nucleotide sequence of SEQ ID
NO: 42 or 44, a
polynucleotide having a nucleotide sequence that encodes a polypeptide having
an amino acid
sequence 98% or more (e.g., 98% or more, 99% or more, or 100%) identical to
the amino acid
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sequence of SEQ ID NO: 43 or 45, and a polynucleotide having a portion of a
nucleotide sequence
96% or more identical to the nucleotide sequence of SEQ ID NO: 42 or 44 and
encodes a
polypeptide having acyl-CoA hydrolase activity.
Also provided herein, is an isolated polynucleotide having a polynucleotide
sequence identical to
the polynucleotide sequence of SEQ ID NO: 45, or fragments thereof and encodes
a polypeptide of
SEQ ID NO:46 having acyl-CoA thioesterase activity.
Provided also herein, is an engineered microorganism capable of producing
adipic acid, the
microorganism including genetic alterations resulting in commitment of
molecular pathways in
directions for production of adipic acid, the pathways and directions include:
(i) fatty acid synthesis
pathway in the direction of acetyl CoA to long-chain fatty acids, and away
from generation of
biomass and/or carbon storage molecules (e.g., starch, lipids,
triacylglycerides) and/or utilization of
fatty acids for energy, (ii) omega oxidation pathway in the direction of long-
chain fatty acids to
diacids and (iii) beta oxidation pathway in the direction of diacids to adipic
acid. Also provided
herein, is an engineered microorganism capable of producing adipic acid, which
microorganism
comprises genetic alterations resulting in three or more increased activities,
relative to the
microorganism not containing the genetic alterations, selected from the group
consisting of acetyl
CoA carboxylase activity, fatty acid synthase activity, monooxygenase
activity, monooxygenase
reductase activity, acyl-CoA hydrolase activity, acyl-CoA thioesterase
activity and acyl-CoA
oxidase activity.
Also provided herein, is an engineered microorganism capable of producing
adipic acid, the
microorganism including genetic alterations resulting in commitment of
molecular pathways in
directions for production of adipic acid, the pathways and directions include:
(i) hexanoic acid
synthesis pathway in the direction of acetyl CoA to hexanoic acid, and (ii)
omega oxidation
pathway in the direction of hexanoic acid to adipic acid. Provided also
herein, is an engineered
microorganism capable of producing adipic acid, which microorganism comprises
genetic
alterations resulting in three or more increased activities, relative to the
microorganism not
containing the genetic alterations, selected from the group consisting of
acetyl CoA carboxylase
activity, acyl-CoA hydrolase activity, acyl-CoA thioesterase activity,
hexanoate synthase activity,
monooxygenase activity and monooxygenase reductase activity.
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Provided also herein, is an engineered microorganism capable of producing
adipic acid, the
microorganism includes genetic alterations resulting in commitment of
molecular pathways in
directions for production of adipic acid, the pathways and directions include:
(i) gluconeogenesis
pathway in the direction of triacyl glycerides to 6-phosphoglucono-lactone and
nicotinamide
microorganism not containing the genetic alterations, independently in each
pathway. In certain
embodiments, an engineered microorganism includes an increased acetyl CoA
carboxylase activity
in the fatty acid synthesis pathway. In some embodiments, an engineered
microorganism includes
an increased fatty acid synthase activity in the fatty acid synthesis pathway.
In certain
In certain embodiments, an engineered microorganism includes an increased
acetyl CoA
carboxylase activity in the hexanoic acid synthesis pathway. In some
embodiments, an
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In some embodiments, an engineered microorganism includes an increased lipase
activity in the
gluconeogenesis pathway. In certain embodiments, an engineered microorganism
includes an
increased glucose-6-phosphate dehydrogenase activity in the gluconeogenesis
pathway. In some
embodiments, an engineered microorganism includes an increased acyl-CoA
oxidase activity in
the beta oxidation pathway. In certain embodiments, an engineered
microorganism includes an
increased acyl-CoA hydrolase and/or an increased acyl-CoA thioesterase in the
fatty acid
synthesis pathway
In some embodiments, one or more enzymes or proteins can provide an activity
(e.g., single
subunit protein, multi subunit protein). In certain embodiments, 2 or more
activities can be
provided by one or more enzymes or proteins (e.g., multifunction single
protein, enzyme complex).
In some embodiments, each of the increased activities independently is
provided by an enzyme
encoded by a gene endogenous to the microorganism. In certain embodiments,
each of the
increased activities independently is provided by an enzyme encoded by a gene
exogenous to the
microorganism. In some embodiments, each of the increased activities
independently is provided
by an increased amount of an enzyme from a yeast. In some embodiments, the
microorganism is
a Candida yeast. In certain embodiments, the microorganism is a C. tropicalis
strain, and in some
embodiments, the microorganism is a C. viswanithii strain. In certain
embodiments, the
microorganism is a Yarrowia yeast, and in some embodiments, the microorganism
is a Yarrowia
lipolytica strain.
In some embodiments, each one of the increased activities independently
results from increasing
the copy number of a gene that encodes an enzyme that provides the activity.
In certain
embodiments, each one of the increased activities independently results from
inserting a promoter
in functional proximity to a gene that encodes an enzyme that provides the
activity. In some
embodiments, the gene is in plasmid nucleic acid, and in certain embodiments,
the gene is in
genomic nucleic acid of the microorganism.
In some embodiments, the acetyl CoA carboxylase activity is provided by an
increased amount of
an enzyme comprising (i) the amino acid sequence encoded by SEQ ID NO: 30,
(ii) an amino acid
sequence 90% or more identical to (i), or (iii) an amino acid sequence that
includes 1 to 10 amino
acid substitutions, insertions or deletions with respect to (i).
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In certain embodiments, the fatty acid synthase activity is provided by an
increased amount of a
FAS1-encoded enzyme, a FAS2-encoded enzyme, or FAS1-encoded enzyme and FAS2-
encoded
enzyme. In some embodiments, the FAS1-encoded enzyme comprises (i) the amino
acid
sequence encoded by SEQ ID NO: 32, (ii) an amino acid sequence 90% or more
identical to (i), or
(iii) an amino acid sequence that includes 1 to 10 amino acid substitutions,
insertions or deletions
with respect to (i). In certain embodiments, the FAS2-encoded enzyme comprises
(i) the amino
acid sequence encoded by SEQ ID NO: 31, (ii) an amino acid sequence 90% or
more identical to
(i), or (iii) an amino acid sequence that includes 1 to 10 amino acid
substitutions, insertions or
deletions with respect to (i).
In some embodiments, the monooxygenase activity is provided by an increased
amount of a
cytochrome P450 enzyme. In certain embodiments, the monooxygenase activity is
provided by an
exogenous cytochrome P450 enzyme. In some embodiments, the exogenous
cytochrome P450
enzyme is from Bacillus megaterium. In certain embodiments, the exogenous
cytochrome P450
enzyme comprises (i) the amino acid sequence of SEQ ID NO: 41, (ii) an amino
acid sequence
90% or more identical to (i), or (iii) an amino acid sequence that includes 1
to 10 amino acid
substitutions, insertions or deletions with respect to (i).
In some embodiments, two or more endogenous cytochrome P450 enzymes are
expressed in
increased amounts. In certain embodiments, all endogenous cytochrome P450
enzymes are
expressed in increased amounts. In some embodiments, the endogenous cytochrome
P450
enzymes comprise (i) an amino acid sequence encoded by a polynucleotide
selected from the
group consisting of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22,
(ii) an amino acid
sequence 90% or more identical to (i), or (iii) an amino acid sequence that
includes 1 to 10 amino
acid substitutions, insertions or deletions with respect to (i).
In certain embodiments, the monooxygenase reductase activity is provided by an
increased
amount of an enzyme comprising (i) the amino acid sequence encoded by any one
of SEQ ID
NOS: 23 to 26, (ii) an amino acid sequence 90% or more identical to (i), or
(iii) an amino acid
sequence that includes 1 to 10 amino acid substitutions, insertions or
deletions with respect to (i).
In some embodiments, the monooxygenase reductase activity is provided by an
increased amount
of a cytochrome P450:NADPH P450 reductase-encoded enzyme, a CPR-encoded
enzyme, a
CPRA-encoded enzyme, a CPRB-encoded enzyme, or a cytochrome P450:NADPH P450
reductase-encoded enzyme, a CPR-encoded enzyme, a CPRA-encoded enzyme, and/or
a CPRB-
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encoded enzyme. In certain embodiments, the cytochrome P450:NADPH P450
reductase-
encoded enzyme comprises (i) the amino acid sequence of SEQ ID NO: 41 (ii) an
amino acid
sequence 90% or more identical to (i), or (iii) an amino acid sequence that
includes 1 to 10 amino
acid substitutions, insertions or deletions with respect to (i). In some
embodiments, the CPR-,
CPRA and/or CPRB encoded enzymes comprise (i)an amino acid sequence encoded by
any one
of SEQ ID NOS: 24 to 26, (ii) an amino acid sequence 90% or more identical to
(i), or (iii) an amino
acid sequence that includes 1 to 10 amino acid substitutions, insertions or
deletions with respect to
(i).
In some embodiments, the acyl-CoA oxidase activity is provided by an increased
amount of a
PDX4-encoded enzyme, a PDX5-encoded enzyme, or a PDX4-encoded enzyme and a
PDX5-
encoded enzyme an enzyme. In certain embodiments, the PDX4-encoded enzyme
comprises (i)
the amino acid sequence of SEQ ID NO: 39, (ii) an amino acid sequence 90% or
more identical to
(i), or (iii) an amino acid sequence that includes 1 to 10 amino acid
substitutions, insertions or
deletions with respect to (i). In some embodiments, the PDX5-encoded enzyme
comprises (i) the
amino acid sequence of SEQ ID NO: 40, (ii) an amino acid sequence 90% or more
identical to (i),
or (iii) an amino acid sequence that includes 1 to 10 amino acid
substitutions, insertions or
deletions with respect to (i). In certain embodiments, the microorganism lacks
an enzyme
providing an acyl-CoA oxidase activity. In certain embodiments, the enzyme is
a PDX4-encoded
enzyme or a PDX5-encoded enzyme. In certain embodiments, the PDX4
polynucleotide that
encodes the PDX4-encoded enzyme comprises (i) the polynucleotide of SEQ ID NO:
37, (ii) a
polynucleotide 90% or more identical to (i), or (iii) polynucleotide that
includes 1 to 10 nucleotide
substitutions, insertions or deletions with respect to (i). In some
embodiments, the PDX5
polynucleotide that encodes the PDX5-encoded enzyme comprises (i) the
polynucleotide of SEQ
ID NO: 38, (ii) a polynucleotide 90% or more identical to (i), or (iii) a
polynucleotide that includes 1
to 10 amino acid substitutions, insertions or deletions with respect to (i).
In certain embodiments,
the microorganism lacks an enzyme providing an acyl-CoA oxidase activity, and
sometimes lacks a
PDX4-encoded enzyme or a PDX5-encoded enzyme.
In certain embodiments, the hexanoate synthase activity is provided by an
increased amount of a
HEXA-encoded protein, a HEXB-encoded protein, or HEXA-encoded protein and HEXB-
encoded
protein. In some embodiments, the HEXA-encoded protein comprises (i) the amino
acid sequence
encoded by SEQ ID NO: 35, (ii) an amino acid sequence 90% or more identical to
(i), or (iii) an
amino acid sequence that includes 1 to 10 amino acid substitutions, insertions
or deletions with
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respect to (i). In certain embodiments, the HEXB-encoded protein comprises (i)
the amino acid
sequence encoded by SEQ ID NO: 36, (ii) an amino acid sequence 90% or more
identical to (i), or
(iii) an amino acid sequence that includes 1 to 10 amino acid substitutions,
insertions or deletions
with respect to (i).
In some embodiments, the lipase activity is provided by an increased amount of
an enzyme
comprising (i) the amino acid sequences of SEQ ID NO: 28 or 29, (ii) an amino
acid sequence 90%
or more identical to (i), or (iii) an amino acid sequence that includes 1 to
10 amino acid
substitutions, insertions or deletions with respect to (i). In some
embodiments, the lipase activity is
provided by an increased amount of an enzyme encoded by a polynucleotide
comprising (i) the
polynucleotide of SEQ ID NO: 27, (ii) a polynucleotide 90% or more identical
to (i), or (iii) a
polynucleotide that includes 1 to 10 nucleotide substitutions, insertions or
deletions with respect to
(i).
In certain embodiments, the glucose-6-phosphate dehydrogenase activity is
provided by an
increased amount of an enzyme comprising (i) the amino acid sequence of SEQ ID
NO: 34, (ii) an
amino acid sequence 90% or more identical to (i), or (iii) an amino acid
sequence that includes 1 to
10 amino acid substitutions, insertions or deletions with respect to (i). In
some embodiments, the
glucose-6-phosphate dehydrogenase activity is provided by an increased amount
of an enzyme
encoded by a polynucleotide comprising (i) the polynucleotide of SEQ ID NO:
33, (ii) a
polynucleotide 90% or more identical to (i), or (iii) a polynucleotide that
includes 1 to 10 nucleotide
substitutions, insertions or deletions with respect to (i).
In some embodiments, the acyl-CoA hydrolase activity is provided by an
increased amount of an
enzyme comprising (i) the amino acid sequence encoded by SEQ ID NO: 43 or 45,
(ii) an amino
acid sequence 98% or more identical to (i), or (iii) an amino acid sequence
that includes 1 to 10
amino acid substitutions, insertions or deletions with respect to (i).
In some embodiments, the acyl-CoA thioesterase activity is provided by an
increased amount of an
enzyme comprising (i) the amino acid sequence encoded by SEQ ID NO: 47, or
(ii) an amino acid
sequence that includes 1 to 10 amino acid substitutions, insertions or
deletions with respect to (i).
In some embodiments, the microorganism is a yeast. In some embodiments, the
microorganism is
a Candida yeast. In certain embodiments, the microorganism is a C. tropicalis
strain, and in some
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embodiments, the microorganism is a C. viswanithii strain. In certain
embodiments, the
microorganism is a Yarrowia yeast, and in some embodiments, the microorganism
is a Yarrowia
lipolytica strain. In certain embodiments, the microorganism is a haploid and
in some
embodiments, the microorganism is a diploid
In certain embodiments, an expression vector includes a polynucleotide
sequence or expresses an
amino acid sequence of any one of SEQ ID NOS: 1 to 59. In some embodiments, an
integration
vector includes a polynucleotide sequence or expresses an amino acid sequence
of any one of
SEQ ID NOS: 1 to 59. In certain embodiments, a microorganism includes an
expression vector, an
integration vector, or an expression vector and an integration vector that
includes a polynucleotide
sequence of SEQ ID NOS: 1 to 59. In some embodiments, a culture includes a
microorganism that
includes an expression vector, an integration vector, or an expression vector
and an integration
vector that includes a polynucleotide sequence or expresses an amino acid
sequence of any one
of SEQ ID NOS: 1 to 59. In certain embodiments, a fermentation device includes
a microorganism
that includes an expression vector, an integration vector, or an expression
vector and an
integration vector that includes a polynucleotide sequence or expresses an
amino acid sequence
of any one of SEQ ID NOS: 1 to 59. In some embodiments an integration vector
is used to disrupt
a polynucleotide sequence. Also provided herein is a polypeptide or a
polypeptide encoded by a
polynucleotide sequence of any one of SEQ ID NOS: 1 to 47 or produced by an
expression vector
that includes a polynucleotide sequence of, or expresses an amino acid
sequence of any one of
SEQ ID NOS: 1 to 47. Provided also herein is an antibody that specifically
binds to a polypeptide
of, or is encoded by a polynucleotide sequence or expresses an amino acid
sequence of any one
of SEQ ID NOS: 1 to 59 or produced by an expression vector that includes a
polynucleotide
sequence or expresses an amino acid sequence of any one of SEQ ID NOS: 1 to
59.
Also provided herein is a method for producing adipic acid, the method
including culturing an
engineered microorganism described herein under conditions in which adipic
acid is produced. In
some embodiments, the culture conditions include fermentation conditions. In
certain
embodiments, the culture conditions include introduction of biomass. In some
embodiments, the
culture conditions include introduction of a feedstock comprising glucose. In
certain embodiments,
the culture conditions include introduction of a feedstock comprising hexane.
In some
embodiments, the culture conditions include introduction of a feedstock
comprising an oil.
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In certain embodiments, the adipic acid (and/or adipate) is produced with a
yield of greater than
about 0.15 grams per gram of the glucose, hexane or oil. In some embodiments,
the adipic acid
(and/or adipate) is produced at between about 20% and about 100`)/0 of maximum
theoretical yield
of any introduced feedstock (e.g., about 25%, about 30%, about 35%, about 40%,
about 45%,
about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,
about 85%,
about 90%, about 95%, about 99%, or about 100`)/0 of theoretical maximum
yield) for the feedstock
utilized. In certain embodiments, the adipic acid (and/or adipate) is produced
in a concentration
range of between about 1 g/L (grams per liter) to about 1000g/L of culture
media, fermentation
medium or fermentation broth (e.g., about 2 g/L, about 3 g/L, about 4 g/L,
about 5 g/L, about 10
g/L, about 15 g/L, about 20 g/L, about 25 g/L, about 30 g/L, about 35 g/L,
about 40 g/L, about 45
g/L, about 50 g/L, about 55 g/L, about 60 g/L, about 65 g/L, about 70 g/L,
about 75 g/L, about 80
g/L, about 85 g/L, about 90 g/L, about 95 g/L, about 100 g/L, about 110 g/L,
about 120 g/L, about
130 g/L, about 140 g/L, about 150 g/L, about 160 g/L, about 170 g/L, about 180
g/L, about 190 g/L,
about 200 g/L, about 225 g/L, about 250 g/L, about 275 g/L, about 300 g/L,
about 325 g/L, about
350 g/L, about 375 g/L, about 400 g/L, about 425 g/L, about 450 g/L, about 475
g/L, about 500 g/L,
about 550 g/L, about 600 g/L, about 650 g/L, about 700 g/L, about 750 g/L,
about 800 g/L, about
850 g/L, about 900 g/L, about 950 g/L, or about 1000 g/L).
In some embodiments, the adipic acid (and/or adipate) is produced at a rate of
between about 0.5
g/L/hour to about 5 g/L/hour (e.g., about 0.5 g/L/hour, about 0.6 g/L/hour,
about 0.7 g/L/hour, about
0.8 g/L/hour, about 0.9 g/L/hour, about 1.0 g/L/hour, about 1.1 g/L/hour,
about 1.2 g/L/hour, about
1.3 g/L/hour, about 1.4 g/L/hour, about 1.5 g/L/hour, about 1.6 g/L/hour,
about 1.7 g/L/hour, about
1.8 g/L/hour, about 1.9 g/L/hour, about 2.0 g/L/hour, about 2.25 g/L/hour,
about 2.5 g/L/hour, about
2.75 g/L/hour, about 3.0 g/L/hour, about 3.25 g/L/hour, about 3.5 g/L/hour,
about 3.75 g/L/hour,
about 4.0 g/L/hour, about 4.25 g/L/hour, about 4.5 g/L/hour, about 4.75
g/L/hour, or about 5.0
g/L/hour.) In certain, embodiments, the engineered organism comprises between
about a 5-fold to
about a 500-fold increase in adipic acid production (and/or adipate
production) when compared to
wild-type or partially engineered organisms of the same strain, under
identical fermentation
conditions (e.g., about a 5-fold increase, about a 10-fold increase, about a
15-fold increase, about
a 20-fold increase, about a 25-fold increase, about a 30-fold increase, about
a 35-fold increase,
about a 40-fold increase, about a 45-fold increase, about a 50-fold increase,
about a 55-fold
increase, about a 60-fold increase, about a 65-fold increase, about a 70-fold
increase, about a 75-
fold increase, about a 80-fold increase, about a 85-fold increase, about a 90-
fold increase, about a
95-fold increase, about a 100-fold increase, about a 125-fold increase, about
a 150-fold increase,
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about a 175-fold increase, about a 200-fold increase, about a 250-fold
increase, about a 300-fold
increase, about a 350-fold increase, about a 400-fold increase, about a 450-
fold increase, or about
a 500-fold increase).
In some embodiments, a method includes purifying the adipic acid from the
cultured
microorganisms. In certain embodiments, the method includes modifying the
adipic acid, thereby
producing modified adipic acid. In some embodiments, the method includes
placing the cultured
microorganisms, the adipic acid or the modified adipic acid in a container,
and in certain
embodiments, the method includes shipping the container.
In some embodiments, provided is a chimera exhibiting a fatty acid synthase
and/or hexanoate
synthase activity. In some embodiments, the chimera is encoded by a nucleotide
sequence that
includes a donor sequence in a base sequence. In certain embodiments, a donor
sequence
replaces a sequence earlier excised from the base sequence (excised sequence).
There can be
one or more donor sequences, and optionally one or more excised sequences, in
a particular
polynucleotide that encodes a chimera. In certain embodiments, the base
sequence and donor
sequence are from fatty acid synthase polynucleotides from different
organisms. In some
embodiments, each fatty acid synthase polynucleotide independently is obtained
from a fungus or
yeast (e.g., Candida yeast (e.g., C. tropicalis, C. viswanithii)). In certain
embodiments, the donor
sequence and/or base sequence is from a hexanoate synthase subunit A or B
(e.g., HEXA,
HEXB), such as a HEXA or HEXB sequence described herein. In some embodiments,
the donor
sequence or base sequence is from a fatty acid synthase gene of a fungus or
yeast (e.g., Candida
(e.g., C. tropicalis, C. viswanithii).
In certain chimera embodiments, the excised sequence and/or the donor sequence
encodes a
functional polypeptide, or portion thereof, that adds malonyl units to a
growing fatty acid chain,
and/or removes a grown fatty acid chain (e.g., palmitoyl fatty acid or
derivative) from the
polypeptide or portion thereof. In some embodiments, the donor sequence and/or
excised
sequence encodes a malonyl-palmitoyl transferase domain (MPT domain) from a
hexanoate
synthase gene or fatty acid synthase gene. In certain embodiments, the donor
sequence and/or
excised sequence encodes all or part of the functional polypeptide or domain
described above, and
optionally includes one or more additional nucleotides or stretches of
contiguous polynucleotides.
In some embodiments, the donor sequence or excised sequence is about 900
contiguous
nucleotides to about 1500 contiguous nucleotides in length, and sometimes
about 1200 contiguous
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nucleotides in length. In certain embodiments, the base sequence and/or donor
sequence
independently are (i) identical to a native sequence, (ii) 90% or more
identical to a native
sequence, or (iii) include 1 to 10 insertions, deletions or substitutions with
respect to a native
sequence. In some embodiments, the native donor sequence is from HEXB and the
native base
sequence is from FAS1. Examples of each of these sequences are provided
herein.
In some chimera embodiments, the donor sequence and/or excised sequence
encodes a ketoacyl
synthase domain (KS domain) from a hexanoate synthase gene or fatty acid
synthase gene. In
certain embodiments, the donor sequence and/or excised sequence encodes all or
part of the
domain described above, and optionally includes one or more additional
nucleotides or stretches of
contiguous polynucleotides. In some embodiments, the donor sequence or excised
sequence is
about 900 contiguous nucleotides to about 1700 contiguous nucleotides in
length, and sometimes
about 1350 contiguous nucleotides in length. In certain embodiments, the base
sequence and/or
donor sequence independently are (i) identical to a native sequence, (ii) 90%
or more identical to a
native sequence, or (iii) include 1 to 10 insertions, deletions or
substitutions with respect to a native
sequence. In some embodiments, the native donor sequence is from HEXA and the
native base
sequence is from FAS2. Examples of each of these sequences are provided
herein.
In certain chimera embodiments, donor sequences from FAS1 are used to fill in
regions of a base
HexB sequence that are not present in HexB. Donor sequences may be selected,
in some
embodiments, based upon an alignment of HexB and FAS1 sequences and
identifying sequences
present in FAS1 that do not align with, and/or appear to be inserted with
respect to, HexB. In
some embodiments, the donor sequence is about 100 contiguous nucleotides or
less from FAS1.
In certain embodiments, the base sequence and/or donor sequence independently
are (i) identical
to a native sequence from HexB and FAS1, respectively, (ii) 90% or more
identical to the native
sequence, or (iii) include 1 to 10 insertions, deletions or substitutions with
respect to the native
sequence.
Provided also herein are genetically modified microorganisms including one or
more increased
activities, with respect to the activity level in an unmodified or parental
strain, which increased
activities are chosen from: a monooxygenase activity, a monooxygenase
reductase activity, an
acyl-CoA oxidase activity, an acyl-CoA hydrolase activity, an acyl-CoA
thioesterase activity and
combinations of the forgoing. In some embodiments, the monooxygenase activity
includes a
cytochrome P450 A19 (e.g., CYP52A19) activity. In certain embodiments, the
monooxygenase
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activity is a cytochrome P450 A19 (e.g., CYP52A19) activity. In some
embodiments, the
monooxygenase reductase activity includes one or more activities selected from
CPR, CPRA,
CPRB, and combinations of the foregoing.
In certain embodiments, the acyl-CoA oxidase activity includes a P0X5
activity. In some
embodiments, the acyl-CoA oxidase activity is a PDX5 activity. In certain
embodiments, the acyl-
CoA hydrolase activity includes one or more activities selected from ACHA
activity, ACHB activity,
and ACHA activity and ACHB activity. In some embodiments, the acyl-CoA
thioesterase activity
includes a TESA activity.
In some embodiments, the one or more increased activities are three or more
increased activities.
In certain embodiments, the one or more increased activities are four or more
increased activities,
and in some embodiments, the one or more increased activities are five or more
increased
activities. In certain embodiments, the three or more increased activities
include an increased
monooxygenase activity, an increased monooxygenase reductase activity, and an
increased acyl-
CoA hydrolase activity. In some embodiments, the three or more increased
activities include an
increased monooxygenase activity, an increased monooxygenase reductase
activity, and an
increased acyl-CoA thioesterase activity. In certain embodiments, the four or
more increased
activities include an increased monooxygenase activity, an increased
monooxygenase reductase
activity, an increased acyl-CoA oxidase activity and an increased acyl-CoA
hydrolase activity. In
some embodiments, the four or more increased activities include an increased
monooxygenase
activity, an increased monooxygenase reductase activity, an increased acyl-CoA
hydrolase activity
and an increased acyl-CoA thioesterase activity. In certain embodiments, the
five or more
increased activities include an increased monooxygenase activity, an increased
monooxygenase
reductase activity, an increased acyl-CoA oxidase activity, and increased acyl-
CoA thioesterase
activity and an increased acyl-CoA hydrolase activity.
In some embodiments, genetically modified microorganisms further include one
or more reduced
activities, with respect to the activity level in an unmodified or parental
strain, which reduced
activities are chosen from: acyl-CoA synthetase activity, long chain acyl-CoA
synthetase activity,
acyl-CoA sterol acyl transferase activity, acyltransferase activity, and
combinations of the
foregoing. In certain embodiments, the acyl-CoA synthetase activity includes
an ACS1 activity. In
some embodiments, the long chain acyl-CoA synthetase activity includes a FAT1
activity. In
certain embodiments, the acyl-CoA sterol acyl transferase activity includes
one or more activities
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selected from an ARE1 activity, an ARE2 activity, and an ARE1 activity and an
ARE2 activity. In
some embodiments, the acyltransferase activity is a diacylglycerol
acyltransferase activity, and in
certain embodiments, the diacylglycerol acyltransferase activity includes one
or more activities
selected from a DGA1 activity, a LRO1 activity and a DGA1 activity and a LRO1
activity.
In certain embodiments, the one or more reduced activities is three or more
reduced activities. In
some embodiments, the three or more reduced activities are four or more
reduced activities. In
certain embodiments, the three or more reduced activities are five or more
reduced activities. In
some embodiments, genetically modified microorganisms include a reduced ACS1
activity, a
reduced FAT1 activity, a reduced ARE1 activity, a reduced ARE2 activity, a
reduced DGA1 activity
and a reduced LRO1 activity.
Also provided herein, is a method for preparing a microorganism that produces
adipic acid, which
includes: (a) introducing one or more genetic modifications to a host organism
that add or increase
one or more activities selected from the group consisting of 6-oxohexanoic
acid dehydrogenase
activity, omega oxo fatty acid dehydrogenase activity, 6-hydroxyhexanoic acid
dehydrogenase
activity, omega hydroxyl fatty acid dehydrogenase activity, glucose-6-
phosphate dehydrogenase,
hexanoate synthase activity, lipase activity, fatty acid synthase activity,
acetyl CoA carboxylase
activity, acyl-CoA hydrolase activity, acyl-CoA thioesterase activity,
monooxygenase activity, and
monooxygenase reductase activity, thereby producing engineered microorganisms,
and (b)
selecting for engineered microorganisms that produce adipic acid. In some
embodiments a
method for preparing a microorganism that produces adipic acid further
includes selecting for
engineered microorganisms having one or more detectable and/or increased
activities selected
from the group consisting of 6-oxohexanoic acid dehydrogenase activity, omega
oxo fatty acid
dehydrogenase activity, 6-hydroxyhexanoic acid dehydrogenase activity, omega
hydroxyl fatty acid
dehydrogenase activity, glucose-6-phosphate dehydrogenase, hexanoate synthase
activity, lipase
activity, fatty acid synthase activity, acetyl CoA carboxylase activity, acyl-
CoA hydrolase activity,
acyl-CoA thioesterase activity, monooxygenase activity, and monooxygenase
reductase activity,
relative to the host microorganism. In certain embodiments, the method
includes introducing a
genetic modification that reduces one or more activities selected from acyl-
CoA oxidase, acyl-CoA
synthetase activity, long chain acyl-CoA synthetase activity, acyl-CoA sterol
acyl transferase
activity, acyltransferase activity, and 6-hydroxyhexanoic acid conversion
activity, thereby producing
engineered microorganisms, and selecting for engineered microorganisms having
one or more
reduced activities selected from acyl-CoA oxidase, acyl-CoA synthetase
activity, long chain acyl-
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CoA synthetase activity, acyl-CoA sterol acyl transferase activity,
acyltransferase activity, and 6-
hydroxyhexanoic acid conversion activity relative to the host microorganism.
Provided also herein is a method for producing adipic acid, including:
contacting an engineered
microorganism with a feedstock including one or more sugars, cellulose, fatty
acids,
triacylglycerides or combinations of the forgoing, wherein the engineered
microorganism includes:
(a) a genetic alteration that partially blocks beta oxidation activity, (b) a
genetic alteration that adds
or increases a monooxygenase activity, (c) a genetic alteration that adds or
increases a
monooxygenase reductase activity, and (d) a genetic alteration that adds or
increases an acyl-CoA
hydrolase and/or an acyl-CoA thioesterase activity, and culturing the
engineered microorganism
under conditions in which adipic acid is produced. In some embodiments, the
engineered
microorganism further includes one or more genetic alterations that reduce an
activity selected
from an acyl-CoA oxidase activity, an acyl-CoA synthetase activity, a long
chain acyl-CoA
synthetase activity, an acyl-CoA sterol acyl transferase activity, and an
acyltransferase activity.
In certain embodiments, the acyltransferase activity is a diacyl-glycerol
acyltransferase activity.
In some embodiments, the engineered microorganism includes a heterologous
polynucleotide
encoding a polypeptide having acyl-CoA thioesterase activity. In certain
embodiments, the
heterologous polynucleotide independently is selected from a bacterium. In
some embodiments,
the bacterium is an Enteric bacterium, and in certain embodiments, the Enteric
bacterium is E. coli.
In some embodiments, the genetically modified microorganism is a yeast. In
certain embodiments,
the genetically modified microorganism is a Candida yeast. In some
embodiments, the Candida
yeast is C. tropicalis, and in certain embodiments, the C. tropicalis is C.
tropicalis strain 20336. In
some embodiments, the Candida yeast is C. viswanithii.
In certain embodiments, the engineered microorganism includes a heterologous
polynucleotide
encoding a polypeptide having acyl-CoA thioesterase activity, and the
polynucleotide sequence
has been codon optimized for expression in C. tropicalis or C. viswanithii. In
some embodiments,
the genetic alteration that partially blocks beta oxidation activity reduces
or eliminates PDX4
activity. In certain embodiments, the genetic alteration that adds or
increases monooxygenase
activity, increases CYP52A19 activity. In some embodiments, the genetic
alteration that adds or
increases monooxygenase reductase activity increases a CPR activity, a CPRA
activity, a CPRB
activity, or combinations thereof.
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In some embodiments, the genetic alteration that adds or increases acyl-CoA
hydrolase activity
increases an ACHA activity, an ACHB activity or an ACHA activity and an ACHB
activity. In certain
embodiments, the genetic alteration that adds or increases acyl-CoA
thioesterase activity adds an
E. coli derived TESA activity. In some embodiments, the genetic alteration
that reduces an acyl-
CoA synthetase activity, reduces or eliminates an ACS1 activity. In certain
embodiments, the
genetic alteration that reduces a long chain acyl-CoA synthetase activity,
reduces or eliminates an
FAT1 activity. In some embodiments, the genetic alteration that reduces an
acyl-CoA sterol acyl
transferase activity, reduces or eliminates an ARE1 activity, an ARE2
activity, or an ARE1 activity
and an ARE2 activity. In certain embodiments, the genetic alteration that
reduces an
acyltransferase activity, reduces or eliminates a DGA1 activity, a LRO1
activity or a DGA1 activity
and a LRO1 activity.
In certain embodiments, the maximum theoretical yield (Y.) is about 0.6 grams
of adipic acid
produced per gram of coconut oil added, the percentage of Y. for the
engineered microorganism
under conditions in which adipic acid is produced is calculated as ( inY Y
= max, = = p/s = = Y
max *100,
where (Ypis) = [adipic acid (g/L] *final volume of culture in flask (L)] /
[feedstock added to flask (g)].
In some embodiments, the engineered microorganism produces adipic acid at
about 10% to about
100% of maximum theoretical yield.
Also provided herein is an isolated polynucleotide selected from the group
consisting of: a
polynucleotide having a nucleotide sequence 96% or more identical to the
nucleotide sequence of
SEQ ID NO: 42 or 44: a polynucleotide having a nucleotide sequence that
encodes a polypeptide
having an amino acid sequence 98% or more identical to the amino acid sequence
of SEQ ID NO:
43 or 45; and a polynucleotide having a portion of a nucleotide sequence 96%
or more identical to
the nucleotide sequence of SEQ ID NO: 42 or 44 and encodes a polypeptide
having acyl-coA
hydrolase activity. Provided also herein is an isolated polynucleotide
selected from the group
consisting of: a polynucleotide having a nucleotide sequence identical to the
nucleotide sequence
of SEQ ID NO: 46: a polynucleotide having a nucleotide sequence that encodes a
polypeptide of
SEQ ID NO: 47; and a polynucleotide having a portion of a nucleotide sequence
identical to the
nucleotide sequences of SEQ ID NO: 46 and encodes a polypeptide having acyl-
CoA thioesterase
activity. Also provided herein are expression vectors including a
polynucleotide sequence 96% or
more identical to the nucleotide sequence of SEQ ID NOS: 42 and 44. Provided
also herein are
expression vectors including a polynucleotide having the nucleotide sequence
of SEQ ID NO: 46.
Provided also herein are integration vectors including a polynucleotide
sequence 96% or more
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identical to the nucleotide sequence of SEQ ID NOS: 42 and 44. Provided also
herein are
integration vectors including a polynucleotide having the nucleotide sequence
of SEQ ID NO: 46.
Also provided herein are microorganisms including an expression vector and/or
an integration
vector including a polynucleotide sequence 96% or more identical to the
nucleotide sequence of
SEQ ID NOS: 42 and 44. Provided also here are microorganisms including an
expression vector
and/or an integration vector including a polynucleotide having the nucleotide
sequence of SEQ ID
NO: 46. Provided also herein is a culture including a microorganism that
includes an expression
vector and/or integration described herein. Also provided herein is a
fermentation device including
a microorganism that includes an expression vector and/or integration
described herein. Also
provided in some embodiments are antibodies that specifically bind to a
polypeptide produced from
an expression vector described herein. Provided also in some embodiments are
polypeptides
encoded by a polynucleotide SEQ ID NO: 42, 44, and 46, expressed by an
engineered
microorganism.
Also provided herein is an engineered microorganism capable of producing
adipic acid, which
microorganism includes genetic alterations resulting in one or more increased
activities and further
resulting in commitment of molecular pathways in directions for production of
adipic acid, which
pathways and directions include: (i) fatty acid synthesis pathway in the
direction of acetyl CoA to
long-chain fatty acids and away from synthesis or generation of biomass and/or
carbon storage
molecules, (ii) omega oxidation pathway in the direction of long-chain fatty
acids to diacids and (iii)
beta oxidation pathway in the direction of diacids to adipic acid. In some
embodiments, carbon
storage molecules include storage starches, storage lipids and combinations
thereof. In certain
embodiments, an engineered microorganism includes an increased activity,
relative to the
microorganism not containing the genetic alterations, independently in each
pathway.
In some embodiments, an engineered microorganism includes an increased
monooxygenase
activity. In certain embodiments, an engineered microorganism includes an
increased
monooxygenase reductase activity. In some embodiments, an engineered
microorganism includes
an increased acyl-CoA oxidase activity. In certain embodiments, an engineered
microorganism
includes an increased acetyl CoA carboxylase activity in the fatty acid
synthesis pathway. In some
embodiments, an engineered microorganism includes an increased fatty acid
synthase activity in
the fatty acid synthesis pathway. In certain embodiments, an engineered
microorganism includes
an increased acyl-CoA hydrolase activity in the fatty acid synthesis pathway.
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In some embodiments, the increased activities independently is provided by an
enzyme encoded
by a gene endogenous to the microorganism. In certain embodiments, each of the
increased
activities independently is provided by an increased amount of an enzyme from
a yeast. In some
embodiments, the microorganism is a Candida yeast. In certain embodiments, the
microorganism
is a C. tropicalis strain, and in some embodiments, the microorganism is a C.
viswanithii strain.
In certain embodiments, an engineered microorganism includes an added acyl-CoA
thioesterase
activity in the fatty acid synthesis pathway. In some embodiments, the added
activity
independently is provided by an enzyme encoded by a gene exogenous to the
microorganism. In
certain embodiments an engineered microorganism further includes one or more
reduced activities
selected from an acyl-CoA oxidase activity, an acyl-CoA synthetase activity, a
long chain acyl-CoA
synthetase activity, an acyl-CoA sterol acyl transferase activity, and an
acyltransferase activity. In
some embodiments, the acyltransferase activity is a diacyl-glycerol
acyltransferase activity.
Provided also herein is an engineered yeast including genetic modifications
that (a) reduce an
acyl-CoA oxidase activity and (b) reduce an acyl-CoA synthetase activity,
reduce a long chain acyl-
CoA synthetase activity, or reduce an acyl-CoA synthetase activity and reduce
a long chain acyl-
CoA synthetase activity. In some embodiments, an engineered yeast strain
further includes a
genetic modification that partially blocks beta oxidation. In certain
embodiments, the genetic
modification reduces the expression level of a polypeptide having an acyl-CoA
oxidase activity. In
some embodiments, the reduced acyl-CoA oxidase activity is a reduced PDX4
activity. In certain
embodiments, the reduced PDX4 activity results from a reduction in the level
of expression of a
polypeptide including (i) the amino acid sequence of SEQ ID NO: 39, (ii) an
amino acid sequence
90% or more identical to (i), or (iii) an amino acid sequence that includes 1
to 10 amino acid
substitutions, insertions or deletions with respect to (i).
In certain embodiments, an engineered yeast strain further includes genetic
modifications that
reduce the long chain acyl-CoA synthetase activity and do not reduce the acyl-
CoA synthetase
activity. In some embodiments, the reduced long chain acyl-CoA synthetase
activity is a FAT1
activity. In certain embodiments, the reduced FAT1 activity results from a
reduction in the level of
expression of a polypeptide including (i) the amino acid sequence of SEQ ID
NO: 51, (ii) an amino
acid sequence 90% or more identical to (i), or (iii) an amino acid sequence
that includes 1 to 10
amino acid substitutions, insertions or deletions with respect to (i). In some
embodiments, an
engineered yeast strain further includes genetic modifications that reduce the
acyl-CoA synthetase
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activity and do not reduce the long chain acyl-CoA synthetase activity. In
certain embodiments,
the reduced acyl-CoA synthetase activity is an ACS1 activity. In some
embodiments, the reduced
ACS1 activity results from a reduction in the level of expression of a
polypeptide including (i) the
amino acid sequence of SEQ ID NO: 49, (ii) an amino acid sequence 90% or more
identical to (i),
or (iii) an amino acid sequence that includes 1 to 10 amino acid
substitutions, insertions or
deletions with respect to (i). In certain embodiments, an engineered yeast
strain further includes
genetic modifications that reduce the long chain acyl-CoA synthetase activity
and the acyl-CoA
synthetase activity. In some embodiments, the reduced ACS1 activity results
from a reduction in
the level of expression of a polypeptide including (i) the amino acid sequence
of SEQ ID NO: 49,
(ii) an amino acid sequence 90% or more identical to (i), or (iii) an amino
acid sequence that
includes 1 to 10 amino acid substitutions, insertions or deletions with
respect to (i), and the
reduced FAT1 activity results from a reduction in the level of expression of a
polypeptide including
(iv) the amino acid sequence of SEQ ID NO: 51, (v) an amino acid sequence 90%
or more identical
to (iv), or (vi) an amino acid sequence that includes 1 to 10 amino acid
substitutions, insertions or
deletions with respect to (iv).
In some embodiments, an engineered yeast strain further includes one or more
genetic
modifications that increase one or more activities, with respect to the
activity level in an unmodified
or parental strain, which increased activities are chosen from: an acyl-CoA
oxidase activity, one or
more monooxygenase activities, a monooxygenase reductase activity and an acyl-
CoA hydrolase
activity. In certain embodiments, a genetic modification that increases an
acyl-CoA oxidase activity
increases a P0X5 activity. In some embodiments, the PDX5 activity is provided
by an increased
amount of an enzyme including (i) the amino acid sequence of SEQ ID NO: 40,
(ii) an amino acid
sequence 90% or more identical to (i), or (iii) an amino acid sequence that
includes 1 to 10 amino
acid substitutions, insertions or deletions with respect to (i). In certain
embodiments, a genetic
modification that increases an acyl-CoA hydrolase activity increases an ACH
activity. In some
embodiments, the ACH activity is an ACHA activity, an ACHB activity or an ACHA
and an ACHB
activity. In certain embodiments, the ACHA activity is provided by an
increased amount of an
enzyme including (i) the amino acid sequence of SEQ ID NO: 43, (ii) an amino
acid sequence 90%
or more identical to (i), or (iii) an amino acid sequence that includes 1 to
10 amino acid
substitutions, insertions or deletions with respect to (i), and the ACHB
activity is provided by an
increased amount of an enzyme including (iv) the amino acid sequence of SEQ ID
NO: 45, (v) an
amino acid sequence 90% or more identical to (iv), or (vi) an amino acid
sequence that includes 1
to 10 amino acid substitutions, insertions or deletions with respect to (iv).
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In some embodiments, the one or more monooxygenase activities includes one or
more
cytochrome P450 activities chosen from: a CYP52A13 activity, a CYP52A14
activity, a CYP52A15
activity, a CYP52A16 activity, and a CYP52A19 activity. In certain
embodiments, the CYP52A13
activity is provided by an increased amount of an enzyme including (i) the
amino acid sequence of
SEQ ID NO: 63, (ii) an amino acid sequence 90% or more identical to (i), or
(iii) an amino acid
sequence that includes 1 to 10 amino acid substitutions, insertions or
deletions with respect to (i).
In some embodiments, the CYP52A14 activity is provided by an increased amount
of an enzyme
including (i) the amino acid sequence of SEQ ID NO: 65, (ii) an amino acid
sequence 90% or more
identical to (i), or (iii) an amino acid sequence that includes 1 to 10 amino
acid substitutions,
insertions or deletions with respect to (i). In certain embodiments, the
CYP52A15 activity is
provided by an increased amount of an enzyme including (i) the amino acid
sequence of SEQ ID
NO: 67, (ii) an amino acid sequence 90% or more identical to (i), or (iii) an
amino acid sequence
that includes 1 to 10 amino acid substitutions, insertions or deletions with
respect to (i). In some
embodiments, the CYP52A16 activity is provided by an increased amount of an
enzyme including
(i) the amino acid sequence of SEQ ID NO: 69, (ii) an amino acid sequence 90%
or more identical
to (i), or (iii) an amino acid sequence that includes 1 to 10 amino acid
substitutions, insertions or
deletions with respect to (i). In certain embodiments, the CYP52A19 activity
is provided by an
increased amount of an enzyme including (i) the amino acid sequence of SEQ ID
NO: 75, (ii) an
amino acid sequence 90% or more identical to (i), or (iii) an amino acid
sequence that includes 1 to
10 amino acid substitutions, insertions or deletions with respect to (i).
In certain embodiments, a genetic modification that increases a monooxygenase
reductase activity
increases a CPR activity. In some embodiments, the CPR activity is a CPRB
activity. In certain
embodiments, the CPRB activity is provided by an increased amount of an enzyme
including (i) the
amino acid sequence of SEQ ID NO: 81, (ii) an amino acid sequence 90% or more
identical to (i),
or (iii) an amino acid sequence that includes 1 to 10 amino acid
substitutions, insertions or
deletions with respect to (i).
In some embodiments, the one or more increased activities are three or more
increased activities.
In certain embodiments, the three or more increased activities are four or
more increased activities.
In some embodiments, the three or more increased activities are five or more
increased activities.
In certain embodiments, the three or more increased activities are six or more
increased activities.
In some embodiments, the three or more increased activities include an
increased
monooxygenase activity, an increased monooxygenase reductase activity, and an
increased acyl-
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CoA oxidase activity. In certain embodiments, the four or more increased
activities include an
increased monooxygenase activity, an increased monooxygenase reductase
activity, an increase
acyl-CoA oxidase, and an increased acyl-CoA hydrolase activity. In some
embodiments, the five
or more increased activities include at least two increased monooxygenase
activities, an increased
monooxygenase reductase activity, an increased acyl-CoA oxidase activity, and
an increased acyl-
CoA hydrolase activity. In certain embodiments, the six or more increased
activities include at
least two increased monooxygenase activities, an increased monooxygenase
reductase activity,
an increased acyl-CoA oxidase activity, and at least two increased acyl-CoA
hydrolase activities.
In certain embodiments, the one or more increased activities independently is
provided by an
increased amount of an enzyme from a yeast. In some embodiments, each of the
one or more
increased activities independently results from increasing the copy number of
a gene that encodes
an enzyme that provides the activity. In certain embodiments, the one or more
increased activities
independently results from inserting a promoter in functional proximity to a
gene that encodes an
enzyme that provides the activity.
In some embodiments, the yeast is a Candida yeast. In certain embodiments, the
Candida yeast is
a Candida tropicalis yeast, and in some embodiments, the Candida yeast is a
Candida viswanithii
yeast. In certain embodiments, the yeast is a Yarrowia yeast, and in some
embodiments, the
Yarrowia yeast is a Yarrowia lipolytica yeast. In certain embodiments, the
genetically modified
yeast is derived from an ancestral yeast cell line chosen from: ATCC 20362,
ATCC 8862, ATCC
18944, ATCC 20228, ATCC 76982, LGAM S(7)1, ATCC 20336, ATCC20913, SU-2 (ura3-
/ura3-),
ATCC 20962, ATCC 24690, ATCC 38163, H5343, ATCC 8661, ATCC 8662, ATCC 9773,
ATCC
15586, ATCC 16617, ATCC 16618, ATCC 18942, ATCC 18943, ATCC 18944, ATCC 18945,
ATCC 20114, ATCC 20177, ATCC 20182, ATCC 20225, ATCC 20226, ATCC 20228, ATCC
20237, ATCC 20255, ATCC 20287, ATCC 20297, ATCC 20306, ATCC 20315, ATCC 20320,
ATCC 20324, ATCC 20341, ATCC 20346, ATCC 20348, ATCC 20362, ATCC 20363, ATCC
20364, ATCC 20372, ATCC 20373, ATCC 20383, ATCC 20390, ATCC 20400, ATCC 20460,
ATCC 20461, ATCC 20462, ATCC 20496, ATCC 20510, ATCC 20628, ATCC 20688, ATCC
20774, ATCC 20775, ATCC 20776, ATCC 20777, ATCC 20778, ATCC 20779, ATCC 20780,
ATCC 20781, ATCC 20794, ATCC 20795, ATCC 20875, ATCC 22421, ATCC 22422, ATCC
22423, ATCC 22969, ATCC 32338, ATCC 32339, ATCC 32340, ATCC 32341, ATCC 32342,
ATCC 32343, ATCC 32935, ATCC 34017, ATCC 34018, ATCC 34088, ATCC 34922, ATCC
38295, ATCC 42281, ATCC 44601, ATCC 46025, ATCC 46026, ATCC 46027, ATCC 46028,
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ATCC 46067, ATCC 46068, ATCC 46069, ATCC 46070, ATCC 46330, ATCC 46482, ATCC
46483, ATCC 46484, ATCC 48436, ATCC 60594, ATCC 62385, ATCC 64042, ATCC 74234,
ATCC 76598, ATCC 76861, ATCC 76862, ATCC 90716, ATCC 90806, ATCC 90811, ATCC
90812, ATCC 90813, ATCC 90814, ATCC 90903, ATCC 90904, ATCC 90905, ATCC 96028,
ATCC 201089, ATCC 201241, ATCC 201242, ATCC 201243, ATCC 201244, ATCC 201245,
ATCC 201246, ATCC 201247, ATCC 201248, ATCC 201249, ATCC 201847, ATCC MYA-165,
ATCC MYA-166, ATCC MYA-2613, and ATCC MYA-4467.
Also provided in certain aspects are methods for producing adipic acid,
comprising: (a) contacting
a Candida yeast with a feedstock comprising a vegetable oil, wherein the
Candida yeast
comprises: (1) a genetic alteration that reduces or eliminates PDX4 activity
but not P0X5 activity,
(2) a genetic alteration that reduces or eliminates an ACS1 acyl-CoA
synthetase activity, and (3)
a genetic alteration that reduces or eliminates a FAT1 long chain acyl-CoA
synthetase activity; and
(b) culturing the Candida yeast under conditions in which adipic acid is
produced at a yield of 2.20
grams per liter of culture medium or greater.
Certain embodiments are described further in the following description,
examples, claims and
drawings.
Brief Description of the Drawings
The drawings illustrate embodiments of the technology and are not limiting.
For clarity and ease of
illustration, the drawings are not made to scale and, in some instances,
various aspects may be
shown exaggerated or enlarged to facilitate an understanding of particular
embodiments.
Figure 1 depicts a metabolic pathway for making adipic acid. The pathway can
be engineered into
a eukaryotic microorganism to generate a microorganism capable of producing
adipic acid.
Figure 2 depicts an embodiment for a method of generating an adipic acid
producing
microorganism. The method comprises expressing one or more genes catalyzing
the omega
oxidation of fatty acids to dicarboxylic acids in a host microorganism that
produces hexanoate. In
the method depicted, the host organism, for example A. parasiticus or A.
nidulans, endogenously
includes HEXA and HEXB (or STCJ and STCK) genes. In one embodiment the method
comprises
knocking out or otherwise disabling the gene coding for diversion of hexanoate
into an endogenous
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pathway such as mycotoxin production. Certain embodiments of the method
further comprise
inserting a heterologous cytochrome P450 gene. Some embodiments of the method
comprise
growing the culture on hexane and screening for increased P450 expression. The
copy number of
hexanoate induced P450 may in certain embodiments be increased. In some
embodiments the
microorganism may be altered to increase the flux of six carbon substrate
through the final two
oxidation steps.
Figure 3 depicts an embodiment for a method of generating an adipic acid
producing organism.
The method comprises expressing one or more genes encoding hexanoate synthase
in a host
microorganism that produces dicarboxylic acids via an omega-oxidation pathway.
Such
microorganisms may include, without limitation, C. tropicalis, C. viswanithii
and C. maltosa. As
depicted, the method comprises inserting HEXA and HEXB genes into the host
microorganism.
The genes may be isolated from Aspergillus, or another appropriate organism.
In some
embodiments, the genes are synthesized from an alternative sequence as
described herein to
produce the amino acid sequence of the donor mircroorganism enzyme through a
non-standard
translation mechanism of C. tropicalis. In some embodiments the method
comprises inserting a
heterologous cytochrome P450 gene into the host organism. In certain
embodiments the
microorganism may be altered to increase the flux of a six-carbon substrate
through the final two
oxidation steps.
Figure 4 depicts an embodiment of a method for generating an adipic acid
producing organism.
The method comprises expressing one or more genes encoding hexanoate synthase
in a host
microorganism that produces dicarboxylic acids via an omega-oxidation pathway.
The
microorganisms can include, without limitation, C. tropicalis, C. viswanithii
and C. maltosa. In
some embodiments, the method comprises growing a host microorganism on hexane
and
screening for increased P450 expression. In certain embodiments, copy number
of hexane-
induced P450 may be increased. HEXA and HEXB genes may be inserted into the
host
microorganism. In certain embodiments, the host microorganism may be altered
to increase the
flux of a six-carbon substrate through the final two oxidation steps.
Figure 5 depicts a plasmid diagram for inserting Aspergillus hexanoate
synthase genes HEXA and
HEXB into C. tropicalis or Y. lipolytica.
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Figure 6 depicts a plasmid diagram for inserting a heterologous cytochrome
P450 monooxygenase
gene and cytochrome P450 reductase gene into C. tropicalis or Y. lipolytica.
Figure 7 depicts a plasmid diagram for inserting a heterologous cytochrome
P450 monooxygenase
gene and cytochrome P450 reductase gene into A. parasiticus or A. nidulans.
Figure 8 depicts a system for biological production of a target product. As
depicted, a fermenter is
populated with microorganisms engineered for target product production. A
flexible feedstock
supplies the fermenter with an energy and nutrition source for the
microorganisms. In some
embodiments the feedstock comprises a sugar. In certain embodiments the
feedstock comprises
fatty acids. The feedstock may also include biomass, industrial waste products
and other sources
of carbon. Vitamins, minerals, enzymes and other growth or production
enhancers may be added
to the feedstock. In certain embodiments the fermentation produces adipic
acid. The fermentation
process may produce other novel chemicals.
Figure 9 depicts a metabolic pathway for making adipic acid from saccharide or
polysaccharide
carbon sources, similar to the pathway depicted in Figure 1, with additional
activities that aid in
metabolism of, or enhance metabolism of, pathway intermediates, thereby
potentially increasing
the yield of adipic acid. The additional activities are a monooxygenase
reductase activity
(cytochrome P450 reductase or CPR) and a fatty alcohol oxidase activity (FAO).
Part, or all, of the
pathway can be engineered into a eukaryotic microorganism to generate a
microorganism capable
of producing adipic acid.
Figure 10 depicts a non-limiting example of a metabolic pathway for making
adipic acid from
paraffins, fats, oils, fatty acids or dicarboxylic acids, as described in
Figure 2. Part, or all, of the
pathway can be engineered (e.g., added, altered to increase or decrease copy
number, or increase
or decrease promoter activity, depending on the desired effect) into a
microorganism, depending
on the activities already present in the host organism, to generate a
microorganism capable of
producing adipic acid.
Figure 11A and 11B depict omega and beta oxidation pathways useful for
producing adipic acid
from various carbon sources. Adipic acid can be produced from paraffins, fats,
oils and
intermediates of sugar metabolism, using omega oxidation, as shown in Figure
11A. Adipic acid
also can be produced from long chain fatty acids or dicarboxylic acids using
beta oxidation, as
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shown in Figure 11A. Figure 11B shows a common intermediate from the
metabolism of fats and
sugars entering the omega oxidation pathway to ultimately produce adipic acid.
Figure 12 shows results of immunodetection of 6xHis-tagged proteins expressed
in S. cerevisiae
BY4742. Strains sAA061, sAA140, sAA141, sAA142 contain 6xHis-tagged HEXA and
HEXB
proteins. Strain sAA144 contains 6xHis-tagged STCJ and STCK proteins. Strain
sAA048 contains
only vectors p425GPD and p426GPD.
Figure 13 shows results of immunodetection of 6xHis-tagged proteins expressed
in either S.
cerevisiae (5AA144) or in C. tropicalis (5AA103, sAA270, 5AA269). 6xHis tagged
HEXA and HEXB
expressed in strains sAA269 and sAA270 are indicated with arrows. 6xHis tagged
STCJ and
STCK from strain sAA144 were included as a positive control. Strain sAA103 is
the parent strain
for sAA269 and sAA270 and does not contain integrated vectors for the
expression of 6xHis-
tagged HEXA and HEXB.
Figure 14 shows results of RT-PCR from cultures of C. tropicalis strain sAA003
exposed to glucose
only (Glc), hexane only (Hex), or hexanoic acid only (HA). PCR products of A15
and A16 alleles
show hexane and hexanoic acid specific induction.
Figures 15A-15C illustrate results of acyl-CoA oxidase (PDX) enzymatic
activity assays on
substrates of various carbon lengths, using acyl-CoA enzyme preparations from
Candida tropicalis
strains with no PDX genes disrupted (see Figure 15A), PDX4 genes disrupted
(see Figure 15C) or
PDX5 genes disrupted (see Figure 15B). Experimental results and conditions are
given in the
Detailed Description and Examples sections.
Figures 16-34 illustrate various plasmids for cloning, expression, or
integration of various activities
described herein, into a host organism or engineered organism. Figure 16
depicts a plasmid
diagram for inserting a heterologous HEXA gene into S. cerevisiae. Figure 17
depicts a plasmid
diagram for inserting a heterologous HEXB gene into S. cerevisiae. Figure 18
depicts a plasmid
diagram for inserting a heterologous HEXA-6xHis gene into S. cerevisiae.
Figure 19 depicts a
plasmid diagram for inserting a heterologous HEXB-6xHis gene into S.
cerevisiae.
Figure 20 depicts a plasmid diagram for inserting a heterologous STCJ gene
into S. cerevisiae.
Figure 21 depicts a plasmid diagram for inserting a heterologous STCK gene
into S. cerevisiae.
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Figure 22 depicts a plasmid diagram for inserting a heterologous STCJ-6xHis
gene into S.
cerevisiae. Figure 23 depicts a plasmid diagram for inserting a heterologous
STCK-6xHis gene
into S. cerevisiae.
Figure 24 depicts a plasmid diagram for inserting a heterologous alternative
genetic code (AGC)
HEXA gene into C. tropicalis. Figure 25 depicts a plasmid diagram for
inserting a heterologous
AGC-HEXB gene into C. tropicalis. Figure 26 depicts a plasmid diagram for
inserting a
heterologous AGC-HEXA-6xHis gene into C. tropicalis. Figure 27 depicts a
plasmid diagram for
inserting a heterologous AGC-HEXB-6xHis gene into C. tropicalis.
Figure 28 depicts a diagram of a plasmid used for cloning the PDX5 gene from
C. tropicalis.
Figure 29 depicts a diagram of a plasmid used for cloning the PDX4 gene from
C. tropicalis.
Figure 30 illustrates a plasmid constructed for use of URA selection in C.
tropicalis. Figure 31
depicts a plasmid containing the PGK promoter and terminator from C.
tropicalis. Figure 32
depicts a plasmid used for integration of the CPR gene in C. tropicalis.
Figure 33 depicts a
plasmid used for integration of the CYP52A15 gene in C. tropicalis. Figure 34
depicts a plasmid
used for integration of the CYP52A16 gene in C. tropicalis.
Figure 35 depicts a metabolic pathway for making adipic acid from saccharide
or polysaccharide
carbon sources, similar to the pathways depicted in Figures 1 and 9, with
additional activities that
aid in metabolism of, or enhance metabolism of, pathway intermediates, thereby
potentially
increasing the yield of adipic acid. The additional activities are an acetyl-
CoA carboxylase activity
(ACC), a fatty acid synthase (FAS1, FAS2), a monooxygenase (P450) activity, a
monooxygenase
reductase activity (CPR) and an acyl-CoA oxidase activity (P0X5). Part, or
all, of the pathway can
be engineered into a eukaryotic microorganism to generate a microorganism
capable of producing
adipic acid.
Figure 36 depicts a metabolic pathway for making adipic acid from saccharide
or polysaccharide
carbon sources, similar to the pathways depicted in Figures 1 and 9, with
additional activities that
aid in metabolism of, or enhance metabolism of, pathway intermediates, thereby
potentially
increasing the yield of adipic acid. The additional activities are an acetyl-
CoA carboxylase activity
(ACC), a hexanoate synthase (HEXA, HEXB), a monooxygenase (P450) activity, and
a
monooxygenase reductase activity (CPR). Part, or all, of the pathway can be
engineered into a
eukaryotic microorganism to generate a microorganism capable of producing
adipic acid.
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Figure 37 depicts a non-limiting example of a metabolic pathway for making
adipic acid from
paraffins, fats, oils, fatty acids or dicarboxylic acids, as described in
Figures 2 and 10, with
additional activities that aid in metabolism of, or enhance metabolism of,
pathway intermediates,
thereby potentially increasing the yield of adipic acid. The additional
activities are a lipase, a fatty
acid synthase, a glucose-6-phosphate dehydrogenase (ZWF1), a monooxygenase
(P450) activity,
and a monooxygenase reductase activity (CPR). Part, or all, of the pathway can
be engineered
(e.g., added, altered to increase or decrease copy number, or increase or
decrease promoter
activity, depending on the desired effect) into a microorganism, depending on
the activities already
present in the host organism, to generate a microorganism capable of producing
adipic acid.
Figure 38 graphically illustrates the ratio of C6 diacids/(C6 + C8 diacids)
produced in yeast cultures
grown in shake flasks using coconut oil as the feedstock (e.g., carbon
source). Experimental
results and conditions are given in Example 32.
Figure 39 graphically illustrates the effect of increased lipase activity on
the conversion of coconut
oil to adipic acid. Experimental results and conditions are given in Example
33.
Figure 40 shows the results of immunodetection of over expressed polypeptides
coding FAS2 and
FAS1 activities after denaturing polyacrylamide gel electrophoresis (SDS-
PAGE). Figure 41 shows
the results of immunodetection of over expressed polypeptides coding FAS2 and
FAS1 activities
after native polyacrylamide gel electrophoresis (Native-PAGE). Experimental
results and
conditions are given in Example 35.
FIG. 42A depicts naturally occurring metabolic pathways which together combine
to produce adipic
acid, acetyl-CoA, and phospholipids, triacylglycerides and steryl esters from
a number of different
feedstocks (e.g., triacylglycerides, fatty acids, sugars, cellulose).
Production of adipic acid is
accompanied by the production of energy and carbon dioxide. Production of
acetyl-CoA is
accompanied by the production of biomass, energy and carbon dioxide.
Production of
phospholipids, triacylglycerides and steryl esters is accompanied by the
production of biomass and
carbon storage moieties (e.g., triacylglycerides and steryl esters). FIG. 42B
depicts modifications
to the various metabolic pathways, illustrated in FIG. 42A, which alter the
host organism's carbon
flux towards the production of adipic acid through increased fatty acid
production, increased omega
oxidation and increased beta oxidation. The altered activities are highlighted
by a + for activities
that are added or increased and by an X for activities that are reduced or
eliminated.
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FIG. 43 illustrates a plasmid used for cloning the TESA gene from E. coli,
which also was codon
optimized for proper functionality in C. tropicalis. The codon optimization
included altering CTG
codons which are translated differently in C. tropicalis with respect to E.
coli, as noted herein. FIG.
44 depicts a plasmid used to donate the PDX4 promoter which is used to drive
transcription of
various genes described herein. For example, the PDX4 promoter is the promoter
that drives
transcription of the TESA gene included in the plasmid shown in FIG. 43. FIG.
45 depicts a
plasmid used for integration of the codon optimized TESA gene into C.
tropicalis. The plasmid
depicted in FIG. 45 is assembled from pieces of the plasmids depicted in FIGS.
43 and 44. See
Example 40 for experimental details and results.
FIG. 46 depicts a plasmid used to donate a "knockout cassette", for disrupting
various genes
described herein. FIG. 47 depicts a plasmid used for cloning the ACS1 gene
from C. tropicalis, for
use in generating an ACS1 knockout construct. FIGS. 48 and 49 depict the ACS1
knockout
constructs generated from pieces of the plasmids depicted in FIGS. 46 and 47.
The difference
between the constructs illustrated in FIGS. 48 and 49 is the orientation of
the URA3 cassette (e.g.,
Promoter URA3-URA3-Terminator URA3-Promoter URA3). See Example 43 for
experimental
details and results.
FIG. 50 illustrates the plasmid used to amplify the number of copies of
cytochrome P450 A19 (e.g.,
CYP52A19). See Examples 48 and 49 for experimental details and results.
FIG. 51 illustrates a plasmid used for cloning the ACHA allele from C.
tropicalis. FIG. 52 illustrates
a plasmid used for cloning ACHB from C. tropicalis. See Example 37 for
experimental details.
FIG 53. illustrates a plasmid used for cloning the FAT1 gene from C.
tropicalis. See Example 38
for experimental details. The cloned FAT1 DNA sequence are used to construct
FAT1 "knock out"
constructs.
FIG. 54 illustrates a plasmid used for cloning the ARE1 gene from C.
tropicalis. FIG. 55 illustrates
a plasmid used for cloning the ARE2 gene from C. tropicalis. See Example 39
for experimental
details. The cloned ARE1 and ARE2 DNA sequence are used to construct ARE1 and
ARE2
"knock out" constructs.
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FIG. 56 illustrates a plasmid used for cloning the DGA1 gene from C.
tropicalis. See Example 41
for experimental details. The cloned DGA1 DNA sequence are used to construct
DGA1 "knock
out" constructs.
FIG. 57 illustrates a plasmid used for cloning the LRO1 gene from C.
tropicalis. See Example 42
for experimental details. The cloned LRO1 DNA sequence are used to construct
LRO1 "knock out"
constructs.
FIG. 58 graphically illustrates the percent of theoretical maximum yield for
production of adipic acid
from various parental and engineered strains of C. tropicalis. See Example 50
for experimental
details and results.
FIG. 59 and FIG. 60 graphically illustrate nucleic acid design features for
certain C. tropicalis
strains addressed in Examples 52 and 53. FIG. 61 shows the yield of adipic
acid produced by
these strains.
Detailed Description
Adipic acid is a six-carbon organic molecule that is a chemical intermediate
in manufacturing
processes used to make certain polyamides, polyurethanes and plasticizers, all
of which have wide
applications in producing items such as carpets, coatings, adhesives,
elastomers, food packaging,
and lubricants, for example. Some large-scale processes for making adipic acid
include (i) liquid
phase oxidation of ketone alcohol oil (KA oil); (ii) air oxidation/hydration
of cyclohexane with boric
acid to make cyclohexanol, followed by oxidation with nitric acid; and (iii)
hydrocyanation of
butadiene to a pentenenitrile mixture, followed by hydroisomerization of
adiponitrile, followed by
hydrogenation. Each of the latter processes requires use of noxious chemicals
and/or solvents,
some require high temperatures, and all require significant energy input. In
addition, some of the
processes emit toxic byproducts (such as nitrous oxide) and give rise to
environmental concerns.
Provided herein are methods for producing adipic acid and other organic
chemical intermediates
using biological systems. Such production systems may have significantly less
environmental
impact and could be economically competitive with current manufacturing
systems. Thus, provided
herein are methods for manufacturing adipic acid by engineered microorganisms.
In some
embodiments microorganisms are engineered to contain at least one heterologous
gene encoding
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an enzyme, where the enzyme is a member of a novel pathway engineered into the
microorganism. In certain embodiments, an organism may be selected for
elevated activity of a
native enzyme.
Microorganisms
A microorganism selected often is suitable for genetic manipulation and often
can be cultured at
cell densities useful for industrial production of a target product. A
microorganism selected often
can be maintained in a fermentation device.
The term "engineered microorganism" as used herein refers to a modified
microorganism that
includes one or more activities distinct from an activity present in a
microorganism utilized as a
starting point (hereafter a "host microorganism"). An engineered microorganism
includes a
heterologous polynucleotide in some embodiments, and in certain embodiments,
an engineered
organism has been subjected to selective conditions that alter an activity, or
introduce an activity,
relative to the host microorganism. Thus, an engineered microorganism has been
altered directly
or indirectly by a human being. A host microorganism sometimes is a native
microorganism, and
at times is a microorganism that has been engineered to a certain point. In
some embodiments,
an engineered microorganism is derived from any one of the following cell
lines: ATCC 20362,
ATCC 8862, ATCC 18944, ATCC 20228, ATCC 76982, LGAM S(7)1, ATCC 20336,
ATCC20913,
SU-2 (ura3-/ura3-), ATCC 20962, ATCC 24690, ATCC 38163, H5343, ATCC 8661, ATCC
8662,
ATCC 9773, ATCC 15586, ATCC 16617, ATCC 16618, ATCC 18942, ATCC 18943, ATCC
18944,
ATCC 18945, ATCC 20114, ATCC 20177, ATCC 20182, ATCC 20225, ATCC 20226, ATCC
20228, ATCC 20237, ATCC 20255, ATCC 20287, ATCC 20297, ATCC 20306, ATCC 20315,
ATCC 20320, ATCC 20324, ATCC 20341, ATCC 20346, ATCC 20348, ATCC 20362, ATCC
20363, ATCC 20364, ATCC 20372, ATCC 20373, ATCC 20383, ATCC 20390, ATCC 20400,
ATCC 20460, ATCC 20461, ATCC 20462, ATCC 20496, ATCC 20510, ATCC 20628, ATCC
20688, ATCC 20774, ATCC 20775, ATCC 20776, ATCC 20777, ATCC 20778, ATCC 20779,
ATCC 20780, ATCC 20781, ATCC 20794, ATCC 20795, ATCC 20875, ATCC 22421, ATCC
22422, ATCC 22423, ATCC 22969, ATCC 32338, ATCC 32339, ATCC 32340, ATCC 32341,
ATCC 32342, ATCC 32343, ATCC 32935, ATCC 34017, ATCC 34018, ATCC 34088, ATCC
34922, ATCC 38295, ATCC 42281, ATCC 44601, ATCC 46025, ATCC 46026, ATCC 46027,
ATCC 46028, ATCC 46067, ATCC 46068, ATCC 46069, ATCC 46070, ATCC 46330, ATCC
46482, ATCC 46483, ATCC 46484, ATCC 48436, ATCC 60594, ATCC 62385, ATCC 64042,
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ATCC 74234, ATCC 76598, ATCC 76861, ATCC 76862, ATCC 90716, ATCC 90806, ATCC
90811, ATCC 90812, ATCC 90813, ATCC 90814, ATCC 90903, ATCC 90904, ATCC 90905,
ATCC 96028, ATCC 201089, ATCC 201241, ATCC 201242, ATCC 201243, ATCC 201244,
ATCC
201245, ATCC 201246, ATCC 201247, ATCC 201248, ATCC 201249, ATCC 201847, ATCC
MYA-165, ATCC MYA-166, ATCC MYA-2613, and ATCC MYA-4467. That is, an
engineered
microorganism described herein is generated from one or more of the
aforementioned ancestral
cell lines, in certain embodiments.
In some embodiments an engineered microorganism is a single cell organism,
often capable of
dividing and proliferating. A microorganism can include one or more of the
following features:
aerobe, anaerobe, filamentous, non-filamentous, monoploid, dipoid, auxotrophic
and/or non-
auxotrophic. In certain embodiments, an engineered microorganism is a
prokaryotic
microorganism (e.g., bacterium), and in certain embodiments, an engineered
microorganism is a
non-prokaryotic microorganism. In some embodiments, an engineered
microorganism is a
eukaryotic microorganism (e.g., yeast, fungi, amoeba).
Any suitable yeast may be selected as a host microorganism, engineered
microorganism or source
for a heterologous polynucleotide. Yeast include, but are not limited to,
Yarrowia yeast (e.g., Y.
lipolytica (formerly classified as Candida lipolytica)), Candida yeast (e.g.,
C. revkaufi, C.
pulcherrima, C. tropicalis, C. utilis, C. viswanithii), Rhodotorula yeast
(e.g., R. glutinus, R.
graminis), Rhodosporidium yeast (e.g., R. toruloides), Saccharomyces yeast
(e.g., S. cerevisiae, S.
bayanus, S. pastorianus, S. carlsbergensis), Cryptococcus yeast, Trichosporon
yeast (e.g., T.
pullans, T. cutaneum), Pichia yeast (e.g., P. pastoris) and Lipomyces yeast
(e.g., L. starkeyii, L.
lipoferus). In some embodiments, a yeast is a Y. lipolytica strain that
includes, but is not limited to,
ATCC20362, ATCC8862, ATCC18944, ATCC20228, ATCC76982 and LGAM S(7)1 strains
(Papanikolaou S., and Aggelis G., Bioresour. Technol. 82(1):43-9 (2002)). In
certain
embodiments, a yeast is a C. tropicalis strain, a C. viswanithii strain, a Y.
lipolytica strain or a yeast
strain that includes, but is not limited to, ATCC20336, ATCC20913, SU-2 (ura3-
/ura3-),
ATCC20962, H5343 (beta oxidation blocked; US Patent No. 5648247) ATCC 20362,
ATCC 8862,
ATCC 18944, ATCC 20228, ATCC 76982, LGAM S(7)1, ATCC 8661, ATCC 8662, ATCC
9773,
ATCC 15586, ATCC 16617, ATCC 16618, ATCC 18942, ATCC 18943, ATCC 18944, ATCC
18945, ATCC 20114, ATCC 20177, ATCC 20182, ATCC 20225, ATCC 20226, ATCC 20228,
ATCC 20237, ATCC 20255, ATCC 20287, ATCC 20297, ATCC 20306, ATCC 20315, ATCC
20320, ATCC 20324, ATCC 20341, ATCC 20346, ATCC 20348, ATCC 20362, ATCC 20363,
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ATCC 20364, ATCC 20372, ATCC 20373, ATCC 20383, ATCC 20390, ATCC 20400, ATCC
20460, ATCC 20461, ATCC 20462, ATCC 20496, ATCC 20510, ATCC 20628, ATCC 20688,
ATCC 20774, ATCC 20775, ATCC 20776, ATCC 20777, ATCC 20778, ATCC 20779, ATCC
20780, ATCC 20781, ATCC 20794, ATCC 20795, ATCC 20875, ATCC 22421, ATCC 22422,
ATCC 22423, ATCC 22969, ATCC 32338, ATCC 32339, ATCC 32340, ATCC 32341, ATCC
32342, ATCC 32343, ATCC 32935, ATCC 34017, ATCC 34018, ATCC 34088, ATCC 34922,
ATCC 38295, ATCC 42281, ATCC 44601, ATCC 46025, ATCC 46026, ATCC 46027, ATCC
46028, ATCC 46067, ATCC 46068, ATCC 46069, ATCC 46070, ATCC 46330, ATCC 46482,
ATCC 46483, ATCC 46484, ATCC 48436, ATCC 60594, ATCC 62385, ATCC 64042, ATCC
74234, ATCC 76598, ATCC 76861, ATCC 76862, ATCC 90716, ATCC 90806, ATCC 90811,
ATCC 90812, ATCC 90813, ATCC 90814, ATCC 90903, ATCC 90904, ATCC 90905, ATCC
96028, ATCC 201089, ATCC 201241, ATCC 201242, ATCC 201243, ATCC 201244, ATCC
201245, ATCC 201246, ATCC 201247, ATCC 201248, ATCC 201249, ATCC 201847, ATCC
MYA-165, ATCC MYA-166, ATCC MYA-2613, and ATCC MYA-4467.
Any suitable fungus may be selected as a host microorganism, engineered
microorganism or
source for a heterologous polynucleotide. Non-limiting examples of fungi
include, but are not
limited to Aspergillus fungi (e.g., A. parasiticus, A. nidulans),
Thraustochytrium fungi,
Schizochytrium fungi and Rhizopus fungi (e.g., R. arrhizus, R. oryzae, R.
nigricans), and the
aforementioned yeast strains. In some embodiments, a fungus is one of the
aforementioned yeast
strains, an A. parasiticus strain that includes, but is not limited to, strain
ATCC24690, and in certain
embodiments, a fungus is an A. nidulans strain that includes, but is not
limited to, strain
ATCC38163.
Any suitable prokaryote may be selected as a host microorganism, engineered
microorganism or
source for a heterologous polynucleotide. A Gram negative or Gram positive
bacteria may be
selected. Examples of bacteria include, but are not limited to, Bacillus
bacteria (e.g., B. subtilis, B.
megaterium), Acinetobacter bacteria, Norcardia baceteria, Xanthobacter
bacteria, Escherichia
bacteria (e.g., E. coli (e.g., strains DH10B, StbI2, DH5-alpha, DB3, DB3.1),
DB4, DB5, JDP682 and
ccdA-over (e.g., U.S. Application No. 09/518,188))), Streptomyces bacteria,
Erwinia bacteria,
Klebsiella bacteria, Serratia bacteria (e.g., S. marcessans), Pseudomonas
bacteria (e.g., P.
aeruginosa), Salmonella bacteria (e.g., S. typhimurium, S. typhi), Megasphaera
bacteria (e.g.,
Megasphaera elsdenii). Bacteria also include, but are not limited to,
photosynthetic bacteria (e.g.,
green non-sulfur bacteria (e.g., Choroflexus bacteria (e.g., C. aurantiacus),
Chloronema bacteria
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(e.g., C. gigateum)), green sulfur bacteria (e.g., Chlorobium bacteria (e.g.,
C. limicola), Pelodictyon
bacteria (e.g., P. luteolum), purple sulfur bacteria (e.g., Chromatium
bacteria (e.g., C. okenii)), and
purple non-sulfur bacteria (e.g., Rhodospirillum bacteria (e.g., R. rubrum),
Rhodobacter bacteria
(e.g., R. sphaeroides, R. capsulatus), and Rhodomicrobium bacteria (e.g., R.
vanellii)).
Cells from non-microbial organisms can be utilized as a host microorganism,
engineered
microorganism or source for a heterologous polynucleotide. Examples of such
cells, include, but
are not limited to, insect cells (e.g., Drosophila (e.g., D. melanogaster),
Spodoptera (e.g., S.
frugiperda Sf9 or Sf21 cells) and Trichoplusa (e.g., High-Five cells);
nematode cells (e.g., C.
elegans cells); avian cells; amphibian cells (e.g., Xenopus laevis cells);
reptilian cells; and
mammalian cells (e.g., NIH3T3, 293, CHO, COS, VERO, C127, BHK, Per-C6, Bowes
melanoma
and HeLa cells).
Microorganisms or cells used as host organisms or source for a heterologous
polynucleotide are
commercially available. Microoganisms and cells described herein, and other
suitable
microorganisms and cells are available, for example, from Invitrogen
Corporation, (Carlsbad, CA),
American Type Culture Collection (Manassas, Virginia), and Agricultural
Research Culture
Collection (NRRL; Peoria, Illinois).
Host microorganisms and engineered microorganisms may be provided in any
suitable form. For
example, such microorganisms may be provided in liquid culture or solid
culture (e.g., agar-based
medium), which may be a primary culture or may have been passaged (e.g.,
diluted and cultured)
one or more times. Microorganisms also may be provided in frozen form or dry
form (e.g.,
lyophilized). Microorganisms may be provided at any suitable concentration.
Carbon Processing Pathways and Activities
Figures 1, 9, 35, 36, 42A and 42B depict embodiments of a biological pathways
for making adipic
acid, using a sugar as the carbon source starting material. Any suitable sugar
can be used as the
feedstock for the organism, (e.g., 6-carbon sugars (e.g., glucose, fructose),
5-carbon sugars (e.g.,
xylose), the like or combinations thereof). The sugars are initially
metabolized using naturally
occurring and/or engineered pathways to yield malonyl CoA, which is depicted
as the molecule
entering the omega oxidation pathway shown in Figure 9. Malonyl-CoA sometimes
is generated
by the activity of an acyl-CoA carboxylase activity (e.g., ACC), as depicted
in Figures 35 and 36,
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and sometimes is formed by the metabolism of sugars or paraffins, yielding
Malonyl-CoA as a
direct or indirect metabolic product.
An acetyl-CoA carboxylase activity (e.g., EC 6.4.1.2) catalyzes the
irreversible carboxylation of
acetyl-CoA to produce malonyl-CoA through its two catalytic activities, biotin
carboxylase and
carboxyltransferase. The reaction can be represented as;
ATP + acetyl-CoA + HCO3- = ADP + phosphate + malonyl-CoA
Production of malonyl-CoA is the committed step in fatty acid biosynthesis.
Acetyl-CoA
carboxylase activity sometimes is present in a variety of organisms (e.g.,
prokaryotes, plants,
algae) as a large multi-subunit protein, and often located in the endoplasmic
reticulum of
eukaryotes. Acetyl-CoA carboxylase activity in some plants also can
carboxylate propanoyl-CoA
and butanoyl-CoA. ACC sometimes is also referred to as "acetyl-CoA:carbon-
dioxide ligase (ADP-
forming)" and "acetyl coenzyme A carboxylase". The reverse activity (e.g.,
decarboxylation of
malonyl-CoA) is carried out by a separate enzyme, malonyl-CoA decarboxylase.
In some
embodiments, to further increase carbon flux through a particular reaction or
through a metabolic
pathway, one or more reverse activities in the pathway can be altered to
inhibit the back
conversion of a desired product into its starting reactants. In certain
embodiments, a malonyl-CoA
decarboxylase activity is reduced or eliminated to further increase the carbon
flux through an
acetyl-CoA carboxylase activity in the direction of malonyl-CoA production.
An ACC activity in yeast may be amplified by over-expression of the ACC gene
by any suitable
method. Non-limiting examples of methods suitable to amplify or over express
ACC include
amplifying the number of ACC genes in yeast following transformation with a
high-copy number
plasmid (e.g., such as one containing a 2u origin of replication), integration
of multiple copies of
ACC into the yeast genome, over-expression of the ACC gene directed by a
strong promoter, the
like or combinations thereof. The ACC gene may be native to C. tropicalis or
C. viswanithii, for
example, or it may be obtained from a heterologous source.
A fatty acid synthase (e.g., FAS) activity catalyzes a series of
decarboxylative Claisen
condensation reactions from acetyl-CoA and malonyl-CoA. Without being limited
by any theory, it
is believed that following each round of elongation the beta keto group is
reduced to the fully
saturated carbon chain by the sequential action of a ketoreductase activity, a
dehydratase activty,
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and an enol reductase activity. In the case of Type I FAS's, the growing fatty
acid chain is carried
between these active sites while attached covalently to the phosphopantetheine
prosthetic group of
an acyl carrier protein (ACP), and is released by the action of a thioesterase
(TE) upon reaching a
carbon chain length of 16 (e.g., palmitic acid). Thus, a fatty acid synthase
activity comprises a
collection of activities (e.g., an enzymatic system) that perform functions
associated with the
production of fatty acids. Therefore, the terms "fatty acid synthase
activity", "fatty acid synthase",
"FAS", and "FAS activity", as used herein refer to a collection of activities,
or an enzymatic system,
that perform functions associated with the production of fatty acids. In some
embodiments, the
collection of activities is found in a multifunctional, multi-subunit protein
complex (e.g., Type I FAS
activity).
Fatty acid synthases typically produce fatty acids with longer chain carbon
chain lengths, however
fatty acids with carbon chain lengths of 6C or 8C often are found in
organisms. In some instances,
the shorter fatty acids are the result of metabolic activities (e.g., beta-
oxidation) that shorten the
carbon chain length to a desired shorter number of carbon units. However, in
certain instances,
shorter chain fatty acids are produced directly by the activity of a
specialized fatty acid synthase
activity, hexanoate synthase.
As depicted in Figures 1, 9, and 36, the enzyme hexanoate synthase converts
two molecules of
malonyl-CoA and one molecule of acetyl-CoA to one molecule of hexanoic acid.
As depicted in
Figure 36, fatty acid synthase converts malonyl-CoA to a long chain fatty acyl-
CoA by repeated
condensation with acetyl-CoA. In some embodiments a cytochrome P450 enzyme
converts
hexanoic acid to 6-hydroxyhexanoic acid, which may be oxidized to 6-
oxohexanoic acid via 6-
hydroxyhexanoic acid dehydrogenase, or fatty alcohol oxidase. 6-oxohexanoic
acid may be
converted to adipic acid by 6-oxohexanoic acid dehydrogenase. In certain
embodiments, a
cytochrome P450 enzyme converts medium-, or long-chain fatty acids to
dicarboxylic acids (e.g.,
diacids), which may be further metabolized by natural or engineered pathways
described herein to
yield adipic acid.
A fatty acid synthase enzyme (FAS) is coded by fatty acid synthase subunit
alpha (FAS2) and fatty
acid synthase subunit beta (FAS1) genes. In some embodiments, the FAS enzyme
is endogenous
to the host microorganism. Fatty acid synthase activity in yeast may be
amplified by over-
expression of the FAS2 and FAS1 genes by any suitable method. Non-limiting
examples of
methods suitable to amplify or over express FAS2 and FAS1 genes include
amplifying the number
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of FAS2 and FAS1 genes in yeast following transformation with a high-copy
number plasmid (e.g.,
such as one containing a 2u origin of replication), integration of multiple
copies of FAS2 and FAS1
genes into the yeast genome, over-expression of the FAS2 and FAS1 genes
directed by a strong
promoter, the like or combinations thereof. The FAS2 and FAS1 genes may be
native to C.
tropicalis or C. viswanithii, for example, or they may be obtained from a
heterologous source.
A specialized fatty acid synthase enzyme, hexanoate synthase (HexS) is coded
by hexonate
synthase subunit alpha (HEXA) and hexanoate synthase subunit beta (HEXB)
genes. In some
embodiments, the HexS enzyme is endogenous to the host microorganism. In
certain
embodiments, HEXA and HEXB genes may be isolated from a suitable organism
(e.g., Aspergillus
parasiticus). In some embodiments, HEXA and HEXB orthologs, such as STCJ and
STCK, also
may be isolated from suitable organisms (e.g., Aspergillus nidulans).
Hexanoate is omega-hydroxylated by the activity of cytochrome P450 enzymes,
thereby
generating a six carbon alcohol, in some embodiments. In certain embodiments,
a cytochrome
P450 activity can be increased by increasing the number of copies of a
cytochrome P450 gene
(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more copies of the gene), by
increasing the activity of a
promoter that regulates transcription of a cytochrome P450 gene, or by
increasing the number of
copies of a cytochrome P450 gene and increasing the activity of a promoter
that regulates
transcription of a cytochrome P450 gene, thereby increasing the production of
target product (e.g.,
adipic acid) via increased activity of one or more cytochrome P450 enzymes. In
some
embodiments, a cytochrome P450 enzyme is endogenous to the host microorganism.
In certain
embodiments, the cytochrome P450 gene is isolated from Bacillus megaterium and
codes for a
single subunit, soluble, cytoplasmic enzyme. Soluble or membrane bound
cytochrome P450 from
certain host organisms is specific for 6-carbon substrates and may be used in
some embodiments.
Cytochrome P450 is reduced by the activity of cytochrome P450 reductase (CPR),
thereby
recycling cytochrome P450 to allow further enzymatic activity. In certain
embodiments, the CPR
enzyme is endogenous to the host microorganism. In some embodiments, host CPR
activity can
be increased by increasing the number of copies of a CPR gene (e.g., 2, 3, 4,
5, 6, 7, 8, 9, 10, 15,
20, 25 or more copies of the gene), by increasing the activity of a promoter
that regulates
transcription of a CPR gene, or by increasing the number of copies of a CPR
gene and increasing
the activity of a promoter that regulates transcription of a CPR gene, thereby
increasing the
production of target product (e.g., adipic acid) via increased recycling of
cytochrome P450. In
certain embodiments, the promoter can be a heterologous promoter (e.g.,
endogenous or
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exogenous promoter). In some embodiments, the CPR gene is heterologous and
exogenous and
can be isolated from any suitable organism. Non-limiting examples of organisms
from which a
CPR gene can be isolated include C. tropicalis, C. viswanithii, S. cerevisiae
and Bacillus
megaterium.
Oxidation of the alcohol to an aldehyde may be performed by an enzyme in the
fatty alcohol
oxidase family (e.g., 6-hydroxyhexanoic acid dehydrogenase, omega hydroxyl
fatty acid
dehydrogenase), or an enzyme in the aldehyde dehydrogenase family (e.g., 6-
oxohexanoic acid
dehydrogenase, omega oxo fatty acid dehydrogenase). The enzyme 6-oxohexanoic
acid
dehydrogenase or omega oxo fatty acid dehydrogenase may oxidize the aldehyde
to the carboxylic
acid adipic acid. In some embodiments, the enzymes 6-hydroxyhexanoic acid
dehydrogenase,
omega hydroxyl fatty acid dehydrogenase, fatty alcohol oxidase, 6-oxohexanoic
acid
dehydrogenase, or omega oxo fatty acid dehydrogenase, exist in a host
organism. Flux through
these two steps may sometimes be augmented by increasing the copy number of
the enzymes, or
by increasing the activity of the promoter transcribing the genes. In some
embodiments alcohol
and aldehyde dehydrogenases specific for six carbon substrates may be isolated
from another
organism, for example Acinetobacter, Candida, Saccharomyces or Pseudomonas and
inserted into
the host organism.
Figures 10 and 37 depict embodiments of biological pathways for making adipic
acid, using fats,
oils, dicarboxylic acids, paraffins (e.g., linear, branched, substituted,
saturated, unsaturated, the
like and combinations thereof), fatty alcohols, fatty acids, or the like, as
the carbon source starting
material. Any suitable fatty alcohol, fatty acid, paraffin, dicarboxylic acid,
fat or oil can be used as
the feedstock for the organism, (e.g., hexane, hexanoic acid, oleic acid,
coconut oil, the like or
combinations thereof). Carbon sources with longer chain lengths (e.g., 8
carbons or greater in
length) can be metabolized using naturally occurring and/or engineered
pathways to yield
molecules that can be further metabolized using the beta oxidation pathway
shown in Figure 10
and the lower portion of Figures 11A and 37. In some embodiments, the
activities in the pathway
shown in Figure 10 and 37 also can be engineered (e.g., as described herein)
to enhance
metabolism and target product formation.
In certain embodiments, one or more activities in one or more metabolic
pathways can be
engineered to increase carbon flux through the engineered pathways to produce
a desired product
(e.g., adipic acid). The engineered activities can be chosen to allow
increased production of
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metabolic intermediates that can be utilized in one or more other engineered
pathways to achieve
increased production of a desired product with respect to the unmodified host
organism. This
"carbon flux management" can be optimized for any chosen feedstock, by
engineering the
appropriate activities in the appropriate pathways. A non-limiting example is
given herein using an
oil (e.g. coconut oil) based feedstock (see Figure 37). The engineered
activities increase the
production of adipic acid through the increased activities in a number of
pathways (e.g., fatty acid
degradation, fatty acid synthesis, gluconeogenesis, pentose phosphate pathway,
beta oxidation,
omega oxidation). The pathways utilized in the non-limiting examples presented
herein were
chosen to maximize the production of adipic acid by regenerating or utilizing
metabolic byproducts
to internally generate additional carbon sources that can be further
metabolized to produce adipic
acid. The process of "carbon flux management" through engineered pathways
produces adipic
acid at a level and rate closer to the calculated maximum theoretical yield
for any given feedstock,
in certain embodiments. The terms "theoretical yield" or "maximum theoretical
yield" as used
herein refer to the yield of product of a chemical or biological reaction that
would be formed if the
reaction went to completion. Theoretical yield is based on the stoichiometry
of the reaction and
ideal conditions in which starting material is completely consumed, undesired
side reactions do not
occur, the reverse reaction does not occur, and there no losses in the work-up
procedure. The
overall yield of product depends on the limiting reagent. In the embodiment
depicted in Figure 37,
carbon flux management is achieved by the production of adipic acid from fatty
acids liberated from
triacylglcyerides, and synthesis of glucose through gluconeogenesis which is
subsequently
converted to adipic acid through the engineered pentose phosphate pathway,
omega oxidation
pathway and beta oxidation pathway, as described herein.
Fats, oils, paraffins and the like frequently contain triacylglycerides that
can be converted into one
or more products useful for producing adipic acid utilizing engineered
microorganisms described
herein. Triacylglycerides can be converted into glycerol and fatty acids by a
lipase activity, as
shown in Figure 37. Lipases catalyze the hydrolysis of ester chemical bonds in
water-insoluble
lipid (e.g., fats, oils, paraffins) substrates. The generalized reaction can
be represented by;
triacylglycerol + H(2)0 <=> diacylglycerol + a carboxylate
Lipase activity often is associated with activiation of other activities or
pathways, by its involvement
with protein lipoylation. Certain lipases are involved in the modification of
mitochondria! enzymes.
Increasing lipase activity in an engineered microorganism can enhance the
utilization of fats, oils,
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paraffins and the like as feedstocks for production of adipic acid by
increasing overall carbon flux
through the native and engineered pathways of a host microorganism.
Fatty acids cleaved from a glycerol backbone can be further metabolized
directly, or indirectly via
utilization in synthesis pathways that yield products that subsequently can be
metabolized, by
native and/or engineered (i) omega oxidation pathways, (ii) beta oxidation
pathways, (iii) fatty acid
synthase pathways, (iv) hexanoate synthase pathways, or (v) combinations
thereof, described
herein and shown in Figure 37. In some embodiments, the pathway by which a
fatty acid is further
directly or indirectly metabolized into adipic acid, is determined by fatty
acid chain length.
The glycerol backbone also can be further metabolized (e.g., directly and/or
indirectly) to yield
adipic acid, by entry into the gluconeogenesis pathway to yield glucose. To
further increase
production of adipic acid, metabolism of the increased carbon flux through
gluconeogenesis can be
enhanced by increasing glucose-6-phosphate dehydrogenase activity. Glucose-6-
phosphate
dehydrogenase (EC 1.1.1.49) catalyzes the first step of the pentose phosphate
pathway, and is
encoded by the C. tropicalis gene, ZWF. The reaction for the first step in the
PPP pathway is:
D-glucose 6-phosphate + NADP+ = D-glucono-1,5-lactone 6-phosphate + NADPH + H+
This reaction is irreversible and rate-limiting for efficient fermentation of
sugar via the Entner-
Doudoroff pathway. The enzyme regenerates NADPH from NADP+ and is important
both for
maintaining cytosolic levels of NADPH and protecting yeast against oxidative
stress. Glucoses-6-
phosphate expression in yeast is constitutive, and the activity is inhibited
by NADPH such that
processes that decrease the cytosolic levels of NADPH stimulate the oxidative
branch of the
pentose phosphate pathway. Amplification of glucose-6-phosphate dehydrogenase
activity in
yeast may be desirable to increase the proportion of glucose-6-phosphate
converted to 6-
phosphoglucono-lactone and thereby improve conversion of sugar (e.g., glucose)
to adipic acid via
metabolism into products that can be further metabolized by native and/or
engineered (i) omega
oxidation pathways, (ii) beta oxidation pathways, (iii) fatty acid synthase
pathways, (iv) hexanoate
synthase pathways, or (v) combinations thereof, described herein, and shown in
Figure 37.
Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity in yeast may be
amplified by over-
expression of the ZWF gene by any suitable method. Non-limiting examples of
methods suitable
to amplify or over express ZWF include amplifying the number of ZWF genes in
yeast following
transformation with a high-copy number plasmid (e.g., such as one containing a
2u origin of
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replication), integration of multiple copies of ZWF into the yeast genome,
over-expression of the
ZWF gene directed by a strong promoter, the like or combinations thereof. The
ZWF gene may be
native to C. tropicalis or C. viswanithii, for example, or it may be obtained
from a heterologous
source.
Depicted in the first step of the reaction in Figure 10, the enzyme acyl-CoA
ligase converts a long
chain fatty alcohol, fatty acid or dicarboxylic acid and 1 molecule of acetyl-
CoA into an acyl-CoA
derivative of the long chain fatty alcohol, fatty acid or dicarboxylic acid
with the conversion of ATP
to AMP and inorganic phosphate. The term "beta oxidation pathway" as used
herein, refers to a
series of enzymatic activities utilized to metabolize fatty alcohols, fatty
acids, or dicarboxylic acids.
The activities utilized to metabolize fatty alcohols, fatty acids, or
dicarboxylic acids include, but are
not limited to, acyl-CoA ligase activity, acyl-CoA oxidase activity, acyl-CoA
hydrolase, acyl-CoA
thioesterase enoyl-CoA hydratase activity, 3-hydroxyacyl-CoA dehydrogenase
activity and acetyl-
CoA C-acyltransferase activity. The term "beta oxidation activity" refers to
any of the activities in
the beta oxidation pathway utilized to metabolize fatty alcohols, fatty acids
or dicarboxylic acids.
The term "omega oxidation activity" refers to any of the activities in the
omega oxidation pathway
utilized to metabolize fatty alcohols, fatty acids, dicarboxylic acids, or
sugars.
In certain embodiments, an acyl-CoA ligase enzyme converts a long chain fatty
alcohol, fatty acid
or dicarboxylic acid into the acyl-CoA derivative, which may be oxidized to
yield a trans-2,3-
dehydroacyl-CoA derivative, by the activity of Acyl CoA oxidase (e.g., also
known as acyl-CoA
oxidoreductase and fatty acyl-coenzyme A oxidase). The trans-2,3-dehydroacyl-
CoA derivative
long chain fatty alcohol, fatty acid or dicarboxylic acid may be further
converted to 3-hydroxyacyl-
CoA by the activity of enoyl-CoA hydratase. 3-hydroxyacyl-CoA can be converted
to 3-oxoacyl-
CoA by the activity of 3-hydroxyacyl-CoA dehydrogenase. 3-oxoacyl-CoA may be
converted to an
acyl-CoA molecule, shortened by 2 carbons and an acetyl-CoA, by the activity
of Acetyl-CoA C-
acyltransferase (e.g., also known as beta-ketothiolase and beta-ketothiolase).
In some
embodiments, acyl-CoA molecules may be repeatedly shortened by beta oxidation
until a desired
carbon chain length is generated (e.g., 6 carbons, adipic acid). The shortened
fatty acid can be
further processed using omega oxidation to yield adipic acid.
An acyl-CoA ligase enzyme sometimes is encoded by the host organism and can be
added to
generate an engineered organism. In some embodiments, host acyl-CoA ligase
activity can be
increased by increasing the number of copies of an acyl-CoA ligase gene, by
increasing the activity
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of a promoter that regulates transcription of an acyl-CoA ligase gene, or by
increasing the number
copies of the gene and by increasing the activity of a promoter that regulates
transcription of the
gene, thereby increasing production of target product (e.g., adipic acid) due
to increased carbon
flux through the pathway. In certain embodiments, the acyl-CoA ligase gene can
be isolated from
any suitable organism. Non-limiting examples of organisms that include, or can
be used as donors
for, acyl-CoA ligase enzymes include Candida, Saccharomyces, or Yarrowia.
An enoyl-CoA hydratase enzyme catalyzes the addition of a hydroxyl group and a
proton to the
unsaturated [3-carbon on a fatty-acyl CoA and sometimes is encoded by the host
organism and
sometimes can be added to generate an engineered organism. In certain
embodiments, the enoyl-
CoA hydratase activity is unchanged in a host or engineered organism. In some
embodiments, the
host enoyl-CoA hydratase activity can be increased by increasing the number of
copies of an
enoyl-CoA hydratase gene, by increasing the activity of a promoter that
regulates transcription of
an enoyl-CoA hydratase gene, or by increasing the number copies of the gene
and by increasing
the activity of a promoter that regulates transcription of the gene, thereby
increasing the production
of target product (e.g., adipic acid) due to increased carbon flux through the
pathway. In certain
embodiments, the enoyl-CoA hydratase gene can be isolated from any suitable
organism. Non-
limiting examples of organisms that include, or can be used as donors for,
enoyl-CoA hydratase
enzymes include Candida, Saccharomyces, or Yarrowia.
3-hydroxyacyl-CoA dehydrogenase enzyme catalyzes the formation of a 3-ketoacyl-
CoA by
removal of a hydrogen from the newly formed hydroxyl group created by the
activity of enoyl-CoA
hydratase. In some embodiments, the activity is encoded by the host organism
and sometimes
can be added or increased to generate an engineered organism. In certain
embodiments, the 3-
hydroxyacyl-CoA activity is unchanged in a host or engineered organism. In
some embodiments,
the host 3-hydroxyacyl-CoA dehydrogenase activity can be increased by
increasing the number of
copies of a 3-hydroxyacyl-CoA dehydrogenase gene, by increasing the activity
of a promoter that
regulates transcription of a 3-hydroxyacyl-CoA dehydrogenase gene, or by
increasing the number
copies of the gene and by increasing the activity of a promoter that regulates
transcription of the
gene, thereby increasing production of target product (e.g., adipic acid) due
to increased carbon
flux through the pathway. In certain embodiments, the 3-hydroxyacyl-CoA
dehydrogenase gene
can be isolated from any suitable organism. Non-limiting examples of organisms
that include, or
can be used as donors for, 3-hydroxyacyl-CoA dehydrogenase enzymes include
Candida,
Saccharomyces, or Yarrowia.
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An Acetyl-CoA C-acyltransferase (e.g., beta-ketothiolase) enzyme catalyzes the
formation of a fatty
acyl-CoA shortened by 2 carbons by cleavage of the 3-ketoacyl-CoA by the thiol
group of another
molecule of CoA. The thiol is inserted between C-2 and C-3, which yields an
acetyl CoA molecule
and an acyl CoA molecule that is two carbons shorter. An Acetyl-CoA C-
acyltransferase
sometimes is encoded by the host organism and sometimes can be added to
generate an
engineered organism. In certain embodiments, the acetyl-CoA C-acyltransferase
activity is
unchanged in a host or engineered organism. In some embodiments, the host
acetyl-CoA C-
acyltransferase activity can be increased by increasing the number of copies
of an acetyl-CoA C-
acyltransferase gene, or by increasing the activity of a promoter that
regulates transcription of an
acetyl-CoA C-acyltransferase gene, thereby increasing the production of target
product (e.g.,
adipic acid) due to increased carbon flux through the pathway. In certain
embodiments, the acetyl-
CoA C-acyltransferase gene can be isolated from any suitable organism. Non-
limiting examples of
organisms that include, or can be used as donors for, acetyl-CoA C-
acyltransferase enzymes
include Candida, Saccharomyces, or Yarrowia.
FIGS. 42A and 42B illustrate pathways that can be manipulated to produce
adipic acid from
sugars, cellulose, triacylglycerides and fatty acids. Illustrated in FIG. 42A
are various activities
normally active in a host organism, whereas FIG. 42B illustrates activities,
that when manipulated,
direct the flow of carbon in the host organism towards the production of
adipic acid through
increased fatty acid production and increased omega and beta oxidation
activities. As shown in
FIG. 42B, activities that are increased or added are shown with a "+" and
activities that are reduced
or eliminated are shown with a "X". In addition to directing the flow of
carbon towards the
production of adipic acid through increased fatty acid production and
increased omega and beta
oxidation activities, the altered activities in the pathways illustrated in
FIGS. 42A and 42B direct the
flow of carbon away from the production of biomass and carbon storage
molecules (e.g., starch,
lipids, triacylglycerides, the like, combinations thereof) and away from the
utilization of fatty acids
for energy. Increased activities shown in FIGS. 42A and 42B (e.g.,
monooxygenase (e.g., P450),
monooxygenase reductase (e.g., CPR), and thioesterase (e.g., ACH and TESA))
are described
herein.
A microorganism may be modified and engineered to include or regulate one or
more activities in
an adipic acid pathway. The term "activity" as used herein refers to the
functioning of a
microorganism's natural or engineered biological pathways to yield various
products including
adipic acid and its precursors. Adipic acid producing activity can be provided
by any non-
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mammalian source in certain embodiments. Such sources include, without
limitation, eukaryotes
such as yeast and fungi and prokaryotes such as bacteria. In some embodiments,
a reverse
activity in a pathway described herein can be altered (e.g., disrupted,
reduced) to increase carbon
flux through a beta oxidation pathway, an omega oxidation pathway, or a beta
oxidation and
omega oxidation pathway, towards the production of target product (e.g.,
adipic acid). In some
embodiments, a genetic modification disrupts an activity in the beta oxidation
pathway, or disrupts
a polynucleotide that encodes a polypeptide that carries out a forward
reaction in the beta
oxidation pathway, which renders beta oxidation activity undetectable. The
term "undetectable" as
used herein refers to an amount of an analyte that is below the limits of
detection, using detection
methods or assays known (e.g., described herein). In certain embodiments, the
genetic
modification partially reduces beta oxidation activity. The term "partially
reduces beta oxidation
activity" as used here refers to a level of activity in an engineered organism
that is lower than the
level of activity found in the host or starting organism.
An activity within an engineered microorganism provided herein can include one
or more (e.g., 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or all) of the following
activities: 6-oxohexanoic acid
dehydrogenase activity; 6-hydroxyhexanoic acid dehydrogenase activity;
hexanoate synthase
activity; cytochrome P450 activity; cytochrome P450 reductase activity; fatty
alcohol oxidase
activity; acyl-CoA ligase activity, acyl-CoA oxidase activity; enoyl-CoA
hydratase activity, 3-
hydroxyacyl-CoA dehydrogenase activity, fatty acid synthase activity, lipase
activity, acyl-CoA
carboxylase activity, glucose-6-phosphate dehydrogenase, and thioesterase
activity (e.g., acyl-
CoA hydrolase, acyl-CoA thioesterase, acetyl-CoA C-acyltransferase, beta-
ketothiolase). In
certain embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all)
of the foregoing activities
is altered by way of a genetic modification. In some embodiments, one or more
(e.g., 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14 or all) of the foregoing activities is altered
by way of (i) adding a
heterologous polynucleotide that encodes a polypeptide having the activity,
and/or (ii) altering or
adding a regulatory sequence that regulates the expression of a polypeptide
having the activity.
The term "6-oxohexanoic acid dehydrogenase activity" as used herein refers to
conversion of 6-
oxohexanoic acid to adipic acid. The 6-oxohexanoic acid dehydrogenase activity
can be provided
by a polypeptide. In some embodiments, the polypeptide is encoded by a
heterologous nucleotide
sequence introduced to a host microorganism. In certain embodiments, an
endogenous
polypeptide having the 6-oxohexanoic acid dehydrogenase activity is identified
in the host
microorganism, and the host microorganism is genetically altered to increase
the amount of the
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polypeptide produced (e.g., a heterologous promoter is introduced in operable
linkage with a
polynucleotide that encodes the polypeptide; the copy number of a
polynucleotide that encodes the
polypeptide is increased (e.g., by introducing a plasmid that includes the
polynucleotide)). Nucleic
acid sequences conferring 6-oxohexanoic acid dehydrogenase activity can be
obtained from a
number of sources, including Actinobacter, Norcardia, Pseudomonas and
Xanthobacter bacteria.
Examples of an amino acid sequence of a polypeptide having 6-oxohexanoic acid
dehydrogenase
activity, and a nucleotide sequence of a polynucleotide that encodes the
polypeptide, are
presented herein. Presence, absence or amount of 6-oxohexanoic acid
dehydrogenase activity
can be detected by any suitable method known in the art. For an example of a
detection method
for alcohol oxidase or alcohol dehydrogenase activity (see Appl. Environ.
Microbiol. 70: 4872). In
some embodiments, 6-oxohexanoic acid dehydrogenase activity is not altered in
a host
microorganism, and in certain embodiments, the activity is added or increased
in the engineered
microorganism relative to the host microorganism.
The term "omega oxo fatty acid dehydrogenase activity" as used herein refers
to conversion of an
omega oxo fatty acid to a dicarboxylic acid. The omega oxo fatty acid
dehydrogenase activity can
be provided by a polypeptide. In some embodiments, the polypeptide is encoded
by a
heterologous nucleotide sequence introduced to a host microorganism. In
certain embodiments,
an endogenous polypeptide having the omega oxo fatty acid dehydrogenase
activity is identified in
the host microorganism, and the host microorganism is genetically altered to
increase the amount
of the polypeptide produced (e.g., a heterologous promoter is introduced in
operable linkage with a
polynucleotide that encodes the polypeptide; the copy number of a
polynucleotide that encodes the
polypeptide is increased (e.g., by introducing a plasmid that includes the
polynucleotide)). Nucleic
acid sequences conferring omega oxo fatty acid dehydrogenase activity can be
obtained from a
number of sources, including Actinobacter, Norcardia, Pseudomonas and
Xanthobacter bacteria.
Examples of an amino acid sequence of a polypeptide having omega oxo fatty
acid
dehydrogenase activity and a nucleotide sequence of a polynucleotide that
encodes the
polypeptide, are presented herein. Presence, absence or amount of omega oxo
fatty acid
dehydrogenase activity can be detected by any suitable method known in the
art. In some
embodiments, omega oxo fatty acid dehydrogenase activity is not altered in a
host microorganism,
and in certain embodiments, the activity is added or increased in the
engineered microorganism
relative to the host microorganism.
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The term "6-hydroxyhexanoic acid dehydrogenase activity" as used herein refers
to conversion of
6-hydroxyhexanoic acid to 6-oxohexanoic acid. The 6-hydroxyhexanoic acid
dehydrogenase
activity can be provided by a polypeptide. In some embodiments, the
polypeptide is encoded by a
heterologous nucleotide sequence introduced to a host microorganism. In
certain embodiments,
an endogenous polypeptide having the 6-hydroxyhexanoic acid dehydrogenase
activity is identified
in the host microorganism, and the host microorganism is genetically altered
to increase the
amount of the polypeptide produced (e.g., a heterologous promoter is
introduced in operable
linkage with a polynucleotide that encodes the polypeptide; the copy number of
a polynucleotide
that encodes the polypeptide is increased (e.g., by introducing a plasmid that
includes the
polynucleotide)). Nucleic acid sequences conferring 6-hydroxohexanoic acid
dehydrogenase
activity can be obtained from a number of sources, including Actinobacter,
Norcardia,
Pseudomonas, and Xanthobacter. Examples of an amino acid sequence of a
polypeptide having
6-hydroxyhexanoic acid dehydrogenase activity, and a nucleotide sequence of a
polynucleotide
that encodes the polypeptide, are presented herein. Presence, absence or
amount of 6-
hydroxyhexanoic acid dehydrogenase activity can be detected by any suitable
method known in
the art. An example of such a method is described in Methods in Enzymology,
188: 176. In some
embodiments, 6-hydroxyhexanoic acid dehydrogenase activity is not altered in a
host
microorganism, and in certain embodiments, the activity is added or increased
in the engineered
microorganism relative to the host microorganism.
The term "omega hydroxyl fatty acid dehydrogenase activity" as used herein
refers to conversion of
an omega hydroxyl fatty acid to an omega oxo fatty acid. The omega hydroxyl
fatty acid
dehydrogenase activity can be provided by a polypeptide. In some embodiments,
the polypeptide
is encoded by a heterologous nucleotide sequence introduced to a host
microorganism. In certain
embodiments, an endogenous polypeptide having the omega hydroxyl fatty acid
dehydrogenase
activity is identified in the host microorganism, and the host microorganism
is genetically altered to
increase the amount of the polypeptide produced (e.g., a heterologous promoter
is introduced in
operable linkage with a polynucleotide that encodes the polypeptide; the copy
number of a
polynucleotide that encodes the polypeptide is increased (e.g., by introducing
a plasmid that
includes the polynucleotide)). Nucleic acid sequences conferring omega
hydroxyl fatty acid
dehydrogenase activity can be obtained from a number of sources, including
Actinobacter,
Norcardia, Pseudomonas and Xanthobacter bacteria. Examples of an amino acid
sequence of a
polypeptide having omega hydroxyl fatty acid dehydrogenase activity and a
nucleotide sequence of
a polynucleotide that encodes the polypeptide are presented herein. Presence,
absence or
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amount of omega hydroxyl fatty acid dehydrogenase activity can be detected by
any suitable
method known in the art. In some embodiments, omega hydroxyl fatty acid
dehydrogenase activity
is not altered in a host microorganism, and in certain embodiments, the
activity is added or
increased in the engineered microorganism relative to the host microorganism.
The term "lipase activity" as used herein refers to the hydrolysis of
triacylglycerol to produce a
diacylglycerol and a fatty acid anion. The lipase activity can be provided by
a polypeptide. In
certain embodiments, an endogenous polypeptide having the lipase activity is
identified in the host
microorganism, and the host microorganism is genetically altered to increase
the amount of the
polypeptide produced (e.g., a heterologous promoter is introduced in operable
linkage with a
polynucleotide that encodes the polypeptide; the copy number of a
polynucleotide that encodes the
polypeptide is increased (e.g., by introducing a plasmid that includes the
polynucleotide)).
Examples of a nucleotide sequence of a polynucleotide that encodes a
polypeptide having lipase
activity, and amino acid sequences that code a lipase activity are presented
herein. In some
embodiments, lipase activity is not altered in a host microorganism, and in
certain embodiments,
the activity is added or increased in the engineered microorganism relative to
the host
microorganism. Presence, absence or amount of lipase activity can be detected
by any suitable
method known in the art, including western blot analysis.
The term "glucose-6-phosphate dehydrogenase activity" as used herein refers to
the conversion of
D-glucose 6-phosphate into D-glucono-1,5-lactone 6-phosphate. Glucose-6-
phosphate
dehydrogenase is an activity that forms part of the pentose phosphate pathway.
Glycerol
backbones liberated from fatty acids by a lipase activity can ultimately be
converted to glucose by
the action of the gluconeogenesis pathway, where glycerol is first converted
to dihydroxyacetone
phosphate. Glucose can be preferentially metabolized by the pentose phosphate
pathway by
increasing one or more activities in the pentose phosphate pathway (e.g.,
glucose-6-phosphate
dehydrogenase). In addition to increasing the conversion of D-glucose 6-
phosphate into D-
glucono-1,5-lactone 6-phosphate, increasing the level of glucose-6-phosphate
dehydrogenase
activity also may yield advantageous benefits due to the additional reducing
power generated by
the increased activity of glucose-6-phosphate dehydrogenase. In some
embodiments, increasing
the activity of glucose-6-phosphate dehydrogenase increases the activity of a
gluconeogenesis
pathway, a pentose phosphate pathway or a gluconeogenesis pathway and a
pentose phosphate
pathway due to a forward biased increased carbon flux through the pathways.
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A glucose-6-phosphate dehydrogenase (G6PD) activity may be provided by an
enzyme. The
glucose-6-phosphate dehydrogenase activity can be provided by a polypeptide.
In certain
embodiments, an endogenous polypeptide having the glucose-6-phosphate
dehydrogenase
activity is identified in the host microorganism, and the host microorganism
is genetically altered to
increase the amount of the polypeptide produced (e.g., a heterologous promoter
is introduced in
operable linkage with a polynucleotide that encodes the polypeptide; the copy
number of a
polynucleotide that encodes the polypeptide is increased (e.g., by introducing
a plasmid that
includes the polynucleotide)). Examples of a nucleotide sequence of a
polynucleotide that
encodes a polypeptide having glucose-6-phosphate dehydrogenase activity, is
presented herein.
In some embodiments, glucose-6-phosphate dehydrogenase activity is not altered
in a host
microorganism, and in certain embodiments, the activity is added or increased
in the engineered
microorganism relative to the host microorganism. Presence, absence or amount
of glucose-6-
phosphate dehydrogenase activity can be detected by any suitable method known
in the art
including western blot analysis
The term "acetyl-CoA carboxylase activity" as used herein refers to the
irreversible carboxylation of
acetyl-CoA to produce malonyl-CoA. Acetyl-CoA carboxylase activity may be
provided by an
enzyme that includes one or two subunits, depending on the source organism.
The acetyl-CoA
carboxylase synthase activity can be provided by a polypeptide. In certain
embodiments, an
endogenous polypeptide having the acetyl-CoA carboxylase activity is
identified in the host
microorganism, and the host microorganism is genetically altered to increase
the amount of the
polypeptide produced (e.g., a heterologous promoter is introduced in operable
linkage with a
polynucleotide that encodes the polypeptide; the copy number of a
polynucleotide that encodes the
polypeptide is increased (e.g., by introducing a plasmid that includes the
polynucleotide)). An
example of a nucleotide sequence of a polynucleotide that encodes a
polypeptide having acetyl-
CoA carboxylase activity, is presented herein. In some embodiments, acetyl-CoA
carboxylase
activity is not altered in a host microorganism, and in certain embodiments,
the activity is added or
increased in the engineered microorganism relative to the host microorganism.
Presence,
absence or amount of acetyl-CoA carboxylase activity can be detected by any
suitable method
known in the art including western blot analysis
The term "fatty acid synthase activity" as used herein refers to conversion of
acetyl-CoA and
malonyl-CoA to fatty acids. Fatty acid synthase activity may be provided by an
enzyme that
includes one or two subunits (referred to hereafter as "subunit alpha" and/or
"subunit beta"). The
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fatty acid synthase activity can be provided by a polypeptide. In certain
embodiments,
endogenous polypeptides having the fatty acid synthase activity are identified
in the host
microorganism, and the host microorganism is genetically altered to increase
the amount of the
polypeptide produced (e.g., a heterologous promoter is introduced in operable
linkage with a
polynucleotide that encodes the polypeptide; the copy number of a
polynucleotide that encodes the
polypeptide is increased (e.g., by introducing a plasmid that includes the
polynucleotide)).
Examples of a nucleotide sequence of a polynucleotide that encodes a
polypeptide having fatty
acid synthase activity, is presented herein. In some embodiments, fatty acid
synthase activity is
not altered in a host microorganism, and in certain embodiments, the activity
is added or increased
in the engineered microorganism relative to the host microorganism. Presence,
absence or
amount of fatty acid synthase activity can be detected by any suitable method
known in the art,
including western blot analysis.
The term "hexanoate synthase activity" as used herein refers to conversion of
acetyl-CoA and
malonyl-CoA to hexanoic acid. Hexanoate synthase activity may be provided by
an enzyme that
includes one or two subunits (referred to hereafter as "subunit A" and/or
"subunit B"). The
hexanoate synthase activity can be provided by a polypeptide. In some
embodiments, the
polypeptide is encoded by a heterologous nucleotide sequence introduced to a
host
microorganism. Nucleic acid sequences conferring hexanoate synthase activity
can be obtained
from a number of sources, including Aspergillus parasiticus, for example.
Examples of an amino
acid sequence of a polypeptide having hexanoate synthase activity, and a
nucleotide sequence of
a polynucleotide that encodes the polypeptide, are presented herein. In some
embodiments,
hexanoate synthase activity is not altered in a host microorganism, and in
certain embodiments,
the activity is added or increased in the engineered microorganism relative to
the host
microorganism.
Presence, absence or amount of hexanoate synthase activity can be detected by
any suitable
method known in the art. An example of such a method is described in Hexanoate
synthase +
thioesterase (Chemistry and Biology 9: 981-988). Briefly, an indicator strain
may be prepared. An
indicator strain may be Bacillus subtilis containing a reporter gene (beta-
galactosidase, green
fluorescent protein, etc.) under control of the promoter regulated by LiaR,
for example. An
indicator strain also may be Candida tropicalis or Candida viswanithii
containing either the LiaR
regulatable promoter from Bacillus subtilis or the alkane inducible promoter
for the native gene for
the peroxisomal 3-ketoacyl coenzyme A thiolase gene (CT-T3A), for example.
Mutants with an
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improved functionality of HexS, thereby producing more hexanoic acid, can be
plated onto a lawn
of indicator strain. Upon incubation and growth of both the test mutant and
the indicator strain, the
appearance of a larger halo, which correlates to the induction of the reporter
strain compared to
control strains, indicates a mutant with improved activity. In alternative
approach, mutants are
grown in conditions favoring production of hexanoyl CoA or hexanoic acid and
lysed. Cell lysates
are treated with proteases which may release hexanoic acid from the PKS.
Clarified lysates may
be spotted onto lawns of indicator strains to assess improved production. In
another alternative
approach, indicator strains are grown under conditions suitable to support
expression of the
reporter gene when induced by hexanoic acid. Dilutions of a known
concentration of hexanoic acid
are used to determine a standard curve. Lysates of the test strain grown under
conditions favoring
production of hexanoic acid are prepared and dilutions of the lysate added to
the indicator strain.
Indicator strains with lysates are placed under identical conditions as used
to determine the
standard curve. The lysate dilutions that minimally support induction can be
used to determine,
quantitatively, the amount produced when compared to the standard curve.
The term "monooxygenase activity" as used herein refers to inserting one atom
of oxygen from 02
into an organic substrate (RH) and reducing the other oxygen atom to water. In
some
embodiments, monooxygenase activity refers to incorporation of an oxygen atom
onto a six-carbon
organic substrate. In certain embodiments, monooxygenase activity refers to
conversion of
hexanoate to 6-hydroxyhexanoic acid. Monooxygenase activity can be provided by
any suitable
polypeptide, such as a cytochrome P450 polypeptide (hereafter "CYP450") in
certain
embodiments. Nucleic acid sequences conferring CYP450 activity can be obtained
from a number
of sources, including Bacillus megaterium and may be induced in organisms
including but not
limited to Candida tropicalis, Candida viswanithii, Yarrowia lipolytica,
Aspergillus nidulans, and
Aspergillus parasiticus. Examples of oligonucleotide sequences utilized to
isolate a polynucleotide
sequence encoding a polypeptide having CYP450 activity (e.g., CYP52Al2
polynucleotide, a
CYP52A13 polynucleotide, a CYP52A14 polynucleotide, a CYP52A15 polynucleotide,
a
CYP52A16 polynucleotide, a CYP52A17 polynucleotide, a CYP52A18 polynucleotide,
a
CYP52A19 polynucleotide, a CYP52A20 polynucleotide, a CYP52D2 polynucleotide,
and/or a BM3
polynucleotide) are presented herein. In some embodiments, monooxygenase
activity is not
altered in a host microorganism, and in certain embodiments, the activity is
added or increased in
the engineered microorganism relative to the host microorganism. In some
embodiments, the
altered monooxygenase activity is an endogenous activity, and in certain
embodiments, the altered
monooxygenase activity is an exogenous activity. In some embodiments, the
exogenous activity is
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a single polypeptide with both monooxygenase and monooxygenase reductase
activities (e.g., B.
megaterium cytochrome P450:NADPH P450 reductase).
Presence, absence or amount of cytochrome P450 activity can be detected by any
suitable method
known in the art. For example, detection can be performed by assaying a
reaction containing
cytochrome P450 (CYP52A family) and NADPH - cytochrome P450 reductase (see
Appl Environ
Microbiol 69: 5983 and 5992). Briefly, cells are grown under standard
conditions and harvested
for production of microsomes, which are used to detect CYP activity.
Microsomes are prepared by
lysing cells in Tris-buffered sucrose (10mM Tris-HCI pH 7.5, 1mM EDTA, 0.25M
sucrose).
Differential centrifugation is performed first at 25,000xg then at 100,000xg
to pellet cell debris then
microsomes, respectively. The microsome pellet is resuspended in 0.1M
phosphate buffer (pH
7.5), 1mM EDTA to a final concentration of approximately 10mg protein/mL. A
reaction mixture
containing approximately 0.3mg microsomes, 0.1mM sodium hexanoate, 0.7mM
NADPH, 50mM
Tris-HCI pH 7.5 in 1mL is initiated by the addition of NADPH and incubated at
37 C for 10 minutes.
The reaction is terminated by addition of 0.25mL 5M HCI and 0.25mL 2.5ug/mL 10-
hydroxydecanoic acid is added as an internal standard (3.3 nmol). The mixture
is extracted with
4.5mL diethyl ether under NaCI-saturated conditions. The organic phase is
transferred to a new
tube and evaporated to dryness. The residue is dissolved in acetonitrile
containing 10mM 3-
bromomethy1-7-methoxy-1,4-benzoxazin-2-one (BrMB) and 0.1mL of 15mg/mL 18-
crown-6 in
acetonitril saturated with K2CO3. The solution is incubated at 40 C for 30
minutes before addition
of 0.05mL 2% acetic acid. The fluorescently labeled omega-hydroxy fatty acids
are resolved via
HPLC with detection at 430nm and excitation at 355nm (Yamada et al., 1991,
AnalBiochem 199:
132-136). Optionally, specifically induced CYP gene(s) may be detected by
Northern blotting
and/or quantitative RT-PCR. (Craft et al., 2003, App. Environ. Micro. 69: 5983-
5991).
The term "monooxygenase reductase activity" as used herein refers to the
transfer of an electron
from NAD(P)H, FMN, or FAD by way of an electron transfer chain, reducing the
ferric heme iron of
cytochrome P450 to the ferrous state. The term "monooxygenase reductase
activity" as used
herein also can refer to the transfer of a second electron via the electron
transport system,
reducing a dioxygen adduct to a negatively charged peroxo group. In some
embodiments, a
monooxygenase activity can donate electrons from the two-electron donor
NAD(P)H to the heme
of cytochrome P450 (e.g., monooxygenase activity) in a coupled two-step
reaction in which
NAD(P)H can bind to the NAD(P)H-binding domain of the polypeptide having the
monooxygenase
reductase activity and electrons are shuttled from NAD(P)H through FAD and FMN
to the heme of
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the monooxygenase activity, thereby regenerating an active monooxygenase
activity (e.g.,
cytochrome P450). Monooxygenase reductase activity can be provided by any
suitable
polypeptide, such as a cytochrome P450 reductase polypeptide (hereafter "CPR")
in certain
embodiments. Nucleic acid sequences conferring CPR activity can be obtained
from and/or
induced in a number of sources, including but not limited to Bacillus
megaterium, Candida
tropicalis, Candida viswanithii, Yarrowia lipolytica, Aspergillus nidulans,
and Aspergillus parasiticus.
Examples of oligonucleotide sequences utilized to isolate a polynucleotide
sequence encoding a
polypeptide having CPR activity are presented herein. In some embodiments,
monooxygenase
reductase activity is not altered in a host microorganism, and in certain
embodiments, the activity is
added or increased in the engineered microorganism relative to the host
microorganism. In some
embodiments, the altered monooxygenase reductase activity is an endogenous
activity, and in
certain embodiments, the altered monooxygenase reductase activity is an
exogenous activity. In
some embodiments, the exogenous activity is a single polypeptide with both
monooxygenase and
monooxygenase reductase activities (e.g., B. megaterium cytochrome P450:NADPH
P450
reductase).
Presence, absence or amount of CPR activity can be detected by any suitable
method known in
the art. For example, an engineered microorganism having an increased number
of genes
encoding a CPR activity, relative to the host microorganism, could be detected
using quantitative
nucleic acid detection methods (e.g., southern blotting, PCR, primer
extension, the like and
combinations thereof). An engineered microorganism having increased expression
of genes
encoding a CPR activity, relative to the host microorganism, could be detected
using quantitative
expression based analysis (e.g., RT-PCR, western blot analysis, northern blot
analysis, the like
and combinations thereof). Alternately, an enzymatic assay can be used to
detect Cytochrome
P450 reductase activity, where the enzyme activity alters the optical
absorbance at 550
nanometers of a substrate solution (Masters, B.S.S., Williams, C.H., Kamin, H.
(1967) Methods in
Enzymology, X, 565-573).
The term "fatty alcohol oxidase activity" as used herein refers to inserting
one atom of oxygen from
02 into an organic substrate and reducing the other oxygen atom to peroxide.
Fatty alcohol
oxidase activity sometimes also is referred to as "long-chain-alcohol oxidase
activity", "long-chain-
alcohol :oxygen oxidoreductase activity", "fatty alcohol :oxygen
oxidoreductase activity" and "long-
chain fatty acid oxidase activity". In some embodiments, fatty alcohol oxidase
activity refers to
incorporation of an oxygen atom onto a six-carbon organic substrate. In
certain embodiments,
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fatty alcohol oxidase activity refers to the conversion of 6-hydroxyhexanoic
acid into 6-oxohexanoic
acid. In some embodiments, fatty alcohol oxidase activity refers to the
conversion of an omega
hydroxyl fatty acid into an omega oxo fatty acid. A Fatty alcohol oxidase
(FAO) activity can be
provided by any suitable polypeptide, such as a fatty alcohol oxidase peptide,
a long-chain-alcohol
oxidase peptide, a long-chain-alcohol:oxygen oxidoreductase peptide, a fatty
alcohol:oxygen
oxidoreductase peptide and a long-chain fatty acid oxidase peptide. Nucleic
acid sequences
conferring FAO activity can be obtained from a number of sources, including
but not limited to
Candida tropicalis, Candida viswanithii, Candida cloacae, Yarrowia lipolytica,
and Arabidopsis
thaliana. Examples of amino acid sequences of polypeptides having FAO
activity, and nucleotide
sequences of polynucleotides that encode the polypeptides, are presented
herein. In some
embodiments, fatty alcohol oxidase activity is not altered in a host
microorganism, and in certain
embodiments, the activity is added or increased in the engineered
microorganism relative to the
host microorganism.
Presence, absence or amount of FAO activity can be detected by any suitable
method known in
the art. For example, an engineered microorganism having an increased number
of genes
encoding an FAO activity, relative to the host microorganism, could be
detected using quantitative
nucleic acid detection methods (e.g., southern blotting, PCR, primer
extension, the like and
combinations thereof). An engineered microorganism having increased expression
of genes
encoding an FAO activity, relative to the host microorganism, could be
detected using quantitative
expression based analysis (e.g., RT-PCR, western blot analysis, northern blot
analysis, the like
and combinations thereof). Alternately, an enzymatic assay can be used to
detect fatty alcohol
oxidase activity as described in Eirich et al, 2004, or as modified in the
Examples herein.
The term "acyl-CoA oxidase activity" as used herein refers to the oxidation of
a long chain fatty-
acyl-CoA to a trans-2,3-dehydroacyl-CoA fatty alcohol. In some embodiments,
the acyl-CoA
activity is from a peroxisome. In certain embodiments, the acyl-CoA oxidase
activity is a
peroxisomal acyl-CoA oxidase (PDX) activity, carried out by a PDX polypeptide.
In some
embodiments the acyl-CoA oxidase activity is encoded by the host organism and
sometimes can
be altered to generate an engineered organism. Acyl-CoA oxidase activity is
encoded by the
PDX4 and PDX5 genes of C. tropicalis or C. viswanithii in some embodiments. In
certain
embodiments, endogenous acyl-CoA oxidase activity can be increased. In some
embodiments,
acyl-CoA oxidase activity of the PDX4 polypeptide or the PDX5 polypeptide can
be altered
independently of each other (e.g., increase activity of PDX4 alone, PDX5
alone, increase one and
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disrupt the other, and the like). Increasing the activity of one PDX activity,
while disrupting the
activity of another PDX activity, may alter the specific activity of acyl-CoA
oxidase with respect to
carbon chain length, while maintaining or increasing overall flux through the
beta oxidation
pathway, in certain embodiments.
Figures 15A-15C graphically illustrate the units of acyl-CoA oxidase activity
expressed as units (U)
per milligram of protein (Y axis) in various strains of Candida tropicalis
induced by feedstocks of
specific chain length (Picataggio et al. 1991 Molecular and Cellular Biology
11: 4333-4339).
Isolated protein was assayed for acyl-CoA oxidase activity using carbon chains
of various length (X
axis). The X and Y axes in Figures 15A -15C represent substantially similar
data. Figure 15A
illustrates acyl-CoA oxidase activity as measured in a strain having a full
complement of PDX
genes (e.g., PDX4 and PDX5 are active). Figure 15B illustrates acyl-CoA
oxidase activity as
measured in a strain having a disrupted PDX5 gene. The activity encoded by the
functional PDX4
gene exhibits a higher specific activity for acyl-CoA molecules with shorter
carbon chain lengths
(e.g., less than 10 carbons). The results of the PDX5 disrupted strain also
are presented
numerically in the table in Figure 15B. Figure 15C illustrates acyl-CoA
oxidase activity as
measured in a strain having a disrupted PDX4 gene. The activity encoded by the
functional PDX5
gene exhibits a narrow peak of high specific activity for acyl-CoA molecules
12 carbons in length,
with a lower specific activity for molecules 10 carbons in length. The results
of the PDX4 disrupted
strain are presented numerically in the table in Figure 15C.
In certain embodiments, host acyl-CoA oxidase activity of one of the PDX genes
can be increased
by genetically altering (e.g., increasing) the amount of the polypeptide
produced (e.g., a strongly
transcribed or constitutively expressed heterologous promoter is introduced in
operable linkage
with a polynucleotide that encodes the polypeptide; the copy number of a
polynucleotide that
encodes the polypeptide is increased (e.g., by introducing a plasmid that
includes the
polynucleotide, integration of additional copies in the host genome)). In some
embodiments, the
host acyl-CoA oxidase activity can be decreased by disruption (e.g., knockout,
insertion
mutagenesis, the like and combinations thereof) of an acyl-CoA oxidase gene,
or by decreasing
the activity of the promoter (e.g., addition of repressor sequences to the
promoter or 5'UTR) which
transcribes an acyl-CoA oxidase gene.
A noted above, disruption of nucleotide sequences encoding PDX4, PDX 5, or
PDX4 and PDX5
sometimes can alter pathway efficiency, specificity and/or specific activity
with respect to
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metabolism of carbon chains of different lengths (e.g., carbon chains
including fatty alcohols, fatty
acids, paraffins, dicarboxylic acids of between about 1 and about 60 carbons
in length). In some
embodiments, the nucleotide sequence of PDX4, PDX5, or PDX4 and PDX5 is
disrupted with a
URA3 nucleotide sequence encoding a selectable marker, and introduced to a
host
microorganism, thereby generating an engineered organism deficient in PDX4,
PDX5 or PDX4 and
PDX5 activity. Nucleic acid sequences encoding PDX4 and PDX5 can be obtained
from a number
of sources, including Candida tropicalis, for example. Examples of PDX4 and
PDX5 amino acid
sequences and nucleotide sequences of polynucleotides that encode the
polypeptides, are
presented herein. Example 32 describes experiments conducted to amplify the
activity encoded
by the PDX5 gene.
Presence, absence or amount of PDX4 and/or PDX5 activity can be detected by
any suitable
method known in the art, for example, using enzymatic assays as described in
Shimizu et al, 1979,
and as described herein in the Examples. Alternatively, nucleic acid sequences
representing
native and/or disrupted PDX4 and PDX5 sequences also can be detected using
nucleic acid
detection methods (e.g., PCR, primer extension, nucleic acid hybridization,
the like and
combinations thereof), or quantitative expression based analysis (e.g., RT-
PCR, western blot
analysis, northern blot analysis, the like and combinations thereof), where
the engineered
organism exhibits decreased RNA and/or polypeptide levels as compared to the
host organism.
The term "a genetic modification that results in increased fatty acid
synthesis" as used herein
refers to a genetic alteration of a host microorganism that increases an
endogenous activity and/or
adds a heterologous activity that metabolically synthesizes fatty acids and/or
imports fatty acids
into the microorganism from an external source (e.g., a feedstock, culture
medium, environment,
the like and combinations thereof). In some embodiments, an endogenous
activity that converts
fatty acyl-CoA to fatty acid is increased. In certain embodiments, a
thioesterase activity is added or
increased. Such alterations can advantageously increase yields of end
products, such as adipic
acid.
The term "thioesterase activity" as used herein refers to removal of Coenzyme
A from hexanoate.
The term "thioesterase activity" as used herein also refers to the removal of
Coenzyme A from an
activated fatty acid (e.g., fatty-acyl-CoA). A Non-limiting example of an
enzyme with thioesterase
activity includes acyl-CoA hydrolase (e.g., EC 3.1.2.20; also referred to as
acyl coenzyme A
thioesterase, acyl-CoA thioesterase, acyl coenzyme A hydrolase, thioesterase
B, thioesterase II,
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lecithinase B, lysophopholipase L1, acyl-CoA thioesterase 1, and acyl-CoA
thioesterase).
Thioesterases that remove Coenzyme A from fatty-acyl-CoA molecules catalyze
the reaction,
acyl-CoA + H20 -4 CoA + a carboxylate,
where the carboxylate often is a fatty acid. The released Coenzyme A can then
be reused for other
cellular activities.
The thioesterase activity can be provided by a polypeptide. In certain
embodiments, the
polypeptide is an endogenous nucleotide sequence that is increased in copy
number, operably
linked to a heterologous and/or endogenous promoter, or increased in copy
number and operably
linked to a heterologous and/or endogenous promoter. In some embodiments, the
polypeptide is
encoded by a heterologous nucleotide sequence introduced to a host
microorganism. Nucleic acid
sequences conferring thioesterase activity can be obtained from a number of
sources, including C.
tropicalis (e.g., see SEQ ID NOS: 42 and 44), C. viswanithii, E. coli (e.g.,
see SEQ ID NO: 46) and
Cuphea lanceolata. Examples of such polypeptides include, without limitation,
acyl-CoA
hydrolase (e.g., ACHA and ACHB, see SEQ ID NOS: 43 and 45)) from C.
tropicalis, acyl-CoA
thioesterase (e.g., TESA, see SEQ ID NO: 47) from E. coli, and acyl-(ACP)
thioesterase type B
from Cuphea lanceolata, encoded by the nucleotide sequences referenced by
accession number
CAB60830 at the World Wide Web Uniform Resource Locator (URL) ncbi.nlm.nih.gov
of the
National Center for Biotechnology Information (NCB!).
Presence, absence or amount of thioesterase activity can be detected by any
suitable method
known in the art. An example of such a method is described Chemistry and
Biology 9: 981-988. In
some embodiments, thioesterase activity is not altered in a host
microorganism, and in certain
embodiments, the activity is added or increased in the engineered
microorganism relative to the
host microorganism. In some embodiments, a polypeptide having thioesterase
activity is linked to
another polypeptide (e.g., a hexanoate synthase A or hexanoate synthase B
polypeptide). A non-
limiting example of an amino acid sequence (one letter code sequence) for a
polypeptide having
thioesterase activity is provided hereafter:
MVAAAATSAFFPVPAPGTS PKPGKSGNWPSSLS PT FKPKS I PNAGFQVKANASAHP
KANGSAVNLKSGSLNTQEDTSSSPPPRAFLNQLPDWSMLLTAI TTVFVAAEKQWTM
LDRKSKRPDMLVDSVGLKS IVRDGLVSRQS FL I RS YE I GADRTAS I ET LMNHLQET
S INHCKSLGLLNDGFGRTPGMCKNDLIWVLTKMQIMVNRYPTWGDTVE INTWFSQS
GK I GMAS DWL I SDCNTGE I L I RAT SVWAMMNQKTRRF SRL PYEVRQELT PHFVDS P
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HVIEDNDQKLHKFDVKTGDS I RKGLT PRWNDLDVNQHVSNVKY I GWI LE SMP I EVL
ETQELCSLTVEYRRECGMDSVLESVTAVDPSENGGRSQYKHLLRLEDGTDIVKSRT
EWRPKNAGTNGAI STSTAKTSNGNSAS
Additional examples of polynucleotide sequences encoding thioesterase
activities, and
polypeptides having thioesterase activity are provided in Example 51 (see SEQ
ID NOS: 42-47).
The term "a genetic modification that results in substantial hexanoate usage
by monooxygenase
activity" as used herein refers to a genetic alteration of a host
microorganism that reduces an
endogenous activity that converts hexanoate to another product. In some
embodiments, an
endogenous activity that converts hexanoate to a toxin (e.g., in fungus) is
reduced. In certain
embodiments, a polyketide synthase activity is reduced. Such alterations can
advantageously
increase yields of end products, such as adipic acid.
The term "polyketide synthase activity" as used herein refers to the
alteration of hexanoic acid by
the polyketide synthase enzyme (PKS) as a step in the production of other
products including
mycotoxin. The PKS activity can be provided by a polypeptide. Examples of such
polypeptides
include, without limitation, an Aspergillus parasiticus enzyme referenced by
accession number
AA566004 at the World Wide Web Uniform Resource Locator (URL) ncbi.nlm.nih.gov
of the
National Center for Biotechnology Information (NCB!). In certain embodiments,
a PKS enzyme
uses hexanoic acid generated by hexanoate synthase as a substrate and a
component of the
Aspergillus NorS multienzyme complex, a closely associated gene cluster
involved in the synthesis
of various products including mytoxin. Accordingly, a PKS activity sometimes
is altered to free
hexanoic acid for an engineered adipic acid pathway. In some embodiments PKS
activity is
diminished or blocked. In certain embodiments the PKS enzyme is engineered to
substitute
thioesterase activity for PKS activity. Presence, absence, or amount of PKS
activity can be
detected by any suitable method known in the art, such as that described in
Watanabe C and
Townsend C (2002) Initial characterization of a type I fatty acid synthase and
polyketide synthase
multienzyme complex NorS in the biosynthesis of aflatoxin B1. Chemistry and
Biology 9: 981-988.
A non-limiting example of an amino acid sequence (one letter code sequence) of
a polypeptide
having polyketide synthase activity is provided hereafter:
MAQSRQLFLFGDQTADFVPKLRSLLSVQDS P1 LAAFLDQSHYVVRAQMLQSMNTVD
HKLARTADLRQMVQKYVDGKLT PAFRTALVCLCQLGCF I REYEE SGNMYPQP S DSY
VLGFCMGS LAAVAVSC SRS L SELL P IAVQTVL IAFRLGLCALEMRDRVDGC S DDRG
DPWST IVWGLDPQQARDQ I EVFCRT TNVPQTRRPWI SCI SKNAI TLSGS PSTLRAF
CAMPQMAQHRTAP I P I CL PAHNGALFTQAD I TT I LDT T PT T PWEQL PGQ I PY I SHV
TGNVVQT SNYRDL I EVAL SETLLEQVRLDLVETGL PRLLQSRQVKSVT IVPFLTRM
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NETMSN I L PDS F I STETRTDTGRAI PASGRPGAGKCKLAIVSMSGRFPES PT TE S F
WDLLYKGLDVCKEVPRRRWD INTHVDP SGKARNKGATKWGCWLDF SGDFDPRFFG I
S PKEAPQMDPAQRMALMSTYEAMERAGLVPDTTPSTQRDRIGVFHGVTSNDWMETN
TAQN I DTYF I TGGNRGF I PGRINFCFEFAGPSYTNDTACS S S LAAI HLACNS LWRG
DCDTAVAGGTNMI YT PDGHTGLDKGFFL SRTGNCKPYDDKADGYCRAEGVGTVF I K
RLEDALADNDP I LGVI LDAKTNHSAMSE SMTRPHVGAQ I DNMTAALNT TGLHPNDF
S Y I EMHGTGTQVGDAVEME SVL SVFAP SETARKADQPLFVGSAKANVGHGEGVSGV
TSLIKVLMMMQHDT I PPHCGIKPGSKINRNFPDLGARNVHIAFEPKPWPRTHTPRR
VLINNFSAAGGNTALIVEDAPERHWPTEKDPRS SHIVALSAHVGASMKTNLERLHQ
YLLKNPHTDLAQLSYTTTARRWHYLHRVSVTGASVEEVTRKLEMAIQNGDGVSRPK
SKPKI LFAFTGQGSQYATMGKQVYDAYP S FREDLEKFDRLAQSHGFP S FLHVC T S P
KGDVEEMAPVVVQLAI TCLQMALTNLMT S FG I RPDVTVGHS LGEFAALYAAGVL SA
S DVVYLVGQRAELLQERCQRGTHAMLAVKAT PEAL SQWI QDHDCEVAC INGPEDTV
L SGT TKNVAEVQRAMTDNG I KC TLLKL PFAFHSAQVQP I LDDFEALAQGATFAKPQ
LL I L S PLLRTE I HEQGVVT P S YVAQHCRHTVDMAQALRSAREKGL I DDKTLVI ELG
PKPL I SGMVKMTLGDKI S TL PTLAPNKAIWP S LQKI LT SVYTGGWD INWKKYHAPF
AS SQKVVDL P S YGWDLKDYY I PYQGDWCLHRHQQDCKCAAPGHE I KTADYQVPPE S
TPHRPSKLDPSKEAFPE I KT T T TLHRVVEET TKPLGATLVVETD I SRKDVNGLARG
HLVDG I PLC T PS FYAD IAMQVGQYSMQRLRAGHPGAGAI DGLVDVS DMVVDKALVP
HGKGPQLLRTTLTMEWPPKAAATTRSAKVKFATYFADGKLDTEHASCTVRFTSDAQ
LKS LRRSVSEYKTH I RQLHDGHAKGQFMRYNRKTGYKLMS SMARFNPDYMLLDYLV
LNEAENEAASGVDFSLGS SEGTFAAHPAHVDAI TQVAGFAMNANDNVD I EKQVYVN
HGWDS FQ I YQPLDNSKS YQVYTKMGQAKENDLVHGDVVVLDGEQ IVAFFRGLTLRS
VPRGALRVVLQTTVKKADRQLGFKTMPS PPPPT T TMP I S PYKPANTQVS SQAI PAE
ATHSHTPPQPKHS PVPETAGSAPAAKGVGVSNEKLDAVMRVVSEE SG IALEELTDD
SNFADMG I DS L S SMVI GSRFREDLGLDLGPEF S LF I DC T TVRALKDFMLGSGDAGS
GSNVEDPPP SAT PG INPETDWS S SAS DS I FASEDHGHS SE SGADTGS PPALDLKPY
CRP S T SVVLQGL PMVARKTLFML PDGGGSAF S YAS L PRLKS DTAVVGLNC PYARDP
ENMNCTHGAMIESFCNE I RRRQPRGPYHLGGWS SGGAFAYVVAEALVNQGEEVHSL
I I I DAP I PQAMEQLPRAFYEHCNS I GLFATQPGAS PDGS TE PP S YL I PHFTAVVDV
MLDYKLAPLHARRMPKVG IVWAADTVMDERDAPKMKGMHFMI QKRTEFGPDGWDT I
MPGASFDIVRADGANHFTLMQKEHVS I I S DL I DRVMA
The terms "a genetic modification that reduces 6-hydroxyhexanoic acid
conversion" or "a genetic
modification that reduces omega hydroxyl fatty acid conversion" as used herein
refer to genetic
alterations of a host microorganism that reduce an endogenous activity that
converts 6-
hydroxyhexanoic acid to another product. In some embodiments, an endogenous 6-
hydroxyhexanoic acid dehydrogenase activity is reduced. Such alterations can
advantageously
increase the amount of 6-hydroxyhexanoic acid, which can be purified and
further processed.
The term "a genetic modification that reduces beta-oxidation activity" as used
herein refers to a
genetic alteration of a host microorganism that reduces an endogenous activity
that oxidizes a beta
carbon of carboxylic acid containing organic molecules. In certain
embodiments, the organic
molecule is a six carbon molecule, and sometimes contains one or two
carboxylic acid moieties
located at a terminus of the molecule (e.g., adipic acid). Such alterations
can advantageously
increase yields of end products, such as adipic acid.
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The term "a genetic modification that results in increased fatty acid
synthesis" as used herein also
refers to a genetic alteration of a host microorganism that reduces an
endogenous activity that
converts fatty acids into fatty-acyl-CoA intermediates. In some embodiments,
an endogenous
activity that converts fatty acids into fatty-acyl-CoA intermediates is
reduced. In certain
embodiments, an acyl-CoA synthetase activity is reduced. Such alterations can
advantageously
increase yields of end products, such as adipic acid.
Fatty acids can be converted into fatty-acyl-CoA intermediates by the activity
of an acyl-CoA
synthetase (e.g., ACS1, ACS2; EC 6.2.1.3; also referred to as acyl-CoA
synthetase, acyl-CoA
ligase), in many organisms. Acyl-CoA synthetase has two isoforms encoded by
ACS1 and ACS2,
respectively, in C. tropicalis (e.g., homologous to FAA1, FAA2, FAA3 and FAA4
in S. cerevisiae).
Acyl-CoA synthetase is a member of the ligase class of enzymes and catalyzes
the reaction,
ATP + Fatty Acid + CoA <=> AMP + Pyrophosphate + Fatty-Acyl-CoA.
Fatty acids and Coenzyme A often are utilized in the activation of fatty acids
to fatty-acyl-CoA
intermediates for entry into various cellular processes. Without being limited
by theory, it is
believed that reduction in the amount of fatty-acyl-CoA available for various
cellular processes can
increase the amount of fatty acids available for conversion into adipic acid
by other engineered
pathways in the same host organism (e.g., omega oxidation pathway, beta
oxidation pathway,
omega oxidation pathway and beta oxidation pathway). Acyl-CoA synthetase can
be inactivated
by any suitable means. Described herein are gene knockout methods suitable for
use to disrupt
the nucleotide sequence that encodes a polypeptide having ACS1 activity. A
nucleotide sequence
of ACS1 is provided in Example 51, SEQ ID NO: 48. An example of an
integration/disruption
construct, configured to generate a deletion mutant for ACS1 is described in
Example 43.
The presence, absence or amount of acyl-CoA synthetase activity can be
detected by any suitable
method known in the art. Non-limiting examples of suitable detection methods
include enzymatic
assays (e.g., Lageweg et al "A Fluorometric Assay for Acyl-CoA Synthetase
Activity", Analytical
Biochemistry, 197(2):384-388 (1991)), PCR based assays (e.g., qPCR, RTPCR),
immunological
detection methods (e.g., antibodies specific for acyl-CoA synthetase), the
like and combinations
thereof.
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The term "a genetic modification that results in increased fatty acid
synthesis" as used herein also
refers to a genetic alteration of a host microorganism that reduces an
endogenous activity that
converts long chain and very long chain fatty acids into activated fatty-acyl-
CoA intermediates. In
some embodiments, an endogenous activity that converts long chain and very
long chain fatty
acids into activated fatty-acyl-CoA intermediates is reduced. In certain
embodiments, a long chain
acyl-coA synthetase activity is reduced. Such alterations can advantageously
increase yields of
end products, such as adipic acid.
Long chain fatty acids (e.g., C12-C18 chain lengths) and very long chain fatty
acids (e.g., C20-
C26) often are activated and/or transported by the thioesterification activity
of a long-chain acyl-
CoA synthetase (e.g., FAT1; EC6.2.1.3; also referred to as long-chain fatty
acid-CoA ligase, acyl-
CoA synthetase; fatty acid thiokinase (long chain); acyl-activating enzyme;
palmitoyl-CoA
synthase; lignoceroyl-CoA synthase; arachidonyl-CoA synthetase; acyl coenzyme
A synthetase;
acyl-CoA ligase; palmitoyl coenzyme A synthetase; thiokinase; palmitoyl-CoA
ligase; acyl-
coenzyme A ligase; fatty acid CoA ligase; long-chain fatty acyl coenzyme A
synthetase; oleoyl-CoA
synthetase; stearoyl-CoA synthetase; long chain fatty acyl-CoA synthetase;
long-chain acyl CoA
synthetase; fatty acid elongase; LCFA synthetase; pristanoyl-CoA synthetase;
ACS3; long-chain
acyl-CoA synthetase I; long-chain acyl-CoA synthetase II; fatty acyl-coenzyme
A synthetase; long-
chain acyl-coenzyme A synthetase; and acid:CoA ligase (AMP-forming)), in some
organisms.
Fatty acids also can be transported into the host organism from feedstocks by
the activity of long
chain acyl-CoA synthetase.
Long-chain acyl-CoA synthetase catalyzes the reaction:
ATP + a long-chain carboxylic acid + CoA = AMP + diphosphate + an acyl-CoA,
where "an acyl-CoA" refers to a fatty-acyl-CoA molecule. As noted herein,
activation of fatty acids
is often necessary for entry of fatty acids into various cellular processes
(e.g., as an energy source,
as a component for membrane formation and/or remodeling, as carbon storage
molecules).
Deletion mutants of FAT1 have been shown to accumulate very long chain fatty
acids and exhibit
decreased activation of these fatty acids. Without being limited by theory, it
is believed that
reduction in the activity of long-chain acyl-CoA synthetase may reduce the
amount of long chain
fatty acids converted into fatty-acyl-CoA intermediates, thereby increasing
the amount of fatty acids
available for conversion into adipic acid by other engineered pathways in the
same host organism
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(e.g., omega oxidation pathway, beta oxidation pathway, omega oxidation
pathway and beta
oxidation pathway). Long-chain-acyl-CoA synthetase activity can be reduced or
inactivated by any
suitable means. Described herein are gene knockout methods suitable for
disrupting the
nucleotide sequence that encodes the polypeptide having FAT1 activity. The
nucleotide sequence
of FAT1 is provided in Example 51, SEQ ID NO: 50. DNA vectors suitable for use
in constructing
"knockout" constructs are described herein.
The presence, absence or amount of long-chain-acyl-CoA synthetase activity can
be detected by
any suitable method known in the art. Non-limiting examples of suitable
detection methods include
enzymatic assays, binding assays (e.g., Erland et al, Analytical Biochemistry
295(1):38-44 (2001)),
PCR based assays (e.g., qPCR, RT-PCR), immunological detection methods (e.g.,
antibodies
specific for long-chain-acyl-CoA synthetase), the like and combinations
thereof.
The term "a genetic modification that results in increased fatty acid
synthesis" as used herein also
refers to a genetic alteration of a host microorganism that reduces an
endogenous activity that
converts cholesterol into cholesterol esters. In some embodiments, an
endogenous activity that
converts cholesterol into cholesterol esters is reduced. In certain
embodiments, an acyl-CoA sterol
acyltransferase activity is reduced. Such alterations can advantageously
increase yields of end
products, such as adipic acid.
Cholesterol can be converted into a cholesterol-ester by the activity of acyl-
CoA sterol
acyltransferase (e.g., ARE1, ARE2; EC 2.3.1.26; also referred to as sterol 0-
acyltransferase;
cholesterol acyltransferase; sterol-ester synthase; sterol-ester synthetase;
sterol-ester synthase;
acyl coenzyme A-cholesterol-O-acyltransferase; acyl-CoA:cholesterol
acyltransferase; ACAT;
acylcoenzyme A:cholesterol 0-acyltransferase; cholesterol ester synthase;
cholesterol ester
synthetase; and cholesteryl ester synthetase), in many organisms. Without
being limited by any
theory, cholesterol esterification may be involved in directing cholesterol
away from incorporation
into cell membranes and towards storage forms of lipids. Acyl-CoA sterol
acyltransferase
catalyzes the reaction,
acyl-CoA + cholesterol = CoA + cholesterol ester.
The esterification of cholesterol is believed to limit its solubility in cell
membrane lipids and thus
promotes accumulation of cholesterol ester in the fat droplets (e.g., a form
of carbon storage
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molecule) within cytoplasm. Therefore, without being limited by any theory
esterification of
cholesterol may cause the accumulation of lipid storage molecules, and
disruption of the activity of
acyl-CoA sterol acyltransferase may cause an increase in acyl-CoA levels that
can be converted
into adipic acid by other engineered pathways in the same host organism (e.g.,
omega oxidation
pathway, beta oxidation pathway, omega oxidation pathway and beta oxidation
pathway). Acyl-
CoA sterol acyltransferase can be inactivated by any suitable means. Described
herein are gene
knockout methods suitable for disrupting nucleotide sequences that encode
polypeptides having
ARE1 activity, ARE2 activity or ARE1 activity and ARE2 activity. The
nucleotide sequences of
ARE1 and ARE2 are provided in Example 51, SEQ ID NOS: 52 and 54. DNA vectors
suitable for
use in constructing "knockout" constructs are described herein.
The presence, absence or amount of acyl-CoA sterol acyltransferase activity
can be detected by
any suitable method known in the art. Non-limiting examples of suitable
detection methods include
enzymatic assays (e.g., Chen et al, Plant Physiology 145:974-984 (2007)),
binding assays, PCR
based assays (e.g., qPCR, RT-PCR), immunological detection methods (e.g.,
antibodies specific
for long-chain-acyl-CoA synthetase), the like and combinations thereof.
The term "a genetic modification that results in increased fatty acid
synthesis" as used herein also
refers to a genetic alteration of a host microorganism that reduces an
endogenous activity that
catalyzes diacylglycerol esterification (e.g., addition of acyl group to a
diacylglycerol to form a
triacylglycerol). In some embodiments, an endogenous activity that converts
diacyglycerol into
triacylglycerol is reduced. In certain embodiments, an acyltransferase
activity is reduced. In some
embodiments a diacylglycerol acyltransferase activity is reduced. In some
embodiments a
diacylglycerol acyltransferase (e.g., DGA1) activity and an acyltransferase
(e.g., LR01) activity are
reduced. Such alterations can advantageously increase yields of end products,
such as adipic
acid.
Diacylglycerol can be converted into triacylglycerol by the activity of
diacylglycerol acyltransferase
(e.g., DGA1; EC2.3.1.20; also referred to as diglyceride acyltransferase; 1,2-
diacylglycerol
acyltransferase; diacylglycerol acyltransferase; diglyceride 0-
acyltransferase; palmitoyl-CoA-sn-
1,2-diacylglycerol acyltransferase; acyl-CoA:1,2-diacylglycerol 0-
acyltransferase and acyl-CoA:1,2-
diacyl-sn-glycerol 0-acyltransferase), in many organisms. Diacylglycerol
acyltransferase catalyzes
the reaction,
Acyl-CoA + 1,2-diacyl-sn-glycerol = CoA + triacylglycerol,
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and is generally considered the terminal and only committed step in
triglyceride synthesis. The
product of the DGA1 gene in yeast normally is localized to lipid particles.
In addition to the diacylglycerol esterification activity described for DGA1,
many organisms also can
generate triglycerides by the activity of other acyltransferase activities,
non-limiting examples of
which include lecithin-cholesterol acyltransferase activity (e.g., LRO1; EC
2.3.1.43; also referred to
as phosphatidylcholine-sterol 0-acyltransferase activity; lecithin-cholesterol
acyltransferase
activity; phospholipid-cholesterol acyltransferase activity; LCAT (lecithin-
cholesterol
acyltransferase) activity; lecithin:cholesterol acyltransferase activity; and
lysolecithin
acyltransferase activity) and phospholipid:diacylglycerol acyltransferase
(e.g., EC 2.3.1.158; also
referred to as PDAT activity and phospholipid:1,2-diacyl-sn-glycerol 0-
acyltransferase activity).
Acyltransferases of the families EC 2.3.1.43 and EC 2.3.1.58 catalyze the
general reaction,
phospholipid + 1,2-diacylglycerol = lysophospholipid + triacylglycerol.
Triacylglycerides often are utilized as carbon (e.g., fatty acid or lipid)
storage molecules. Without
being limited by any theory, it is believe that reducing the activity of
acytransferases may reduce
the conversion of diacylglycerol to triacylglycerol, which may cause increased
accumulation of fatty
acid, in conjunction with additional genetic modifications (e.g., lipase to
further remove fatty acids
from the glycerol backbone) that can be converted into adipic acid by other
engineered pathways
in the same host organism (e.g., omega oxidation pathway, beta oxidation
pathway, omega
oxidation pathway and beta oxidation pathway). Acyltransferases can be
inactivated by any
suitable means. Described herein are gene knockout methods suitable for
disrupting nucleotide
sequences that encode polypeptides having DGA1 activity, LRO1 activity or DGA1
activity and
LRO1 activity. The nucleotide sequence of DGA1 is provided in Example 51, SEQ
ID NO: 56. The
nucleotide sequence of LRO1 is provided in Example 51, SEQ ID NO: 58. DNA
vectors suitable
for use in constructing "knockout" constructs are described herein.
The presence, absence or amount of acyltransferase activity can be detected by
any suitable
method known in the art. Non-limiting examples of suitable detection methods
include enzymatic
assays (e.g., Geelen, Analytical Biochemistry 322(2):264-268 (2003), Dahlqvist
et al, PNAS
97(12):6487-6492 (2000)), binding assays, PCR based assays (e.g., qPCR, RT-
PCR),
immunological detection methods (e.g., antibodies specific for a DGA1 or LRO1
acyltransferase),
the like and combinations thereof.
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Polynucleotides and Polypeptides
A nucleic acid (e.g., also referred to herein as nucleic acid reagent, target
nucleic acid, target
nucleotide sequence, nucleic acid sequence of interest or nucleic acid region
of interest) can be
from any source or composition, such as DNA, cDNA, gDNA (genomic DNA), RNA,
siRNA (short
inhibitory RNA), RNAi, tRNA or mRNA, for example, and can be in any form
(e.g., linear, circular,
supercoiled, single-stranded, double-stranded, and the like). A nucleic acid
can also comprise
DNA or RNA analogs (e.g., containing base analogs, sugar analogs and/or a non-
native backbone
and the like). It is understood that the term "nucleic acid" does not refer to
or infer a specific length
of the polynucleotide chain, thus polynucleotides and oligonucleotides are
also included in the
definition. Deoxyribonucleotides include deoxyadenosine, deoxycytidine,
deoxyguanosine and
deoxythymidine. For RNA, the uracil base is uridine.
A nucleic acid sometimes is a plasmid, phage, autonomously replicating
sequence (ARS),
centromere, artificial chromosome, yeast artificial chromosome (e.g., YAC) or
other nucleic acid
able to replicate or be replicated in a host cell. In certain embodiments a
nucleic acid can be from
a library or can be obtained from enzymatically digested, sheared or sonicated
genomic DNA (e.g.,
fragmented) from an organism of interest. In some embodiments, nucleic acid
subjected to
fragmentation or cleavage may have a nominal, average or mean length of about
5 to about
10,000 base pairs, about 100 to about 1,000 base pairs, about 100 to about 500
base pairs, or
about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 200, 300, 400, 500,
600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or
10000 base pairs.
Fragments can be generated by any suitable method in the art, and the average,
mean or nominal
length of nucleic acid fragments can be controlled by selecting an appropriate
fragment-generating
procedure by the person of ordinary skill. In some embodiments, the fragmented
DNA can be size
selected to obtain nucleic acid fragments of a particular size range.
Nucleic acid can be fragmented by various methods known to the person of
ordinary skill, which
include without limitation, physical, chemical and enzymic processes. Examples
of such processes
are described in U.S. Patent Application Publication No. 20050112590
(published on May 26,
2005, entitled "Fragmentation-based methods and systems for sequence variation
detection and
discovery," naming Van Den Boom et al.). Certain processes can be selected by
the person of
ordinary skill to generate non-specifically cleaved fragments or specifically
cleaved fragments.
Examples of processes that can generate non-specifically cleaved fragment
sample nucleic acid
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include, without limitation, contacting sample nucleic acid with apparatus
that expose nucleic acid
to shearing force (e.g., passing nucleic acid through a syringe needle; use of
a French press);
exposing sample nucleic acid to irradiation (e.g., gamma, x-ray, UV
irradiation; fragment sizes can
be controlled by irradiation intensity); boiling nucleic acid in water (e.g.,
yields about 500 base pair
fragments) and exposing nucleic acid to an acid and base hydrolysis process.
Nucleic acid may be specifically cleaved by contacting the nucleic acid with
one or more specific
cleavage agents. The term "specific cleavage agent" as used herein refers to
an agent, sometimes
a chemical or an enzyme that can cleave a nucleic acid at one or more specific
sites. Specific
cleavage agents often will cleave specifically according to a particular
nucleotide sequence at a
particular site. Examples of enzymic specific cleavage agents include without
limitation
endonucleases (e.g., DNase (e.g., DNase I, II); RNase (e.g., RNase E, F, H,
P); CleavaseTM
enzyme; Taq DNA polymerase; E. coli DNA polymerase I and eukaryotic structure-
specific
endonucleases; murine FEN-1 endonucleases; type I, II or III restriction
endonucleases such as
Acc I, Afl III, Alu I, A1w44 I, Apa I, Asn I, Ave I, Ave II, BamH I, Ban II,
Bc1 I, Bgl I. Bgl II, Bln I, Bsm
I, BssH II, BstE II, Cfo I, Cla I, Dde I, Dpn I, Dra I, EcIX I, EcoR I, EcoR
I, EcoR II, EcoR V, Hae II,
Hae II, Hind II, Hind III, Hpa I, Hpa II, Kpn I, Ksp I, Mlu I, MluN I, Msp I,
Nci I, Nco I, Nde I, Nde II,
Nhe I, Not I, Nru I, Nsi I, Pst I, Pvu I, Pvu II, Rsa I, Sac I, Sal I, Sau3A
I, Sca I, ScrF I, Sfi I, Sma I,
Spe I, Sph I, Ssp I, Stu I, Sty I, Swa I, Taq I, Xba I, Xho I); glycosylases
(e.g., uracil-DNA
glycolsylase (UDG), 3-methyladenine DNA glycosylase, 3-methyladenine DNA
glycosylase II,
pyrimidine hydrate-DNA glycosylase, FaPy-DNA glycosylase, thymine mismatch-DNA
glycosylase,
hypoxanthine-DNA glycosylase, 5-Hydroxymethyluracil DNA glycosylase (HmUDG), 5-
Hydroxymethylcytosine DNA glycosylase, or 1,N6-etheno-adenine DNA
glycosylase);
exonucleases (e.g., exonuclease III); ribozymes, and DNAzymes. Sample nucleic
acid may be
treated with a chemical agent, or synthesized using modified nucleotides, and
the modified nucleic
acid may be cleaved. In non-limiting examples, sample nucleic acid may be
treated with (i)
alkylating agents such as methylnitrosourea that generate several alkylated
bases, including N3-
methyladenine and N3-methylguanine, which are recognized and cleaved by alkyl
purine DNA-
glycosylase; (ii) sodium bisulfite, which causes deamination of cytosine
residues in DNA to form
uracil residues that can be cleaved by uracil N-glycosylase; and (iii) a
chemical agent that converts
guanine to its oxidized form, 8-hydroxyguanine, which can be cleaved by
formamidopyrimidine
DNA N-glycosylase. Examples of chemical cleavage processes include without
limitation
alkylation, (e.g., alkylation of phosphorothioate-modified nucleic acid);
cleavage of acid lability of
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P3'-N5'-phosphoroamidate-containing nucleic acid; and osmium tetroxide and
piperidine treatment
of nucleic acid.
As used herein, the term "complementary cleavage reactions" refers to cleavage
reactions that are
carried out on the same nucleic acid using different cleavage reagents or by
altering the cleavage
specificity of the same cleavage reagent such that alternate cleavage patterns
of the same target
or reference nucleic acid or protein are generated. In certain embodiments,
nucleic acids of
interest may be treated with one or more specific cleavage agents (e.g., 1, 2,
3, 4, 5, 6, 7, 8, 9, 10
or more specific cleavage agents) in one or more reaction vessels (e.g.,
nucleic acid of interest is
treated with each specific cleavage agent in a separate vessel).
A nucleic acid suitable for use in the embodiments described herein sometimes
is amplified by any
amplification process known in the art (e.g., PCR, RT-PCR and the like).
Nucleic acid amplification
may be particularly beneficial when using organisms that are typically
difficult to culture (e.g., slow
growing, require specialize culture conditions and the like). The terms
"amplify", "amplification",
"amplification reaction", or "amplifying" as used herein refer to any in vitro
processes for multiplying
the copies of a target sequence of nucleic acid. Amplification sometimes
refers to an "exponential"
increase in target nucleic acid. However, "amplifying" as used herein can also
refer to linear
increases in the numbers of a select target sequence of nucleic acid, but is
different than a one-
time, single primer extension step. In some embodiments, a limited
amplification reaction, also
known as pre-amplification, can be performed. Pre-amplification is a method in
which a limited
amount of amplification occurs due to a small number of cycles, for example 10
cycles, being
performed. Pre-amplification can allow some amplification, but stops
amplification prior to the
exponential phase, and typically produces about 500 copies of the desired
nucleotide sequence(s).
Use of pre-amplification may also limit inaccuracies associated with depleted
reactants in standard
PCR reactions.
In some embodiments, a nucleic acid reagent sometimes is stably integrated
into the chromosome
of the host organism, or a nucleic acid reagent can be a deletion of a portion
of the host
chromosome, in certain embodiments (e.g., genetically modified organisms,
where alteration of the
host genome confers the ability to selectively or preferentially maintain the
desired organism
carrying the genetic modification). Such nucleic acid reagents (e.g., nucleic
acids or genetically
modified organisms whose altered genome confers a selectable trait to the
organism) can be
selected for their ability to guide production of a desired protein or nucleic
acid molecule. When
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desired, the nucleic acid reagent can be altered such that codons encode for
(i) the same amino
acid, using a different tRNA than that specified in the native sequence, or
(ii) a different amino acid
than is normal, including unconventional or unnatural amino acids (including
detectably labeled
amino acids). As described herein, the term "native sequence" refers to an
unmodified nucleotide
sequence as found in its natural setting (e.g., a nucleotide sequence as found
in an organism).
A nucleic acid or nucleic acid reagent can comprise certain elements often
selected according to
the intended use of the nucleic acid. Any of the following elements can be
included in or excluded
from a nucleic acid reagent. A nucleic acid reagent, for example, may include
one or more or all of
the following nucleotide elements: one or more promoter elements, one or more
5' untranslated
regions (5'UTRs), one or more regions into which a target nucleotide sequence
may be inserted
(an "insertion element"), one or more target nucleotide sequences, one or more
3' untranslated
regions (3'UTRs), and one or more selection elements. A nucleic acid reagent
can be provided
with one or more of such elements and other elements may be inserted into the
nucleic acid before
the nucleic acid is introduced into the desired organism. In some embodiments,
a provided nucleic
acid reagent comprises a promoter, 5'UTR, optional 3'UTR and insertion
element(s) by which a
target nucleotide sequence is inserted (i.e., cloned) into the nucleotide acid
reagent. In certain
embodiments, a provided nucleic acid reagent comprises a promoter, insertion
element(s) and
optional 3'UTR, and a 5' UTR/target nucleotide sequence is inserted with an
optional 3'UTR. The
elements can be arranged in any order suitable for expression in the chosen
expression system
(e.g., expression in a chosen organism, or expression in a cell free system,
for example), and in
some embodiments a nucleic acid reagent comprises the following elements in
the 5' to 3'
direction: (1) promoter element, 5'UTR, and insertion element(s); (2) promoter
element, 5'UTR,
and target nucleotide sequence; (3) promoter element, 5'UTR, insertion
element(s) and 3'UTR;
and (4) promoter element, 5'UTR, target nucleotide sequence and 3'UTR.
A promoter element typically is required for DNA synthesis and/or RNA
synthesis. A promoter
element often comprises a region of DNA that can facilitate the transcription
of a particular gene,
by providing a start site for the synthesis of RNA corresponding to a gene.
Promoters generally
are located near the genes they regulate, are located upstream of the gene
(e.g., 5' of the gene),
and are on the same strand of DNA as the sense strand of the gene, in some
embodiments. In
some embodiments, a promoter element can be isolated from a gene or organism
and inserted in
functional connection with a polynucleotide sequence to allow altered and/or
regulated expression.
A non-native promoter (e.g., promoter not normally associated with a given
nucleic acid sequence)
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used for expression of a nucleic acid often is referred to as a heterologous
promoter. In certain
embodiments, a heterologous promoter and/or a 5'UTR can be inserted in
functional connection
with a polynucleotide that encodes a polypeptide having a desired activity as
described herein.
The terms "operably linked" and "in functional connection with" as used herein
with respect to
promoters, refer to a relationship between a coding sequence and a promoter
element. The
promoter is operably linked or in functional connection with the coding
sequence when expression
from the coding sequence via transcription is regulated, or controlled by, the
promoter element.
The terms "operably linked" and "in functional connection with" are utilized
interchangeably herein
with respect to promoter elements.
A promoter often interacts with a RNA polymerase. A polymerase is an enzyme
that catalyses
synthesis of nucleic acids using a preexisting nucleic acid reagent. When the
template is a DNA
template, an RNA molecule is transcribed before protein is synthesized.
Enzymes having
polymerase activity suitable for use in the present methods include any
polymerase that is active in
the chosen system with the chosen template to synthesize protein. In some
embodiments, a
promoter (e.g., a heterologous promoter) also referred to herein as a promoter
element, can be
operably linked to a nucleotide sequence or an open reading frame (ORF).
Transcription from the
promoter element can catalyze the synthesis of an RNA corresponding to the
nucleotide sequence
or ORF sequence operably linked to the promoter, which in turn leads to
synthesis of a desired
peptide, polypeptide or protein.
Promoter elements sometimes exhibit responsiveness to regulatory control.
Promoter elements
also sometimes can be regulated by a selective agent. That is, transcription
from promoter
elements sometimes can be turned on, turned off, up-regulated or down-
regulated, in response to
a change in environmental, nutritional or internal conditions or signals
(e.g., heat inducible
promoters, light regulated promoters, feedback regulated promoters, hormone
influenced
promoters, tissue specific promoters, oxygen and pH influenced promoters,
promoters that are
responsive to selective agents (e.g., kanamycin) and the like, for example).
Promoters influenced
by environmental, nutritional or internal signals frequently are influenced by
a signal (direct or
indirect) that binds at or near the promoter and increases or decreases
expression of the target
sequence under certain conditions.
Non-limiting examples of selective or regulatory agents that can influence
transcription from a
promoter element used in embodiments described herein include, without
limitation, (1) nucleic
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acid segments that encode products that provide resistance against otherwise
toxic compounds
(e.g., antibiotics); (2) nucleic acid segments that encode products that are
otherwise lacking in the
recipient cell (e.g., essential products, tRNA genes, auxotrophic markers);
(3) nucleic acid
segments that encode products that suppress the activity of a gene product;
(4) nucleic acid
segments that encode products that can be readily identified (e.g., phenotypic
markers such as
antibiotics (e.g., [3-lactamase), [3-galactosidase, green fluorescent protein
(GFP), yellow fluorescent
protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP),
and cell surface
proteins); (5) nucleic acid segments that bind products that are otherwise
detrimental to cell
survival and/or function; (6) nucleic acid segments that otherwise inhibit the
activity of any of the
nucleic acid segments described in Nos. 1-5 above (e.g., antisense
oligonucleotides); (7) nucleic
acid segments that bind products that modify a substrate (e.g., restriction
endonucleases); (8)
nucleic acid segments that can be used to isolate or identify a desired
molecule (e.g., specific
protein binding sites); (9) nucleic acid segments that encode a specific
nucleotide sequence that
can be otherwise non-functional (e.g., for PCR amplification of subpopulations
of molecules); (10)
nucleic acid segments that, when absent, directly or indirectly confer
resistance or sensitivity to
particular compounds; (11) nucleic acid segments that encode products that
either are toxic or
convert a relatively non-toxic compound to a toxic compound (e.g., Herpes
simplex thymidine
kinase, cytosine deaminase) in recipient cells; (12) nucleic acid segments
that inhibit replication,
partition or heritability of nucleic acid molecules that contain them; and/or
(13) nucleic acid
segments that encode conditional replication functions, e.g., replication in
certain hosts or host cell
strains or under certain environmental conditions (e.g., temperature,
nutritional conditions, and the
like). In some embodiments, the regulatory or selective agent can be added to
change the existing
growth conditions to which the organism is subjected (e.g., growth in liquid
culture, growth in a
fermentor, growth on solid nutrient plates and the like for example).
In some embodiments, regulation of a promoter element can be used to alter
(e.g., increase, add,
decrease or substantially eliminate) the activity of a peptide, polypeptide or
protein (e.g., enzyme
activity for example). For example, a microorganism can be engineered by
genetic modification to
express a nucleic acid reagent that can add a novel activity (e.g., an
activity not normally found in
the host organism) or increase the expression of an existing activity by
increasing transcription
from a homologous or heterologous promoter operably linked to a nucleotide
sequence of interest
(e.g., homologous or heterologous nucleotide sequence of interest), in certain
embodiments. In
some embodiments, a microorganism can be engineered by genetic modification to
express a
nucleic acid reagent that can decrease expression of an activity by decreasing
or substantially
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eliminating transcription from a homologous or heterologous promoter operably
linked to a
nucleotide sequence of interest, in certain embodiments.
In some embodiments the activity can be altered using recombinant DNA and
genetic techniques
known to the artisan. Methods for engineering microorganisms are further
described herein.
Tables herein provide non-limiting lists of yeast promoters that are up-
regulated by oxygen, yeast
promoters that are down-regulated by oxygen, yeast transcriptional repressors
and their
associated genes, DNA binding motifs as determined using the MEME sequence
analysis
software. Potential regulator binding motifs can be identified using the
program MEME to search
intergenic regions bound by regulators for overrepresented sequences. For each
regulator, the
sequences of intergenic regions bound with p-values less than 0.001 were
extracted to use as
input for motif discovery. The MEME software was run using the following
settings: a motif width
ranging from 6 to 18 bases, the "zoops" distribution model, a 6th order Markov
background model
and a discovery limit of 20 motifs. The discovered sequence motifs were scored
for significance by
two criteria: an E-value calculated by MEME and a specificity score. The motif
with the best score
using each metric is shown for each regulator. All motifs presented are
derived from datasets
generated in rich growth conditions with the exception of a previously
published dataset for
epitope-tagged Ga14 grown in galactose.
In some embodiments, the altered activity can be found by screening the
organism under
conditions that select for the desired change in activity. For example,
certain microorganisms can
be adapted to increase or decrease an activity by selecting or screening the
organism in question
on a media containing substances that are poorly metabolized or even toxic. An
increase in the
ability of an organism to grow a substance that is normally poorly metabolized
would result in an
increase in the growth rate on that substance, for example. A decrease in the
sensitivity to a toxic
substance might be manifested by growth on higher concentrations of the toxic
substance, for
example. Genetic modifications that are identified in this manner sometimes
are referred to as
naturally occurring mutations or the organisms that carry them can sometimes
be referred to as
naturally occurring mutants. Modifications obtained in this manner are not
limited to alterations in
promoter sequences. That is, screening microorganisms by selective pressure,
as described
above, can yield genetic alterations that can occur in non-promoter sequences,
and sometimes
also can occur in sequences that are not in the nucleotide sequence of
interest, but in a related
nucleotide sequences (e.g., a gene involved in a different step of the same
pathway, a transport
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gene, and the like). Naturally occurring mutants sometimes can be found by
isolating naturally
occurring variants from unique environments, in some embodiments.
In addition to the regulated promoter sequences, regulatory sequences, and
coding
polynucleotides provided herein, a nucleic acid reagent may include a
polynucleotide sequence
80% or more identical to the foregoing (or to the complementary sequences).
That is, a nucleotide
sequence that is at least 80% or more, 81% or more, 82% or more, 83% or more,
84% or more,
85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more,
91% or more,
92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more,
98% or more,
or 99% or more identical to a nucleotide sequence described herein can be
utilized. The term
"identical" as used herein refers to two or more nucleotide sequences having
substantially the
same nucleotide sequence when compared to each other. One test for determining
whether two
nucleotide sequences or amino acids sequences are substantially identical is
to determine the
percent of identical nucleotide sequences or amino acid sequences shared.
Calculations of sequence identity can be performed as follows. Sequences are
aligned for optimal
comparison purposes (e.g., gaps can be introduced in one or both of a first
and a second amino
acid or nucleic acid sequence for optimal alignment and non-homologous
sequences can be
disregarded for comparison purposes). The length of a reference sequence
aligned for
comparison purposes is sometimes 30% or more, 40% or more, 50% or more, often
60% or more,
and more often 70% or more, 80% or more, 90% or more, or 100`)/0 of the length
of the reference
sequence. The nucleotides or amino acids at corresponding nucleotide or
polypeptide positions,
respectively, are then compared among the two sequences. When a position in
the first sequence
is occupied by the same nucleotide or amino acid as the corresponding position
in the second
sequence, the nucleotides or amino acids are deemed to be identical at that
position. The percent
identity between the two sequences is a function of the number of identical
positions shared by the
sequences, taking into account the number of gaps, and the length of each gap,
introduced for
optimal alignment of the two sequences.
Comparison of sequences and determination of percent identity between two
sequences can be
accomplished using a mathematical algorithm. Percent identity between two
amino acid or
nucleotide sequences can be determined using the algorithm of Meyers & Miller,
CABIOS 4: 11-17
(1989), which has been incorporated into the ALIGN program (version 2.0),
using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty of 4. Also,
percent identity
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between two amino acid sequences can be determined using the Needleman &
Wunsch, J. Mol.
Biol. 48: 444-453 (1970) algorithm which has been incorporated into the GAP
program in the GCG
software package (available at the http address vvvvvv.gcg.com), using either
a Blossum 62 matrix
or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a
length weight of 1, 2, 3, 4,
5, or 6. Percent identity between two nucleotide sequences can be determined
using the GAP
program in the GCG software package (available at http address vvvvw.gcg.com),
using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length
weight of 1, 2, 3,
4, 5, or 6. A set of parameters often used is a Blossum 62 scoring matrix with
a gap open penalty
of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
Sequence identity can also be determined by hybridization assays conducted
under stringent
conditions. As use herein, the term "stringent conditions" refers to
conditions for hybridization and
washing. Stringent conditions are known to those skilled in the art and can be
found in Current
Protocols in Molecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6 (1989).
Aqueous and non-
aqueous methods are described in that reference and either can be used. An
example of stringent
hybridization conditions is hybridization in 6X sodium chloride/sodium citrate
(SSC) at about 45 C,
followed by one or more washes in 0.2X SSC, 0.1% SDS at 50 C. Another example
of stringent
hybridization conditions are hybridization in 6X sodium chloride/sodium
citrate (SSC) at about
45 C, followed by one or more washes in 0.2X SSC, 0.1 /0 SDS at 55 C. A
further example of
stringent hybridization conditions is hybridization in 6X sodium
chloride/sodium citrate (SSC) at
about 45 C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60 C.
Often, stringent
hybridization conditions are hybridization in 6X sodium chloride/sodium
citrate (SSC) at about
45 C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65 C. More
often, stringency
conditions are 0.5M sodium phosphate, 7% SDS at 65 C, followed by one or more
washes at 0.2X
SSC, 1% SDS at 65 C.
As noted above, nucleic acid reagents may also comprise one or more 5' UTR's,
and one or more
3'UTR's. A 5' UTR may comprise one or more elements endogenous to the
nucleotide sequence
from which it originates, and sometimes includes one or more exogenous
elements. A 5' UTR can
originate from any suitable nucleic acid, such as genomic DNA, plasmid DNA,
RNA or mRNA, for
example, from any suitable organism (e.g., virus, bacterium, yeast, fungi,
plant, insect or mammal).
The artisan may select appropriate elements for the 5' UTR based upon the
chosen expression
system (e.g., expression in a chosen organism, or expression in a cell free
system, for example).
A 5' UTR sometimes comprises one or more of the following elements known to
the artisan:
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enhancer sequences (e.g., transcriptional or translational), transcription
initiation site, transcription
factor binding site, translation regulation site, translation initiation site,
translation factor binding
site, accessory protein binding site, feedback regulation agent binding sites,
Pribnow box, TATA
box, -35 element, E-box (helix-loop-helix binding element), ribosome binding
site, replicon, internal
ribosome entry site (IRES), silencer element and the like. In some
embodiments, a promoter
element may be isolated such that all 5' UTR elements necessary for proper
conditional regulation
are contained in the promoter element fragment, or within a functional
subsequence of a promoter
element fragment.
A 5 'UTR in the nucleic acid reagent can comprise a translational enhancer
nucleotide sequence.
A translational enhancer nucleotide sequence often is located between the
promoter and the target
nucleotide sequence in a nucleic acid reagent. A translational enhancer
sequence often binds to a
ribosome, sometimes is an 18S rRNA-binding ribonucleotide sequence (i.e., a
40S ribosome
binding sequence) and sometimes is an internal ribosome entry sequence (IRES).
An IRES
generally forms an RNA scaffold with precisely placed RNA tertiary structures
that contact a 40S
ribosomal subunit via a number of specific intermolecular interactions.
Examples of ribosomal
enhancer sequences are known and can be identified by the artisan (e.g.,
Mignone et al., Nucleic
Acids Research 33: D141-D146 (2005); Paulous et al., Nucleic Acids Research
31: 722-733
(2003); Akbergenov et al., Nucleic Acids Research 32: 239-247 (2004); Mignone
et al., Genome
Biology 3(3): reviews0004.1-0001.10 (2002); Gallie, Nucleic Acids Research 30:
3401-3411
(2002); Shaloiko et al., http address vvwvv.interscience.wiley.com, DOI:
10.1002/bit.20267; and
Gallie et al., Nucleic Acids Research 15: 3257-3273 (1987)).
A translational enhancer sequence sometimes is a eukaryotic sequence, such as
a Kozak
consensus sequence or other sequence (e.g., hydroid polyp sequence, GenBank
accession no.
U07128). A translational enhancer sequence sometimes is a prokaryotic
sequence, such as a
Shine-Dalgarno consensus sequence. In certain embodiments, the translational
enhancer
sequence is a viral nucleotide sequence. A translational enhancer sequence
sometimes is from a
5' UTR of a plant virus, such as Tobacco Mosaic Virus (TMV), Alfalfa Mosaic
Virus (AMV);
Tobacco Etch Virus (ETV); Potato Virus Y (PVY); Turnip Mosaic (poty) Virus and
Pea Seed Borne
Mosaic Virus, for example. In certain embodiments, an omega sequence about 67
bases in length
from TMV is included in the nucleic acid reagent as a translational enhancer
sequence (e.g.,
devoid of guanosine nucleotides and includes a 25 nucleotide long poly (CAA)
central region).
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A 3' UTR may comprise one or more elements endogenous to the nucleotide
sequence from which
it originates and sometimes includes one or more exogenous elements. A 3' UTR
may originate
from any suitable nucleic acid, such as genomic DNA, plasmid DNA, RNA or mRNA,
for example,
from any suitable organism (e.g., a virus, bacterium, yeast, fungi, plant,
insect or mammal). The
artisan can select appropriate elements for the 3' UTR based upon the chosen
expression system
(e.g., expression in a chosen organism, for example). A 3' UTR sometimes
comprises one or more
of the following elements known to the artisan: transcription regulation site,
transcription initiation
site, transcription termination site, transcription factor binding site,
translation regulation site,
translation termination site, translation initiation site, translation factor
binding site, ribosome
binding site, replicon, enhancer element, silencer element and polyadenosine
tail. A 3' UTR often
includes a polyadenosine tail and sometimes does not, and if a polyadenosine
tail is present, one
or more adenosine moieties may be added or deleted from it (e.g., about 5,
about 10, about 15,
about 20, about 25, about 30, about 35, about 40, about 45 or about 50
adenosine moieties may
be added or subtracted).
In some embodiments, modification of a 5' UTR and/or a 3' UTR can be used to
alter (e.g.,
increase, add, decrease or substantially eliminate) the activity of a
promoter. Alteration of the
promoter activity can in turn alter the activity of a peptide, polypeptide or
protein (e.g., enzyme
activity for example), by a change in transcription of the nucleotide
sequence(s) of interest from an
operably linked promoter element comprising the modified 5' or 3' UTR. For
example, a
microorganism can be engineered by genetic modification to express a nucleic
acid reagent
comprising a modified 5' or 3' UTR that can add a novel activity (e.g., an
activity not normally found
in the host organism) or increase the expression of an existing activity by
increasing transcription
from a homologous or heterologous promoter operably linked to a nucleotide
sequence of interest
(e.g., homologous or heterologous nucleotide sequence of interest), in certain
embodiments. In
some embodiments, a microorganism can be engineered by genetic modification to
express a
nucleic acid reagent comprising a modified 5' or 3' UTR that can decrease the
expression of an
activity by decreasing or substantially eliminating transcription from a
homologous or heterologous
promoter operably linked to a nucleotide sequence of interest, in certain
embodiments.
A nucleotide reagent sometimes can comprise a target nucleotide sequence. A
"target nucleotide
sequence" as used herein encodes a nucleic acid, peptide, polypeptide or
protein of interest, and
may be a ribonucleotide sequence or a deoxyribonucleotide sequence. A target
nucleic acid
sometimes is an untranslated ribonucleic acid and sometimes is a translated
ribonucleic acid. An
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untranslated ribonucleic acid may include, but is not limited to, a small
interfering ribonucleic acid
(siRNA), a short hairpin ribonucleic acid (shRNA), other ribonucleic acid
capable of RNA
interference (RNAi), an antisense ribonucleic acid, or a ribozyme. A
translatable target nucleotide
sequence (e.g., a target ribonucleotide sequence) sometimes encodes a peptide,
polypeptide or
protein, which are sometimes referred to herein as "target peptides," "target
polypeptides" or "target
proteins."
Any peptides, polypeptides or proteins, or an activity catalyzed by one or
more peptides,
polypeptides or proteins may be encoded by a target nucleotide sequence and
may be selected by
a user. Representative proteins include enzymes (e.g., glucose-6-phosphate
dehydrogenase,
lipase, fatty acid synthase acetyl-CoA carboxylase, acyl-CoA oxidase,
hexanoate synthase,
thioesterase, monooxygenase, monooxygenase reductase, fatty alcohol oxidase, 6-
oxohexanoic
acid deydrogenase, 6-hydroxyhexanoic acid dehydrogenase and the like, for
example), antibodies,
serum proteins (e.g., albumin), membrane bound proteins, hormones (e.g.,
growth hormone,
erythropoietin, insulin, etc.), cytokines, etc., and include both naturally
occurring and exogenously
expressed polypeptides. Representative activities (e.g., enzymes or
combinations of enzymes
which are functionally associated to provide an activity) include hexanoate
synthase activity,
thioesterase activity, monooxygenase activity, 6-oxohexanoic acid deydrogenase
activity, 6-
hydroxyhexanoic acid dehydrogenase activity, beta-oxidation activity and the
like, for example.
The term "enzyme" as used herein refers to a protein which can act as a
catalyst to induce a
chemical change in other compounds, thereby producing one or more products
from one or more
substrates.
Specific polypeptides (e.g., enzymes) useful for embodiments described herein
are listed herein.
The term "protein" as used herein refers to a molecule having a sequence of
amino acids linked by
peptide bonds. This term includes fusion proteins, oligopeptides, peptides,
cyclic peptides,
polypeptides and polypeptide derivatives, whether native or recombinant, and
also includes
fragments, derivatives, homologs, and variants thereof. A protein or
polypeptide sometimes is of
intracellular origin (e.g., located in the nucleus, cytosol, or interstitial
space of host cells in vivo)
and sometimes is a cell membrane protein in vivo. In some embodiments
(described above, and in
further detail hereafter in Engineering and Alteration Methods), a genetic
modification can result in
a modification (e.g., increase, substantially increase, decrease or
substantially decrease) of a
target activity.
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A translatable nucleotide sequence generally is located between a start codon
(AUG in ribonucleic
acids and ATG in deoxyribonucleic acids) and a stop codon (e.g., UAA (ochre),
UAG (amber) or
UGA (opal) in ribonucleic acids and TAA, TAG or TGA in deoxyribonucleic
acids), and sometimes
is referred to herein as an "open reading frame" (ORF). A translatable
nucleotide sequence (e.g.,
ORF) sometimes is encoded differently in one organism (e.g., most organisms
encode CTG as
leucine) than in another organism (e.g., C. tropicalis encodes CTG as serine).
In some
embodiments, a translatable nucleotide sequence is altered to correct
alternate genetic code (e.g.,
codon usage) differences between a nucleotide donor organism and an nucleotide
recipient
organism (e.g., engineered organism). In certain embodiments, a translatable
nucleotide
sequence is altered to improve; (i) codon usage, (ii) transcriptional
efficiency, (iii) translational
efficiency, (iv) the like, and combinations thereof.
A nucleic acid reagent sometimes comprises one or more ORFs. An ORF may be
from any
suitable source, sometimes from genomic DNA, mRNA, reverse transcribed RNA or
complementary DNA (cDNA) or a nucleic acid library comprising one or more of
the foregoing, and
is from any organism species that contains a nucleic acid sequence of
interest, protein of interest,
or activity of interest. Non-limiting examples of organisms from which an ORF
can be obtained
include bacteria, yeast, fungi, human, insect, nematode, bovine, equine,
canine, feline, rat or
mouse, for example.
A nucleic acid reagent sometimes comprises a nucleotide sequence adjacent to
an ORF that is
translated in conjunction with the ORF and encodes an amino acid tag. The tag-
encoding
nucleotide sequence is located 3' and/or 5' of an ORF in the nucleic acid
reagent, thereby
encoding a tag at the C-terminus or N-terminus of the protein or peptide
encoded by the ORF. Any
tag that does not abrogate in vitro transcription and/or translation may be
utilized and may be
appropriately selected by the artisan. Tags may facilitate isolation and/or
purification of the desired
ORF product from culture or fermentation media.
A tag sometimes specifically binds a molecule or moiety of a solid phase or a
detectable label, for
example, thereby having utility for isolating, purifying and/or detecting a
protein or peptide encoded
by the ORF. In some embodiments, a tag comprises one or more of the following
elements: FLAG
(e.g., DYKDDDDKG), V5 (e.g., GKPIPNPLLGLDST), c-MYC (e.g., EQKLISEEDL), HSV
(e.g.,
QPELAPEDPED), influenza hemaglutinin, HA (e.g., YPYDVPDYA), VSV-G (e.g.,
YTDIEMNRLGK),
bacterial glutathione-S-transferase, maltose binding protein, a streptavidin-
or avidin-binding tag
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(e.g., pcDNATM6 BioEaseTM Gateway Biotinylation System (Invitrogen)),
thioredoxin, [3-
galactosidase, VSV-glycoprotein, a fluorescent protein (e.g., green
fluorescent protein or one of its
many color variants (e.g., yellow, red, blue)), a polylysine or polyarginine
sequence, a polyhistidine
sequence (e.g., His6) or other sequence that chelates a metal (e.g., cobalt,
zinc, copper), and/or a
cysteine-rich sequence that binds to an arsenic-containing molecule. In
certain embodiments, a
cysteine-rich tag comprises the amino acid sequence CC-Xn-CC, wherein X is any
amino acid and
n is 1 to 3, and the cysteine-rich sequence sometimes is CCPGCC. In certain
embodiments, the
tag comprises a cysteine-rich element and a polyhistidine element (e.g.,
CCPGCC and His6).
A tag often conveniently binds to a binding partner. For example, some tags
bind to an antibody
(e.g., FLAG) and sometimes specifically bind to a small molecule. For example,
a polyhistidine tag
specifically chelates a bivalent metal, such as copper, zinc and cobalt; a
polylysine or polyarginine
tag specifically binds to a zinc finger; a glutathione S-transferase tag binds
to glutathione; and a
cysteine-rich tag specifically binds to an arsenic-containing molecule.
Arsenic-containing
molecules include LUMIOTm agents (Invitrogen, California), such as FIAsHTM
(EDT2[41,51-bis(1,3,2-
dithioarsolan-2-yl)fluorescein-(1,2-ethanedithio1)2]) and ReAsH reagents
(e.g., U.S. Patent
5,932,474 to Tsien et al., entitled "Target Sequences for Synthetic
Molecules;" U.S. Patent
6,054,271 to Tsien et al., entitled "Methods of Using Synthetic Molecules and
Target Sequences;"
U.S. Patents 6,451,569 and 6,008,378; published U.S. Patent Application
2003/0083373, and
published PCT Patent Application WO 99/21013, all to Tsien et al. and all
entitled "Synthetic
Molecules that Specifically React with Target Sequences"). Such antibodies and
small molecules
sometimes are linked to a solid phase for convenient isolation of the target
protein or target
peptide.
A tag sometimes comprises a sequence that localizes a translated protein or
peptide to a
component in a system, which is referred to as a "signal sequence" or
"localization signal
sequence" herein. A signal sequence often is incorporated at the N-terminus of
a target protein or
target peptide, and sometimes is incorporated at the C-terminus. Examples of
signal sequences
are known to the artisan, are readily incorporated into a nucleic acid
reagent, and often are
selected according to the organism in which expression of the nucleic acid
reagent is performed. A
signal sequence in some embodiments localizes a translated protein or peptide
to a cell
membrane. Examples of signal sequences include, but are not limited to, a
nucleus targeting
signal (e.g., steroid receptor sequence and N-terminal sequence of 5V40 virus
large T antigen);
mitochondrial targeting signal (e.g., amino acid sequence that forms an
amphipathic helix);
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peroxisome targeting signal (e.g., C-terminal sequence in YFG from
S.cerevisiae); and a secretion
signal (e.g., N-terminal sequences from invertase, mating factor alpha, PHO5
and SUC2 in
S.cerevisiae; multiple N-terminal sequences of B. subtilis proteins (e.g.,
Tjalsma et al.,
Microbiol.Molec. Biol. Rev. 64: 515-547 (2000)); alpha amylase signal sequence
(e.g., U.S. Patent
No. 6,288,302); pectate lyase signal sequence (e.g., U.S. Patent No.
5,846,818); precollagen
signal sequence (e.g., U.S. Patent No. 5,712,114); OmpA signal sequence (e.g.,
U.S. Patent No.
5,470,719); lam beta signal sequence (e.g., U.S. Patent No. 5,389,529); B.
brevis signal sequence
(e.g., U.S. Patent No. 5,232,841); and P. pastoris signal sequence (e.g., U.S.
Patent No.
5,268,273)).
A tag sometimes is directly adjacent to the amino acid sequence encoded by an
ORF (i.e., there is
no intervening sequence) and sometimes a tag is substantially adjacent to an
ORF encoded amino
acid sequence (e.g., an intervening sequence is present). An intervening
sequence sometimes
includes a recognition site for a protease, which is useful for cleaving a tag
from a target protein or
peptide. In some embodiments, the intervening sequence is cleaved by Factor Xa
(e.g.,
recognition site I (E/D)GR), thrombin (e.g., recognition site LVPRGS),
enterokinase (e.g.,
recognition site DDDDK), TEV protease (e.g., recognition site ENLYFQG) or
PreScission TM
protease (e.g., recognition site LEVLFQGP), for example.
An intervening sequence sometimes is referred to herein as a "linker
sequence," and may be of
any suitable length selected by the artisan. A linker sequence sometimes is
about 1 to about 20
amino acids in length, and sometimes about 5 to about 10 amino acids in
length. The artisan may
select the linker length to substantially preserve target protein or peptide
function (e.g., a tag may
reduce target protein or peptide function unless separated by a linker), to
enhance disassociation
of a tag from a target protein or peptide when a protease cleavage site is
present (e.g., cleavage
may be enhanced when a linker is present), and to enhance interaction of a
tag/target protein
product with a solid phase. A linker can be of any suitable amino acid
content, and often
comprises a higher proportion of amino acids having relatively short side
chains (e.g., glycine,
alanine, serine and threonine).
A nucleic acid reagent sometimes includes a stop codon between a tag element
and an insertion
element or ORF, which can be useful for translating an ORF with or without the
tag. Mutant tRNA
molecules that recognize stop codons (described above) suppress translation
termination and
thereby are designated "suppressor tRNAs." Suppressor tRNAs can result in the
insertion of
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amino acids and continuation of translation past stop codons (e.g., U.S.
Patent Application No.
60/587,583, filed July 14, 2004, entitled "Production of Fusion Proteins by
Cell-Free Protein
Synthesis,"; Eggertsson, et al., (1988) Microbiological Review 52(3):354-374,
and Engleerg-Kukla,
et al. (1996) in Escherichia coli and Salmonella Cellular and Molecular
Biology, Chapter 60, pps
909-921, Neidhardt, et al. eds., ASM Press, Washington, DC). A number of
suppressor tRNAs are
known, including but not limited to, supE, supP, supD, supF and supZ
suppressors, which
suppress the termination of translation of the amber stop codon; supB, gIT,
supL, supN, supC and
supM suppressors, which suppress the function of the ochre stop codon and
glyT, trpT and Su-9
suppressors, which suppress the function of the opal stop codon. In general,
suppressor tRNAs
contain one or more mutations in the anti-codon loop of the tRNA that allows
the tRNA to base pair
with a codon that ordinarily functions as a stop codon. The mutant tRNA is
charged with its
cognate amino acid residue and the cognate amino acid residue is inserted into
the translating
polypeptide when the stop codon is encountered. Mutations that enhance the
efficiency of
termination suppressors (i.e., increase stop codon read-through) have been
identified. These
include, but are not limited to, mutations in the uar gene (also known as the
prfA gene), mutations
in the ups gene, mutations in the sueA, sueB and sueC genes, mutations in the
rpsD (ramA) and
rpsE (spcA) genes and mutations in the rpIL gene.
Thus, a nucleic acid reagent comprising a stop codon located between an ORF
and a tag can yield
a translated ORF alone when no suppressor tRNA is present in the translation
system, and can
yield a translated ORF-tag fusion when a suppressor tRNA is present in the
system. Suppressor
tRNA can be generated in cells transfected with a nucleic acid encoding the
tRNA (e.g., a
replication incompetent adenovirus containing the human tRNA-Ser suppressor
gene can be
transfected into cells, or a YAC containing a yeast or bacterial tRNA
suppressor gene can be
transfected into yeast cells, for example). Vectors for synthesizing
suppressor tRNA and for
translating ORFs with or without a tag are available to the artisan (e.g., Tag-
On-Demand TM kit
(Invitrogen Corporation, California); Tag-On-DemandTm Suppressor Supernatant
Instruction
Manual, Version B, 6 June 2003, at http address
vvww.invitrogen.com/content/sfs/
manuals/tagondemand _supernatant_man.pdf; Tag-On-Demand TM Gateway Vector
Instruction
Manual, Version B, 20 June, 2003 at http address
vvvvvv.invitrogen.com/content/sfs/
manuals/tagondemand_vectors_man.pdf; and Capone et al., Amber, ochre and opal
suppressor
tRNA genes derived from a human serine tRNA gene. EMBO J. 4:213, 1985).
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Any convenient cloning strategy known in the art may be utilized to
incorporate an element, such
as an ORF, into a nucleic acid reagent. Known methods can be utilized to
insert an element into
the template independent of an insertion element, such as (1) cleaving the
template at one or more
existing restriction enzyme sites and ligating an element of interest and (2)
adding restriction
enzyme sites to the template by hybridizing oligonucleotide primers that
include one or more
suitable restriction enzyme sites and amplifying by polymerase chain reaction
(described in greater
detail herein). Other cloning strategies take advantage of one or more
insertion sites present or
inserted into the nucleic acid reagent, such as an oligonucleotide primer
hybridization site for PCR,
for example, and others described herein. In some embodiments, a cloning
strategy can be
combined with genetic manipulation such as recombination (e.g., recombination
of a nucleic acid
reagent with a nucleic acid sequence of interest into the genome of the
organism to be modified,
as described further herein). In some embodiments, the cloned ORF(s) can
produce (directly or
indirectly) adipic acid, by engineering a microorganism with one or more ORFs
of interest, which
microorganism comprises one or more altered activities selected from the group
consisting of 6-
oxohexanoic acid dehydrogenase activity, 6-hydroxyhexanoic acid dehydrogenase
activity,
glucose-6-phosphate dehydrogenase activity, hexanoate synthase activity,
lipase activity, fatty acid
synthase activity, acetyl CoA carboxylase activity, monooxygenase activity and
monooxygenase
reductase activity.
In some embodiments, the nucleic acid reagent includes one or more recombinase
insertion sites.
A recombinase insertion site is a recognition sequence on a nucleic acid
molecule that participates
in an integration/recombination reaction by recombination proteins. For
example, the
recombination site for Cre recombinase is loxP, which is a 34 base pair
sequence comprised of two
13 base pair inverted repeats (serving as the recombinase binding sites)
flanking an 8 base pair
core sequence (e.g., Figure 1 of Sauer, B., Curr. Opin. Biotech. 5:521-527
(1994)). Other
examples of recombination sites include attB, attP, attL, and attR sequences,
and mutants,
fragments, variants and derivatives thereof, which are recognized by the
recombination protein A
Int and by the auxiliary proteins integration host factor (IHF), FIS and
excisionase (Xis) (e.g., U.S.
Patent Nos. 5,888,732; 6,143,557; 6,171,861; 6,270,969; 6,277,608; and
6,720,140; U.S. Patent
Appin. Nos. 09/517,466, filed March 2, 2000, and 09/732,914, filed August 14,
2003, and in U.S.
patent publication no. 2002-0007051-A1; Landy, Curr. Opin. Biotech. 3:699-707
(1993)).
Examples of recombinase cloning nucleic acids are in Gateway systems
(Invitrogen, California),
which include at least one recombination site for cloning a desired nucleic
acid molecule in vivo or
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in vitro. In some embodiments, the system utilizes vectors that contain at
least two different site-
specific recombination sites, often based on the bacteriophage lambda system
(e.g., att1 and att2),
and are mutated from the wild-type (attO) sites. Each mutated site has a
unique specificity for its
cognate partner att site (i.e., its binding partner recombination site) of the
same type (for example
attB1 with attP1, or attL1 with attR1) and will not cross-react with
recombination sites of the other
mutant type or with the wild-type attO site. Different site specificities
allow directional cloning or
linkage of desired molecules thus providing desired orientation of the cloned
molecules. Nucleic
acid fragments flanked by recombination sites are cloned and subcloned using
the Gateway
system by replacing a selectable marker (for example, ccdB) flanked by att
sites on the recipient
plasmid molecule, sometimes termed the Destination Vector. Desired clones are
then selected by
transformation of a ccdB sensitive host strain and positive selection for a
marker on the recipient
molecule. Similar strategies for negative selection (e.g., use of toxic genes)
can be used in other
organisms such as thymidine kinase (TK) in mammals and insects.
A recombination system useful for engineering yeast is outlined briefly. The
system makes use of
the URA3 gene (e.g., for S. cerevisieae and C. albicans, for example) or URA4
and URA5 genes
(e.g., for S. pombe, for example) and toxicity of the nucleotide analogue 5-
Fluoroorotic acid (5-
FOA). The URA3 or URA4 and URA5 genes encode orotine-5'-monophosphate (OMP)
dicarboxylase. Yeast with an active URA3 or URA4 and URA5 gene (phenotypically
Ura+) convert
5-FOA to fluorodeoxyuridine, which is toxic to yeast cells. Yeast carrying a
mutation in the
appropriate gene(s) or having a knock out of the appropriate gene(s) can grow
in the presence of
5-F0A, if the media is also supplemented with uracil.
A nucleic acid engineering construct can be made which may comprise the URA3
gene or cassette
(for S. cerevisieae), flanked on either side by the same nucleotide sequence
in the same
orientation. The URA3 cassette comprises a promoter, the URA3 gene and a
functional
transcription terminator. Target sequences which direct the construct to a
particular nucleic acid
region of interest in the organism to be engineered are added such that the
target sequences are
adjacent to and abut the flanking sequences on either side of the URA3
cassette. Yeast can be
transformed with the engineering construct and plated on minimal media without
uracil. Colonies
can be screened by PCR to determine those transformants that have the
engineering construct
inserted in the proper location in the genome. Checking insertion location
prior to selecting for
recombination of the ura3 cassette may reduce the number of incorrect clones
carried through to
later stages of the procedure. Correctly inserted transformants can then be
replica plated on
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minimal media containing 5-FOA to select for recombination of the URA3
cassette out of the
construct, leaving a disrupted gene and an identifiable footprint (e.g.,
nucleic acid sequence) that
can be use to verify the presence of the disrupted gene. The technique
described is useful for
disrupting or "knocking out" gene function, but also can be used to insert
genes or constructs into a
host organisms genome in a targeted, sequence specific manner.
In certain embodiments, a nucleic acid reagent includes one or more
topoisomerase insertion sites.
A topoisomerase insertion site is a defined nucleotide sequence recognized and
bound by a site-
specific topoisomerase. For example, the nucleotide sequence 5'-(C/T)CCTT-3'
is a
topoisomerase recognition site bound specifically by most poxvirus
topoisomerases, including
vaccinia virus DNA topoisomerase I. After binding to the recognition sequence,
the topoisomerase
cleaves the strand at the 3'-most thymidine of the recognition site to produce
a nucleotide
sequence comprising 5'-(C/T)CCTT-PO4-TOPO, a complex of the topoisomerase
covalently bound
to the 3' phosphate via a tyrosine in the topoisomerase (e.g., Shuman, J.
Biol. Chem. 266:11372-
11379, 1991; Sekiguchi and Shuman, Nucl. Acids Res. 22:5360-5365, 1994; U.S.
Pat. No.
5,766,891; PCT/U595/16099; and PCT/U598/12372). In comparison, the nucleotide
sequence 5'-
GCAACTT-3' is a topoisomerase recognition site for type IA E. coli
topoisomerase III. An element
to be inserted often is combined with topoisomerase-reacted template and
thereby incorporated
into the nucleic acid reagent (e.g., World Wide Web URL
invitrogen.com/downloads/F-
13512_Topo_Flyer.pdf; World Wide Web URL invitrogen.com/content/sfs/brochures/
710_021849%20_B_TOPOCIoning_bro.pdf; TOPO TA Cloning Kit and Zero Blunt
TOPOO
Cloning Kit product information).
A nucleic acid reagent sometimes contains one or more origin of replication
(ORI) elements. In
some embodiments, a template comprises two or more ORls, where one functions
efficiently in one
organism (e.g., a bacterium) and another functions efficiently in another
organism (e.g., a
eukaryote, like yeast for example). In some embodiments, an ORI may function
efficiently in one
species (e.g., S. cerevisieae, for example) and another ORI may function
efficiently in a different
species (e.g., S. pombe, for example). A nucleic acid reagent also sometimes
includes one or
more transcription regulation sites.
A nucleic acid reagent can include one or more selection elements (e.g.,
elements for selection of
the presence of the nucleic acid reagent, and not for activation of a promoter
element which can be
selectively regulated). Selection elements often are utilized using known
processes to determine
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whether a nucleic acid reagent is included in a cell. In some embodiments, a
nucleic acid reagent
includes two or more selection elements, where one functions efficiently in
one organism and
another functions efficiently in another organism. Examples of selection
elements include, but are
not limited to, (1) nucleic acid segments that encode products that provide
resistance against
otherwise toxic compounds (e.g., antibiotics); (2) nucleic acid segments that
encode products that
are otherwise lacking in the recipient cell (e.g., essential products, tRNA
genes, auxotrophic
markers); (3) nucleic acid segments that encode products that suppress the
activity of a gene
product; (4) nucleic acid segments that encode products that can be readily
identified (e.g.,
phenotypic markers such as antibiotics (e.g., [3-lactamase), [3-galactosidase,
green fluorescent
protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein
(RFP), cyan fluorescent
protein (CFP), and cell surface proteins); (5) nucleic acid segments that bind
products that are
otherwise detrimental to cell survival and/or function; (6) nucleic acid
segments that otherwise
inhibit the activity of any of the nucleic acid segments described in Nos. 1-5
above (e.g., antisense
oligonucleotides); (7) nucleic acid segments that bind products that modify a
substrate (e.g.,
restriction endonucleases); (8) nucleic acid segments that can be used to
isolate or identify a
desired molecule (e.g., specific protein binding sites); (9) nucleic acid
segments that encode a
specific nucleotide sequence that can be otherwise non-functional (e.g., for
PCR amplification of
subpopulations of molecules); (10) nucleic acid segments that, when absent,
directly or indirectly
confer resistance or sensitivity to particular compounds; (11) nucleic acid
segments that encode
products that either are toxic or convert a relatively non-toxic compound to a
toxic compound (e.g.,
Herpes simplex thymidine kinase, cytosine deaminase) in recipient cells; (12)
nucleic acid
segments that inhibit replication, partition or heritability of nucleic acid
molecules that contain them;
and/or (13) nucleic acid segments that encode conditional replication
functions, e.g., replication in
certain hosts or host cell strains or under certain environmental conditions
(e.g., temperature,
nutritional conditions, and the like).
A nucleic acid reagent is of any form useful for in vivo transcription and/or
translation. A nucleic
acid sometimes is a plasmid, such as a supercoiled plasmid, sometimes is a
yeast artificial
chromosome (e.g., YAC), sometimes is a linear nucleic acid (e.g., a linear
nucleic acid produced by
PCR or by restriction digest), sometimes is single-stranded and sometimes is
double-stranded. A
nucleic acid reagent sometimes is prepared by an amplification process, such
as a polymerase
chain reaction (PCR) process or transcription-mediated amplification process
(TMA). In TMA, two
enzymes are used in an isothermal reaction to produce amplification products
detected by light
emission (see, e.g., Biochemistry 1996 Jun 25;35(25):8429-38 and http address
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vvvvvv.devicelink.com/ivdt/archive/00/11/007.html). Standard PCR processes are
known (e.g., U. S.
Patent Nos. 4,683,202; 4,683,195; 4,965,188; and 5,656,493), and generally are
performed in
cycles. Each cycle includes heat denaturation, in which hybrid nucleic acids
dissociate; cooling, in
which primer oligonucleotides hybridize; and extension of the oligonucleotides
by a polymerase
(i.e., Taq polymerase). An example of a PCR cyclical process is treating the
sample at 95 C for 5
minutes; repeating forty-five cycles of 95 C for 1 minute, 59 C for 1 minute,
10 seconds, and 72 C
for 1 minute 30 seconds; and then treating the sample at 72 C for 5 minutes.
Multiple cycles
frequently are performed using a commercially available thermal cycler. PCR
amplification
products sometimes are stored for a time at a lower temperature (e.g., at 4 C)
and sometimes are
frozen (e.g., at ¨20 C) before analysis.
In some embodiments, a nucleic acid reagent, protein reagent, protein fragment
reagent or other
reagent described herein is isolated or purified. The term "isolated" as used
herein refers to
material removed from its original environment (e.g., the natural environment
if it is naturally
occurring, or a host cell if expressed exogenously), and thus is altered "by
the hand of man" from
its original environment. The term "purified" as used herein with reference to
molecules does not
refer to absolute purity. Rather, "purified" refers to a substance in a
composition that contains
fewer substance species in the same class (e.g., nucleic acid or protein
species) other than the
substance of interest in comparison to the sample from which it originated.
"Purified," if a nucleic
acid or protein for example, refers to a substance in a composition that
contains fewer nucleic acid
species or protein species other than the nucleic acid or protein of interest
in comparison to the
sample from which it originated. Sometimes, a protein or nucleic acid is
"substantially pure,"
indicating that the protein or nucleic acid represents at least 50% of protein
or nucleic acid on a
mass basis of the composition. Often, a substantially pure protein or nucleic
acid is at least 75% on
a mass basis of the composition, and sometimes at least 95% on a mass basis of
the composition.
Engineering and Alteration Methods
Methods and compositions (e.g., nucleic acid reagents) described herein can be
used to generate
engineered microorganisms. As noted above, the term "engineered microorganism"
as used
herein refers to a modified organism that includes one or more activities
distinct from an activity
present in a microorganism utilized as a starting point for modification
(e.g., host microorganism or
unmodified organism). Engineered microorganisms typically arise as a result of
a genetic
modification, usually introduced or selected for, by one of skill in the art
using readily available
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techniques. Non-limiting examples of methods useful for generating an altered
activity include,
introducing a heterologous polynucleotide (e.g., nucleic acid or gene
integration, also referred to as
"knock in"), removing an endogenous polynucleotide, altering the sequence of
an existing
endogenous nucleic acid sequence ( e.g., site-directed mutagenesis),
disruption of an existing
endogenous nucleic acid sequence (e.g., knock outs and transposon or insertion
element
mediated mutagenesis), selection for an altered activity where the selection
causes a change in a
naturally occurring activity that can be stably inherited (e.g., causes a
change in a nucleic acid
sequence in the genome of the organism or in an epigenetic nucleic acid that
is replicated and
passed on to daughter cells), PCR-based mutagenesis, and the like. The term
"mutagenesis" as
used herein refers to any modification to a nucleic acid (e.g., nucleic acid
reagent, or host
chromosome, for example) that is subsequently used to generate a product in a
host or modified
organism. Non-limiting examples of mutagenesis include, deletion, insertion,
substitution,
rearrangement, point mutations, suppressor mutations and the like. Mutagenesis
methods are
known in the art and are readily available to the artisan. Non-limiting
examples of mutagenesis
methods are described herein and can also be found in Maniatis, T., E. F.
Fritsch and J. Sambrook
(1982) Molecular Cloning: a Laboratory Manual; Cold Spring Harbor Laboratory,
Cold Spring
Harbor, N.Y. Another non-limiting example of mutagenesis can be conducted
using a Stratagene
(San Diego, CA) "QuickChange" kit according to the manufacturer's
instructions.
The term "genetic modification" as used herein refers to any suitable nucleic
acid addition, removal
or alteration that facilitates production of a target product (e.g., adipic
acid, 6-hydroxyhexanoic
acid) in an engineered microorganism. Genetic modifications include, without
limitation, insertion
of one or more nucleotides in a native nucleic acid of a host organism in one
or more locations,
deletion of one or more nucleotides in a native nucleic acid of a host
organism in one or more
locations, modification or substitution of one or more nucleotides in a native
nucleic acid of a host
organism in one or more locations, insertion of a non-native nucleic acid into
a host organism (e.g.,
insertion of an autonomously replicating vector), and removal of a non-native
nucleic acid in a host
organism (e.g., removal of a vector).
The term "heterologous polynucleotide" as used herein refers to a nucleotide
sequence not present
in a host microorganism in some embodiments. In certain embodiments, a
heterologous
polynucleotide is present in a different amount (e.g., different copy number)
than in a host
microorganism, which can be accomplished, for example, by introducing more
copies of a
particular nucleotide sequence to a host microorganism (e.g., the particular
nucleotide sequence
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may be in a nucleic acid autonomous of the host chromosome or may be inserted
into a
chromosome). A heterologous polynucleotide is from a different organism in
some embodiments,
and in certain embodiments, is from the same type of organism but from an
outside source (e.g., a
recombinant source).
In some embodiments, an organism engineered using the methods and nucleic acid
reagents
described herein can produce adipic acid. In certain embodiments, an
engineered microorganism
described herein that produces adipic acid may comprise one ore more altered
activities selected
from the group consisting of 6-oxohexanoic acid dehydrogenase activity, omega
oxo fatty acid
dehydrogenase activity, 6-hydroxyhexanoic acid dehydrogenase activity, omega
hydroxyl fatty acid
dehydrogenase activity, hexanoate synthase activity, lipase activity, fatty
acid synthase activity,
acetyl CoA carboxylase activity, monooxygenase activity and monooxygenase
reductase activity.
In some embodiments, an engineered microorganism as described herein may
comprise a genetic
modification that adds or increases the 6-oxohexanoic acid dehydrogenase
activity, omega oxo
fatty acid dehydrogenase activity, 6-hydroxyhexanoic acid dehydrogenase
activity, omega hydroxyl
fatty acid dehydrogenase activity, glucose-6-phosphate dehydrogenase,
hexanoate synthase
activity, lipase activity, fatty acid synthase activity, acetyl CoA
carboxylase activity,
monooxygenase activity and monooxygenase reductase activity.
In certain embodiments, an engineered microorganism described herein can
comprise an altered
thioesterase activity. In some embodiments, the engineered microorganism may
comprise a
genetic alteration that adds or increases a thioesterase activity. In some
embodiments, the
engineered microorganism comprising a genetic alteration that adds or
increases a thioesterase
activity, may further comprise a heterologous polynucleotide encoding a
polypeptide having
thioesterase activity.
The term "altered activity" as used herein refers to an activity in an
engineered microorganism that
is added or modified relative to the host microorganism (e.g., added,
increased, reduced, inhibited
or removed activity). An activity can be altered by introducing a genetic
modification to a host
microorganism that yields an engineered microorganism having added, increased,
reduced,
inhibited or removed activity.
An added activity often is an activity not detectable in a host microorganism.
An increased activity
generally is an activity detectable in a host microorganism that has been
increased in an
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engineered microorganism. An activity can be increased to any suitable level
for production of a
target product (e.g., adipic acid, 6-hydroxyhexanoic acid), including but not
limited to less than 2-
fold (e.g., about 10% increase to about 99% increase; about 20%, 30%, 40%,
50%, 60%, 70%,
80%, 90% increase), 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-
fold, of 10-fold increase, or
greater than about 10-fold increase. A reduced or inhibited activity generally
is an activity
detectable in a host microorganism that has been reduced or inhibited in an
engineered
microorganism. An activity can be reduced to undetectable levels in some
embodiments, or
detectable levels in certain embodiments. An activity can be decreased to any
suitable level for
production of a target product (e.g., adipic acid, 6-hydroxyhexanoic acid),
including but not limited
to less than 2-fold (e.g., about 10% decrease to about 99% decrease; about
20%, 30%, 40%, 50%,
60%, 70%, 80%, 90% decrease), 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,
8-fold, 9-fold, of 10-fold
decrease, or greater than about 10-fold decrease.
An altered activity sometimes is an activity not detectable in a host organism
and is added to an
engineered organism. An altered activity also may be an activity detectable in
a host organism and
is increased in an engineered organism. An activity may be added or increased
by increasing the
number of copies of a polynucleotide that encodes a polypeptide having a
target activity, in some
embodiments. In certain embodiments an activity can be added or increased by
inserting into a
host microorganism a heterologous polynucleotide that encodes a polypeptide
having the added
activity. In certain embodiments, an activity can be added or increased by
inserting into a host
microorganism a heterologous polynucleotide that is (i) operably linked to
another polynucleotide
that encodes a polypeptide having the added activity, and (ii) up regulates
production of the
polynucleotide. Thus, an activity can be added or increased by inserting or
modifying a regulatory
polynucleotide operably linked to another polynucleotide that encodes a
polypeptide having the
target activity. In certain embodiments, an activity can be added or increased
by subjecting a host
microorganism to a selective environment and screening for microorganisms that
have a
detectable level of the target activity. Examples of a selective environment
include, without
limitation, a medium containing a substrate that a host organism can process
and a medium
lacking a substrate that a host organism can process.
An altered activity sometimes is an activity detectable in a host organism and
is reduced, inhibited
or removed (i.e., not detectable) in an engineered organism. An activity may
be reduced or
removed by decreasing the number of copies of a polynucleotide that encodes a
polypeptide
having a target activity, in some embodiments. In some embodiments, an
activity can be reduced
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or removed by (i) inserting a polynucleotide within a polynucleotide that
encodes a polypeptide
having the target activity (disruptive insertion), and/or (ii) removing a
portion of or all of a
polynucleotide that encodes a polypeptide having the target activity (deletion
or knock out,
respectively). In certain embodiments, an activity can be reduced or removed
by inserting into a
host microorganism a heterologous polynucleotide that is (i) operably linked
to another
polynucleotide that encodes a polypeptide having the target activity, and (ii)
down regulates
production of the polynucleotide. Thus, an activity can be reduced or removed
by inserting or
modifying a regulatory polynucleotide operably linked to another
polynucleotide that encodes a
polypeptide having the target activity.
An activity also can be reduced or removed by (i) inhibiting a polynucleotide
that encodes a
polypeptide having the activity or (ii) inhibiting a polynucleotide operably
linked to another
polynucleotide that encodes a polypeptide having the activity. A
polynucleotide can be inhibited by
a suitable technique known in the art, such as by contacting an RNA encoded by
the
polynucleotide with a specific inhibitory RNA (e.g., RNAi, siRNA, ribozyme).
An activity also can be
reduced or removed by contacting a polypeptide having the activity with a
molecule that specifically
inhibits the activity (e.g., enzyme inhibitor, antibody). In certain
embodiments, an activity can be
reduced or removed by subjecting a host microorganism to a selective
environment and screening
for microorganisms that have a reduced level or removal of the target
activity.
In some embodiments, an untranslated ribonucleic acid, or a cDNA can be used
to reduce the
expression of a particular activity or enzyme. For example, a microorganism
can be engineered by
genetic modification to express a nucleic acid reagent that reduces the
expression of an activity by
producing an RNA molecule that is partially or substantially homologous to a
nucleic acid
sequence of interest which encodes the activity of interest. The RNA molecule
can bind to the
nucleic acid sequence of interest and inhibit the nucleic acid sequence from
performing its natural
function, in certain embodiments. In some embodiments, the RNA may alter the
nucleic acid
sequence of interest which encodes the activity of interest in a manner that
the nucleic acid
sequence of interest is no longer capable of performing its natural function
(e.g., the action of a
ribozyme for example).
In certain embodiments, nucleotide sequences sometimes are added to, modified
or removed from
one or more of the nucleic acid reagent elements, such as the promoter, 5'UTR,
target sequence,
or 3'UTR elements, to enhance, potentially enhance, reduce, or potentially
reduce transcription
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and/or translation before or after such elements are incorporated in a nucleic
acid reagent. In
some embodiments, one or more of the following sequences may be modified or
removed if they
are present in a 5'UTR: a sequence that forms a stable secondary structure
(e.g., quadruplex
structure or stem loop stem structure (e.g., EMBL sequences X12949, AF274954,
AF139980,
AF152961, S95936, U194144, AF116649 or substantially identical sequences that
form such stem
loop stem structures)); a translation initiation codon upstream of the target
nucleotide sequence
start codon; a stop codon upstream of the target nucleotide sequence
translation initiation codon;
an ORF upstream of the target nucleotide sequence translation initiation
codon; an iron responsive
element (IRE) or like sequence; and a 5' terminal oligopyrimidine tract (TOP,
e.g., consisting of 5-
15 pyrimidines adjacent to the cap). A translational enhancer sequence and/or
an internal
ribosome entry site (IRES) sometimes is inserted into a 5'UTR (e.g., EMBL
nucleotide sequences
J04513, X87949, M95825, M12783, AF025841, AF013263, AF006822, M17169, M13440,
M22427, D14838 and M17446 and substantially identical nucleotide sequences).
An AU-rich element (ARE, e.g., AUUUA repeats) and/or splicing junction that
follows a non-sense
codon sometimes is removed from or modified in a 3'UTR. A polyadenosine tail
sometimes is
inserted into a 3'UTR if none is present, sometimes is removed if it is
present, and adenosine
moieties sometimes are added to or removed from a polyadenosine tail present
in a 3'UTR. Thus,
some embodiments are directed to a process comprising: determining whether any
nucleotide
sequences that increase, potentially increase, reduce or potentially reduce
translation efficiency
are present in the elements, and adding, removing or modifying one or more of
such sequences if
they are identified. Certain embodiments are directed to a process comprising:
determining
whether any nucleotide sequences that increase or potentially increase
translation efficiency are
not present in the elements, and incorporating such sequences into the nucleic
acid reagent.
In some embodiments, an activity can be altered by modifying the nucleotide
sequence of an ORF.
An ORF sometimes is mutated or modified (for example, by point mutation,
deletion mutation,
insertion mutation, PCR based mutagenesis and the like) to alter, enhance or
increase, reduce,
substantially reduce or eliminate the activity of the encoded protein or
peptide. The protein or
peptide encoded by a modified ORF sometimes is produced in a lower amount or
may not be
produced at detectable levels, and in other embodiments, the product or
protein encoded by the
modified ORF is produced at a higher level (e.g., codons sometimes are
modified so they are
compatible with tRNA's preferentially used in the host organism or engineered
organism). To
determine the relative activity, the activity from the product of the mutated
ORF (or cell containing
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it) can be compared to the activity of the product or protein encoded by the
unmodified ORF (or cell
containing it).
In some embodiments, an ORF nucleotide sequence sometimes is mutated or
modified to alter the
triplet nucleotide sequences used to encode amino acids (e.g., amino acid
codon triplets, for
example). Modification of the nucleotide sequence of an ORF to alter codon
triplets sometimes is
used to change the codon found in the original sequence to better match the
preferred codon
usage of the organism in which the ORF or nucleic acid reagent will be
expressed. The codon
usage, and therefore the codon triplets encoded by a nucleic acid sequence, in
bacteria may be
different from the preferred codon usage in eukaryotes, like yeast or plants
for example. Preferred
codon usage also may be different between bacterial species. In certain
embodiments an ORF
nucleotide sequences sometimes is modified to eliminate codon pairs and/or
eliminate mRNA
secondary structures that can cause pauses during translation of the mRNA
encoded by the ORF
nucleotide sequence. Translational pausing sometimes occurs when nucleic acid
secondary
structures exist in an mRNA, and sometimes occurs due to the presence of codon
pairs that slow
the rate of translation by causing ribosomes to pause. In some embodiments,
the use of lower
abundance codon triplets can reduce translational pausing due to a decrease in
the pause time
needed to load a charged tRNA into the ribosome translation machinery.
Therefore, to increase
transcriptional and translational efficiency in bacteria (e.g., where
transcription and translation are
concurrent, for example) or to increase translational efficiency in eukaryotes
(e.g., where
transcription and translation are functionally separated), the nucleotide
sequence of a nucleotide
sequence of interest can be altered to better suit the transcription and/or
translational machinery of
the host and/or genetically modified microorganism. In certain embodiments,
slowing the rate of
translation by the use of lower abundance codons, which slow or pause the
ribosome, can lead to
higher yields of the desired product due to an increase in correctly folded
proteins and a reduction
in the formation of inclusion bodies.
Codons can be altered and optimized according to the preferred usage by a
given organism by
determining the codon distribution of the nucleotide sequence donor organism
and comparing the
distribution of codons to the distribution of codons in the recipient or host
organism. Techniques
described herein (e.g., site directed mutagenesis and the like) can then be
used to alter the codons
accordingly. Comparisons of codon usage can be done by hand, or using nucleic
acid analysis
software commercially available to the artisan.
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Modification of the nucleotide sequence of an ORF also can be used to correct
codon triplet
sequences that have diverged in different organisms. For example, certain
yeast (e.g., C.
tropicalis and C. maltosa) use the amino acid triplet CUG (e.g., CTG in the
DNA sequence) to
encode serine. CUG typically encodes leucine in most organisms. In order to
maintain the correct
amino acid in the resultant polypeptide or protein, the CUG codon must be
altered to reflect the
organism in which the nucleic acid reagent will be expressed. Thus, if an ORF
from a bacterial
donor is to be expressed in either Candida yeast strain mentioned above, the
heterologous
nucleotide sequence must first be altered or modified to the appropriate
leucine codon. Therefore,
in some embodiments, the nucleotide sequence of an ORF sometimes is altered or
modified to
correct for differences that have occurred in the evolution of the amino acid
codon triplets between
different organisms. In some embodiments, the nucleotide sequence can be left
unchanged at a
particular amino acid codon, if the amino acid encoded is a conservative or
neutral change in
amino acid when compared to the originally encoded amino acid.
In some embodiments, an activity can be altered by modifying translational
regulation signals, like
a stop codon for example. A stop codon at the end of an ORF sometimes is
modified to another
stop codon, such as an amber stop codon described above. In some embodiments,
a stop codon
is introduced within an ORF, sometimes by insertion or mutation of an existing
codon. An ORF
comprising a modified terminal stop codon and/or internal stop codon often is
translated in a
system comprising a suppressor tRNA that recognizes the stop codon. An ORF
comprising a stop
codon sometimes is translated in a system comprising a suppressor tRNA that
incorporates an
unnatural amino acid during translation of the target protein or target
peptide. Methods for
incorporating unnatural amino acids into a target protein or peptide are
known, which include, for
example, processes utilizing a heterologous tRNA/synthetase pair, where the
tRNA recognizes an
amber stop codon and is loaded with an unnatural amino acid (e.g., World Wide
Web URL
iupac.org/news/prize/2003/wang.pdf).
Depending on the portion of a nucleic acid reagent (e.g., Promoter, 5' or 3'
UTR, ORI, ORF, and
the like) chosen for alteration (e.g., by mutagenesis, introduction or
deletion, for example) the
modifications described above can alter a given activity by (i) increasing or
decreasing feedback
inhibition mechanisms, (ii) increasing or decreasing promoter initiation,
(iii) increasing or
decreasing translation initiation, (iv) increasing or decreasing translational
efficiency, (v) modifying
localization of peptides or products expressed from nucleic acid reagents
described herein, or (vi)
increasing or decreasing the copy number of a nucleotide sequence of interest,
(vii) expression of
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an anti-sense RNA, RNAi, siRNA, ribozyme and the like. In some embodiments,
alteration of a
nucleic acid reagent or nucleotide sequence can alter a region involved in
feedback inhibition (e.g.,
5' UTR, promoter and the like). A modification sometimes is made that can add
or enhance
binding of a feedback regulator and sometimes a modification is made that can
reduce, inhibit or
eliminate binding of a feedback regulator.
In certain embodiments, alteration of a nucleic acid reagent or nucleotide
sequence can alter
sequences involved in transcription initiation (e.g., promoters, 5' UTR, and
the like). A modification
sometimes can be made that can enhance or increase initiation from an
endogenous or
heterologous promoter element. A modification sometimes can be made that
removes or disrupts
sequences that increase or enhance transcription initiation, resulting in a
decrease or elimination of
transcription from an endogenous or heterologous promoter element.
In some embodiments, alteration of a nucleic acid reagent or nucleotide
sequence can alter
sequences involved in translational initiation or translational efficiency
(e.g., 5' UTR, 3' UTR, codon
triplets of higher or lower abundance, translational terminator sequences and
the like, for example).
A modification sometimes can be made that can increase or decrease
translational initiation,
modifying a ribosome binding site for example. A modification sometimes can be
made that can
increase or decrease translational efficiency. Removing or adding sequences
that form hairpins
and changing codon triplets to a more or less preferred codon are non-limiting
examples of genetic
modifications that can be made to alter translation initiation and translation
efficiency.
In certain embodiments, alteration of a nucleic acid reagent or nucleotide
sequence can alter
sequences involved in localization of peptides, proteins or other desired
products (e.g., adipic acid,
for example). A modification sometimes can be made that can alter, add or
remove sequences
responsible for targeting a polypeptide, protein or product to an
intracellular organelle, the
periplasm, cellular membranes, or extracellularly. Transport of a heterologous
product to a
different intracellular space or extracellularly sometimes can reduce or
eliminate the formation of
inclusion bodies (e.g., insoluble aggregates of the desired product).
In some embodiments, alteration of a nucleic acid reagent or nucleotide
sequence can alter
sequences involved in increasing or decreasing the copy number of a nucleotide
sequence of
interest. A modification sometimes can be made that increases or decreases the
number of copies
of an ORF stably integrated into the genome of an organism or on an epigenetic
nucleic acid
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reagent. Non-limiting examples of alterations that can increase the number of
copies of a
sequence of interest include, adding copies of the sequence of interest by
duplication of regions in
the genome (e.g., adding additional copies by recombination or by causing gene
amplification of
the host genome, for example), cloning additional copies of a sequence onto a
nucleic acid
reagent, or altering an ORI to increase the number of copies of an epigenetic
nucleic acid reagent.
Non-limiting examples of alterations that can decrease the number of copies of
a sequence of
interest include, removing copies of the sequence of interest by deletion or
disruption of regions in
the genome, removing additional copies of the sequence from epigenetic nucleic
acid reagents, or
altering an ORI to decrease the number of copies of an epigenetic nucleic acid
reagent.
In certain embodiments, increasing or decreasing the expression of a
nucleotide sequence of
interest can also be accomplished by altering, adding or removing sequences
involved in the
expression of an anti-sense RNA, RNAi, siRNA, ribozyme and the like. The
methods described
above can be used to modify expression of anti-sense RNA, RNAi, siRNA,
ribozyme and the like.
The methods and nucleic acid reagents described herein can be used to generate
genetically
modified microorganisms with altered activities in cellular processes involved
in adipic acid
synthesis. In some embodiments, an engineered microorganism described herein
may comprise a
heterologous polynucleotide encoding a polypeptide having 6-oxohexanoic acid
dehydrogenase
activity, and in certain embodiments, an engineered microorganism described
herein may comprise
a heterologous polynucleotide encoding a polypeptide having omega oxo fatty
acid dehydrogenase
activity. In some embodiments, an engineered microorganism described herein
may comprise a
heterologous polynucleotide encoding a polypeptide having 6-hydroxyhexanoic
acid
dehydrogenase activity, and in certain embodiments, an engineered
microorganism described
herein may comprise a heterologous polynucleotide encoding a polypeptide
having omega
hydroxyl fatty acid dehydrogenase activity. In some embodiments, the
heterologous
polynucleotide can be from a bacterium. In some embodiments, the bacterium can
be an
Acinetobacter, Nocardia, Pseudomonas or Xanthobacter bacterium.
In certain embodiments, an engineered microorganism described herein may
comprise a
heterologous polynucleotide encoding a polypeptide having hexanoate synthase
subunit A activity.
In some embodiments, an engineered microorganism described herein may comprise
a
heterologous polynucleotide encoding a polypeptide having hexanoate synthase
subunit B activity.
In certain embodiments, the heterologous polynucleotide independently is
selected from a fungus.
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In some embodiments, the fungus can be an Aspergillus fungus. In certain
embodiments, the
Aspergillus fungus is A. parasiticus.
In some embodiments, an engineered microorganism described herein may comprise
a
modification that results in primary hexanoate usage by monooxygenase
activity. In certain
embodiments, the genetic modification can reduce a polyketide synthase
activity. In some
embodiments, the engineered microorganism can be a eukaryote. In certain
embodiments, the
eukaryote can be a yeast. In some embodiments, the eukaryote may be a fungus.
In certain
In certain embodiments, an engineered microorganism described herein may
comprise a genetic
modification that reduces 6-hydroxyhexanoic acid conversion. In some
embodiments, the genetic
modification can reduce 6-hydroxyhexanoic acid dehydrogenase activity. In
certain embodiments,
an engineered microorganism described herein may comprise a genetic
modification that reduces
Engineered microorganisms that produce adipic acid, as described herein, can
comprise an altered
monooxygenase activity, in certain embodiments. In some embodiments, the
engineered
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polypeptide having monooxygenase activity. In certain embodiments, the
heterologous
polynucleotide can be from a bacterium. In some embodiments, the bacterium can
be a Bacillus
bacterium. In certain embodiments, the Bacillus bacterium is B. megaterium.
In some embodiments, the engineered microorganism described herein may
comprise an altered
hexanoate synthase activity. In certain embodiments, the altered hexanoate
synthase activity is an
altered hexanoate synthase subunit A activity, altered hexanoate synthase
subunit B activity, or
altered hexanoate synthase subunit A activity and altered hexanoate synthase
subunit B activity.
In some embodiments, the engineered microorganism may comprise a genetic
alteration that adds
or increases hexanoate synthase activity. In certain embodiments, the
engineered microorganism
may comprise a heterologous polynucleotide encoding a polypeptide having
hexanoate synthase
activity. In some embodiments, the heterologous polynucleotide can be from a
fungus. In certain
embodiments, the fungus can be an Aspergillus fungus. In some embodiments, the
Aspergillus
fungus is A. parasiticus.
Engineered microorganisms that produce adipic acid, as described herein, can
comprise an altered
thioesterase activity, in certain embodiments. In some embodiments, the
engineered
microorganism may comprise a genetic modification that adds or increases the
thioesterase
activity. In certain embodiments, the engineered microorganism may comprise a
heterologous
polynucleotide encoding a polypeptide having thioesterase activity.
In some embodiments, the engineered microorganism with an altered thioesterase
activity may
comprise an altered 6-oxohexanoic acid dehydrogenase activity, or an altered
omega oxo fatty
acid dehydrogenase activity. In certain embodiments, the engineered
microorganism with an
altered thioesterase activity may comprise a genetic modification that adds or
increases 6-
oxohexanoic acid dehydrogenase activity, or a genetic modification that adds
or increases omega
oxo fatty acid dehydrogenase activity. In some embodiments, the engineered
microorganism may
comprise a heterologous polynucleotide encoding a polypeptide having altered 6-
oxohexanoic acid
dehydrogenase activity, and in some embodiments, the engineered microorganism
may comprise
a heterologous polynucleotide encoding a polypeptide having altered omega oxo
fatty acid
dehydrogenase activity. In certain embodiments, the heterologous
polynucleotide can be from a
bacterium. In some embodiments, the bacterium can be an Acinetobacter,
Nocardia,
Pseudomonas or Xanthobacter bacterium.
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Engineered microorganisms that produce adipic acid, as described herein, can
comprise an altered
6-hydroxyhexanoic acid dehydrogenase activity, in certain embodiments, and in
some
embodiments, can comprise an altered omega hydroxyl fatty acid dehydrogenase
activity. In
certain embodiments, the engineered microorganism may comprise a genetic
modification that
-- adds or increases the 6-hydroxyhexanoic acid dehydrogenase activity and in
some embodiments
the engineered microorganism may comprise a genetic modification that adds or
increases the
omega hydroxyl fatty acid dehydrogenase activity. In certain embodiments, the
engineered
microorganism may comprise a heterologous polynucleotide encoding a
polypeptide having altered
6-hydroxyhexanoic acid dehydrogenase activity, and in some embodiments, the
engineered
-- microorganism may comprise a heterologous polynucleotide encoding a
polypeptide having altered
omega hydroxyl fatty acid dehydrogenase activity. In some embodiments, the
heterologous
polynucleotide is from a bacterium. In certain embodiments, the bacterium can
be an
Acinetobacter, Nocardia, Pseudomonas or Xanthobacter bacterium. In some
embodiments, the
engineered microorganism can be a eukaryote. In certain embodiments, the
eukaryote can be a
-- yeast. In some embodiments, the eukaryote may be a fungus. In certain
embodiments, the yeast
can be a Candida yeast. In some embodiments, the Candida yeast may be C.
troplicalis. In
certain embodiments, the yeast can be a Yarrowia yeast. In some embodiments
the Yarrowia
yeast may be Y. lipolytica. In certain embodiments, the fungus can be an
Aspergillus fungus. In
some embodiments, the Aspergillus fungus may be A. parasiticus or A. nidulans.
In some embodiments, an engineered microorganism as described above may
comprise a genetic
modification that reduces 6-hydroxyhexanoic acid conversion. In certain
embodiments, the genetic
modification can reduce 6-hydroxyhexanoic acid dehydrogenase activity. In some
embodiments
the genetic may reduce beta-oxidation activity. In certain embodiments, the
genetic modification
-- may reduce a target activity described herein.
Engineered microorganisms can be prepared by altering, introducing or removing
nucleotide
sequences in the host genome or in stably maintained epigenetic nucleic acid
reagents, as noted
above. The nucleic acid reagents use to alter, introduce or remove nucleotide
sequences in the
-- host genome or epigenetic nucleic acids can be prepared using the methods
described herein or
available to the artisan.
Nucleic acid sequences having a desired activity can be isolated from cells of
a suitable organism
using lysis and nucleic acid purification procedures described in a known
reference manual (e.g.,
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Maniatis, T., E. F. Fritsch and J. Sambrook (1982) Molecular Cloning: a
Laboratory Manual; Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) or using commercially
available cell lysis and
DNA purification reagents and kits. In some embodiments, nucleic acids used to
engineer
microorganisms can be provided for conducting methods described herein after
processing of the
organism containing the nucleic acid. For example, the nucleic acid of
interest may be extracted,
isolated, purified or amplified from a sample (e.g., from an organism of
interest or culture
containing a plurality of organisms of interest, like yeast or bacteria for
example). The term
"isolated" as used herein refers to nucleic acid removed from its original
environment (e.g., the
natural environment if it is naturally occurring, or a host cell if expressed
exogenously), and thus is
altered "by the hand of man" from its original environment. An isolated
nucleic acid generally is
provided with fewer non-nucleic acid components (e.g., protein, lipid) than
the amount of
components present in a source sample. A composition comprising isolated
sample nucleic acid
can be substantially isolated (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%
or greater than 99% free of non-nucleic acid components). The term "purified"
as used herein
refers to sample nucleic acid provided that contains fewer nucleic acid
species than in the sample
source from which the sample nucleic acid is derived. A composition comprising
sample nucleic
acid may be substantially purified (e.g., about 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%,
99% or greater than 99% free of other nucleic acid species). The term
"amplified" as used herein
refers to subjecting nucleic acid of a cell, organism or sample to a process
that linearly or
exponentially generates amplicon nucleic acids having the same or
substantially the same
nucleotide sequence as the nucleotide sequence of the nucleic acid in the
sample, or portion
thereof. As noted above, the nucleic acids used to prepare nucleic acid
reagents as described
herein can be subjected to fragmentation or cleavage.
Amplification of nucleic acids is sometimes necessary when dealing with
organisms that are
difficult to culture. Where amplification may be desired, any suitable
amplification technique can
be utilized. Non-limiting examples of methods for amplification of
polynucleotides include,
polymerase chain reaction (PCR); ligation amplification (or ligase chain
reaction (LCR));
amplification methods based on the use of Q-beta replicase or template-
dependent polymerase
(see US Patent Publication Number U520050287592); helicase-dependant
isothermal
amplification (Vincent et al., "Helicase-dependent isothermal DNA
amplification". EMBO reports 5
(8): 795-800 (2004)); strand displacement amplification (SDA); thermophilic
SDA nucleic acid
sequence based amplification (35R or NASBA) and transcription-associated
amplification (TAA).
Non-limiting examples of PCR amplification methods include standard PCR, AFLP-
PCR, Allele-
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specific PCR, Alu-PCR, Asymmetric PCR, Colony PCR, Hot start PCR, Inverse PCR
(IPCR), In
situ PCR (ISH), Intersequence-specific PCR (ISSR-PCR), Long PCR, Multiplex
PCR, Nested PCR,
Quantitative PCR, Reverse Transcriptase PCR (RT-PCR), Real Time PCR, Single
cell PCR, Solid
phase PCR, combinations thereof, and the like. Reagents and hardware for
conducting PCR are
commercially available.
Protocols for conducting the various type of PCR listed above are readily
available to the artisan.
PCR conditions can be dependent upon primer sequences, target abundance, and
the desired
amount of amplification, and therefore, one of skill in the art may choose
from a number of PCR
protocols available (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202; and
PCR Protocols: A
Guide to Methods and Applications, Innis et al., eds, 1990. PCR often is
carried out as an
automated process with a thermostable enzyme. In this process, the temperature
of the reaction
mixture is cycled through a denaturing region, a primer-annealing region, and
an extension
reaction region automatically. Machines specifically adapted for this purpose
are commercially
available. A non-limiting example of a PCR protocol that may be suitable for
embodiments
described herein is, treating the sample at 95 C for 5 minutes; repeating
forty-five cycles of 95 C
for 1 minute, 59 C for 1 minute, 10 seconds, and 72 C for 1 minute 30 seconds;
and then treating
the sample at 72 C for 5 minutes. Additional PCR protocols are described in
the example section.
Multiple cycles frequently are performed using a commercially available
thermal cycler. Suitable
isothermal amplification processes known and selected by the person of
ordinary skill in the art
also may be applied, in certain embodiments. In some embodiments, nucleic
acids encoding
polypeptides with a desired activity can be isolated by amplifying the desired
sequence from an
organism having the desired activity using oligonucleotides or primers
designed based on
sequences described herein.
Amplified, isolated and/or purified nucleic acids can be cloned into the
recombinant DNA vectors
described in Figures herein or into suitable commercially available
recombinant DNA vectors.
Cloning of nucleic acid sequences of interest into recombinant DNA vectors can
facilitate further
manipulations of the nucleic acids for preparation of nucleic acid reagents,
(e.g., alteration of
nucleotide sequences by mutagenesis, homologous recombination, amplification
and the like, for
example). Standard cloning procedures (e.g., enzymic digestion, ligation, and
the like) are known
(e.g., described in Maniatis, T., E. F. Fritsch and J. Sambrook (1982)
Molecular Cloning: a
Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
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In some embodiments, nucleic acid sequences prepared by isolation or
amplification can be used,
without any further modification, to add an activity to a microorganism and
thereby create a
genetically modified or engineered microorganism. In certain embodiments,
nucleic acid
sequences prepared by isolation or amplification can be genetically modified
to alter (e.g., increase
or decrease, for example) a desired activity. In some embodiments, nucleic
acids, used to add an
activity to an organism, sometimes are genetically modified to optimize the
heterologous
polynucleotide sequence encoding the desired activity (e.g., polypeptide or
protein, for example).
The term "optimize" as used herein can refer to alteration to increase or
enhance expression by
preferred codon usage. The term optimize can also refer to modifications to
the amino acid
sequence to increase the activity of a polypeptide or protein, such that the
activity exhibits a higher
catalytic activity as compared to the "natural" version of the polypeptide or
protein.
Nucleic acid sequences of interest can be genetically modified using methods
known in the art.
Mutagenesis techniques are particularly useful for small scale (e.g., 1, 2, 5,
10 or more
nucleotides) or large scale (e.g., 50, 100, 150, 200, 500, or more
nucleotides) genetic modification.
Mutagenesis allows the artisan to alter the genetic information of an organism
in a stable manner,
either naturally (e.g., isolation using selection and screening) or
experimentally by the use of
chemicals, radiation or inaccurate DNA replication (e.g., PCR mutagenesis). In
some
embodiments, genetic modification can be performed by whole scale synthetic
synthesis of nucleic
acids, using a native nucleotide sequence as the reference sequence, and
modifying nucleotides
that can result in the desired alteration of activity. Mutagenesis methods
sometimes are specific or
targeted to specific regions or nucleotides (e.g., site-directed mutagenesis,
PCR-based site-
directed mutagenesis, and in vitro mutagenesis techniques such as
transplacement and in vivo
oligonucleotide site-directed mutagenesis, for example). Mutagenesis methods
sometimes are
non-specific or random with respect to the placement of genetic modifications
(e.g., chemical
mutagenesis, insertion element (e.g., insertion or transposon elements) and
inaccurate PCR based
methods, for example).
Site directed mutagenesis is a procedure in which a specific nucleotide or
specific nucleotides in a
DNA molecule are mutated or altered. Site directed mutagenesis typically is
performed using a
nucleic acid sequence of interest cloned into a circular plasmid vector. Site-
directed mutagenesis
requires that the wild type sequence be known and used a platform for the
genetic alteration. Site-
directed mutagenesis sometimes is referred to as oligonucleotide-directed
mutagenesis because
the technique can be performed using oligonucleotides which have the desired
genetic
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modification incorporated into the complement a nucleotide sequence of
interest. The wild type
sequence and the altered nucleotide are allowed to hybridize and the
hybridized nucleic acids are
extended and replicated using a DNA polymerase. The double stranded nucleic
acids are
introduced into a host (e.g., E. coli, for example) and further rounds of
replication are carried out in
vivo. The transformed cells carrying the mutated nucleic acid sequence are
then selected and/or
screened for those cells carrying the correctly mutagenized sequence. Cassette
mutagenesis and
PCR-based site-directed mutagenesis are further modifications of the site-
directed mutagenesis
technique. Site-directed mutagenesis can also be performed in vivo (e.g.,
transplacement "pop-in
pop-out", In vivo site-directed mutagenesis with synthetic oligonucleotides
and the like, for
example).
PCR-based mutagenesis can be performed using PCR with oligonucleotide primers
that contain
the desired mutation or mutations. The technique functions in a manner similar
to standard site-
directed mutagenesis, with the exception that a thermocycler and PCR
conditions are used to
replace replication and selection of the clones in a microorganism host. As
PCR-based
mutagenesis also uses a circular plasmid vector, the amplified fragment (e.g.,
linear nucleic acid
molecule) containing the incorporated genetic modifications can be separated
from the plasmid
containing the template sequence after a sufficient number of rounds of
thermocycler amplification,
using standard electrophorectic procedures. A modification of this method uses
linear amplification
methods and a pair of mutagenic primers that amplify the entire plasmid. The
procedure takes
advantage of the E. coli Dam methylase system which causes DNA replicated in
vivo to be
sensitive to the restriction endonucleases Dpnl. PCR synthesized DNA is not
methylated and is
therefore resistant to Dpnl. This approach allows the template plasmid to be
digested, leaving the
genetically modified, PCR synthesized plasmids to be isolated and transformed
into a host bacteria
for DNA repair and replication, thereby facilitating subsequent cloning and
identification steps. A
certain amount of randomness can be added to PCR-based sited directed
mutagenesis by using
partially degenerate primers.
Recombination sometimes can be used as a tool for mutagenesis. Homologous
recombination
allows the artisan to specifically target regions of known sequence for
insertion of heterologous
nucleotide sequences using the host organisms natural DNA replication and
repair enzymes.
Homologous recombination methods sometimes are referred to as "pop in pop out"
mutagenesis,
transplacement, knock out mutagenesis or knock in mutagenesis. Integration of
a nucleic acid
sequence into a host genome is a single cross over event, which inserts the
entire nucleic acid
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reagent (e.g., pop in). A second cross over event excises all but a portion of
the nucleic acid
reagent, leaving behind a heterologous sequence, often referred to as a
"footprint" (e.g., pop out).
Mutagenesis by insertion (e.g., knock in) or by double recombination leaving
behind a disrupting
heterologous nucleic acid (e.g., knock out) both server to disrupt or "knock
out" the function of the
gene or nucleic acid sequence in which insertion occurs. By combining
selectable markers and/or
auxotrophic markers with nucleic acid reagents designed to provide the
appropriate nucleic acid
target sequences, the artisan can target a selectable nucleic acid reagent to
a specific region, and
then select for recombination events that "pop out" a portion of the inserted
(e.g., "pop in") nucleic
acid reagent.
Such methods take advantage of nucleic acid reagents that have been
specifically designed with
known target nucleic acid sequences at or near a nucleic acid or genomic
region of interest.
Popping out typically leaves a "foot print" of left over sequences that remain
after the
recombination event. The left over sequence can disrupt a gene and thereby
reduce or eliminate
expression of that gene. In some embodiments, the method can be used to insert
sequences,
upstream or downstream of genes that can result in an enhancement or reduction
in expression of
the gene. In certain embodiments, new genes can be introduced into the genome
of a host
organism using similar recombination or "pop in" methods. An example of a
yeast recombination
system using the ura3 gene and 5-FOA were described briefly above and further
detail is
presented herein.
A method for modification is described in Alani et al., "A method for gene
disruption that allows
repeated use of URA3 selection in the construction of multiply disrupted yeast
strains", Genetics
116(4):541-545 August 1987. The original method uses a Ura3 cassette with 1000
base pairs (bp)
of the same nucleotide sequence cloned in the same orientation on either side
of the URA3
cassette. Targeting sequences of about 50 bp are added to each side of the
construct. The double
stranded targeting sequences are complementary to sequences in the genome of
the host
organism. The targeting sequences allow site-specific recombination in a
region of interest. The
modification of the original technique replaces the two 1000 bp sequence
direct repeats with two
200 bp direct repeats. The modified method also uses 50 bp targeting
sequences. The
modification reduces or eliminates recombination of a second knock out into
the 1000 bp repeat
left behind in a first mutagenesis, therefore allowing multiply knocked out
yeast. Additionally, the
200 bp sequences used herein are uniquely designed, self-assembling sequences
that leave
behind identifiable footprints. The technique used to design the sequences
incorporate design
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features such as low identity to the yeast genome, and low identity to each
other. Therefore a
library of the self-assembling sequences can be generated to allow multiple
knockouts in the same
organism, while reducing or eliminating the potential for integration into a
previous knockout.
As noted above, the URA3 cassette makes use of the toxicity of 5-FOA in yeast
carrying a
functional URA3 gene. Uracil synthesis deficient yeast are transformed with
the modified URA3
cassette, using standard yeast transformation protocols, and the transformed
cells are plated on
minimal media minus uracil. In some embodiments, PCR can be used to verify
correct insertion
into the region of interest in the host genome, and certain embodiments the
PCR step can be
omitted. Inclusion of the PCR step can reduce the number of transformants that
need to be
counter selected to "pop out" the URA3 cassette. The transformants (e.g., all
or the ones
determined to be correct by PCR, for example) can then be counter-selected on
media containing
5-F0A, which will select for recombination out (e.g., popping out) of the URA3
cassette, thus
rendering the yeast ura3 deficient again, and resistant to 5-FOA toxicity.
Targeting sequences
used to direct recombination events to specific regions are presented herein.
A modification of the
method described above can be used to integrate genes in to the chromosome,
where after
recombination a functional gene is left in the chromosome next to the 200bp
footprint.
In some embodiments, other auxotrophic or dominant selection markers can be
used in place of
URA3 (e.g., an auxotrophic selectable marker), with the appropriate change in
selection media and
selection agents. Auxotrophic selectable markers are used in strains deficient
for synthesis of a
required biological molecule (e.g., amino acid or nucleoside, for example).
Non-limiting examples
of additional auxotrophic markers include; HIS3, TRP1, LEU2, LEU2-d, and LYS2.
Certain
auxotrophic markers (e.g., URA3 and LYS2) allow counter selection to select
for the second
recombination event that pops out all but one of the direct repeats of the
recombination construct.
HI53 encodes an activity involved in histidine synthesis. TRP1 encodes an
activity involved in
tryptophan synthesis. LEU2 encodes an activity involved in leucine synthesis.
LEU2-d is a low
expression version of LEU2 that selects for increased copy number (e.g., gene
or plasmid copy
number, for example) to allow survival on minimal media without leucine. LYS2
encodes an
activity involved in lysine synthesis, and allows counter selection for
recombination out of the LYS2
gene using alpha-amino adipate (alpha-amino adipate).
Dominant selectable markers are useful because they also allow industrial
and/or prototrophic
strains to be used for genetic manipulations. Additionally, dominant
selectable markers provide the
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advantage that rich medium can be used for plating and culture growth, and
thus growth rates are
markedly increased. Non-limiting examples of dominant selectable markers
include; Tn903 kanr,
Cmr, Hygr, CUP1, and DHFR. Tn903 kanr encodes an activity involved in
kanamycin antibiotic
resistance (e.g., typically neomycin phosphotransferase II or NPTII, for
example). Cmr encodes
an activity involved in chloramphenicol antibiotic resistance (e.g., typically
chloramphenicol acetyl
transferase or CAT, for example). Hygr encodes an activity involved in
hygromycin resistance by
phosphorylation of hygromycin B (e.g., hygromycin phosphotransferase, or HPT).
CUP1 encodes
an activity involved in resistance to heavy metal (e.g., copper, for example)
toxicity. DHFR
encodes a dihydrofolate reductase activity which confers resistance to
methotrexate and
sulfanilamde compounds.
In contrast to site-directed or specific mutagenesis, random mutagenesis does
not require any
sequence information and can be accomplished by a number of widely different
methods. Random
mutagenesis often is used to create mutant libraries that can be used to
screen for the desired
genotype or phenotype. Non-limiting examples of random mutagenesis include;
chemical
mutagenesis, UV-induced mutagenesis, insertion element or transposon-mediated
mutagenesis,
DNA shuffling, error-prone PCR mutagenesis, and the like.
Chemical mutagenesis often involves chemicals like ethyl methanesulfonate
(EMS), nitrous acid,
mitomycin C, N-methyl-N-nitrosourea (MNU), diepoxybutane (DEB), 1, 2, 7, 8-
diepoxyoctane
(DEO), methyl methane sulfonate (MMS), N-methyl- N'-nitro-N-nitrosoguanidine
(MNNG), 4-
nitroquinoline 1-oxide (4-NQ0), 2-methyloxy-6-chloro-9(34ethyl-
2-chloroethylFaminopropylamino)-acridinedihydrochloride (ICR-170), 2-amino
purine (2AP), and
hydroxylamine (HA), provided herein as non-limiting examples. These chemicals
can cause base-
pair subsitutions, frameshift mutations, deletions, transversion mutations,
transition mutations,
incorrect replication, and the like. In some embodiments, the mutagenesis can
be carried out in
vivo. Sometimes the mutagenic process involves the use of the host organisms
DNA replication
and repair mechanisms to incorporate and replicate the mutagenized base or
bases.
Another type of chemical mutagenesis involves the use of base-analogs. The use
of base-analogs
cause incorrect base pairing which in the following round of replication is
corrected to a
mismatched nucleotide when compared to the starting sequence. Base analog
mutagenesis
introduces a small amount of non-randomness to random mutagenesis, because
specific base
analogs can be chose which can be incorporated at certain nucleotides in the
starting sequence.
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Correction of the mispairing typically yields a known substitution. For
example, Bromo-
deoxyuridine (BrdU) can be incorporated into DNA and replaces T in the
sequence. The host DNA
repair and replication machinery can sometime correct the defect, but
sometimes will mispair the
BrdU with a G. The next round of replication then causes a G-C transversion
from the original A-T
in the native sequence.
Ultra violet (UV) induced mutagenesis is caused by the formation of thymidine
dimers when UV
light irradiates chemical bonds between two adjacent thymine residues.
Excision repair
mechanism of the host organism correct the lesion in the DNA, but occasionally
the lesion is
incorrectly repaired typically resulting in a C to T transition.
Insertion element or transposon-mediated mutagenesis makes use of naturally
occurring or
modified naturally occurring mobile genetic elements. Transposons often encode
accessory
activities in addition to the activities necessary for transposition (e.g.,
movement using a
transposase activity, for example). In many examples, transposon accessory
activities are
antibiotic resistance markers (e.g., see Tn903 kanr described above, for
example). Insertion
elements typically only encode the activities necessary for movement of the
nucleic acid sequence.
Insertion element and transposon mediated mutagenesis often can occur
randomly, however
specific target sequences are known for some transposons. Mobile genetic
elements like IS
elements or Transposons (Tn) often have inverted repeats, direct repeats or
both inverted and
direct repeats flanking the region coding for the transposition genes.
Recombination events
catalyzed by the transposase cause the element to remove itself from the
genome and move to a
new location, leaving behind a portion of an inverted or direct repeat.
Classic examples of
transposons are the "mobile genetic elements" discovered in maize. Transposon
mutagenesis kits
are commercially available which are designed to leave behind a 5 codon insert
(e.g., Mutation
Generation System kit, Finnzymes, World Wide Web URL finnzymes.us, for
example). This allows
the artisan to identify the insertion site, without fully disrupting the
function of most genes.
DNA shuffling is a method which uses DNA fragments from members of a mutant
library and
reshuffles the fragments randomly to generate new mutant sequence
combinations. The
fragments are typically generated using DNasel, followed by random annealing
and re-joining
using self priming PCR. The DNA overhanging ends, from annealing of random
fragments,
provide "primer" sequences for the PCR process. Shuffling can be applied to
libraries generated
by any of the above mutagenesis methods.
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Error prone PCR and its derivative rolling circle error prone PCR uses
increased magnesium and
manganese concentrations in conjunction with limiting amounts of one or two
nucleotides to reduce
the fidelity of the Taq polymerase. The error rate can be as high as 2% under
appropriate
conditions, when the resultant mutant sequence is compared to the wild type
starting sequence.
After amplification, the library of mutant coding sequences must be cloned
into a suitable plasmid.
Although point mutations are the most common types of mutation in error prone
PCR, deletions
and frameshift mutations are also possible. There are a number of commercial
error-prone PCR
kits available, including those from Stratagene and Clontech (e.g., World Wide
Web URL
strategene.com and World Wide Web URL clontech.com, respectively, for
example). Rolling circle
error-prone PCR is a variant of error-prone PCR in which wild-type sequence is
first cloned into a
plasmid, then the whole plasmid is amplified under error-prone conditions.
As noted above, organisms with altered activities can also be isolated using
genetic selection and
screening of organisms challenged on selective media or by identifying
naturally occurring variants
from unique environments. For example, 2-Deoxy-D-glucose is a toxic glucose
analog. Growth of
yeast on this substance yields mutants that are glucose-deregulated. A number
of mutants have
been isolated using 2-Deoxy-D-glucose including transport mutants, and mutants
that ferment
glucose and galactose simultaneously instead of glucose first then galactose
when glucose is
depleted. Similar techniques have been used to isolate mutant microorganisms
that can
metabolize plastics (e.g., from landfills), petrochemicals (e.g., from oil
spills), and the like, either in
a laboratory setting or from unique environments.
Similar methods can be used to isolate naturally occurring mutations in a
desired activity when the
activity exists at a relatively low or nearly undetectable level in the
organism of choice, in some
embodiments. The method generally consists of growing the organism to a
specific density in
liquid culture, concentrating the cells, and plating the cells on various
concentrations of the
substance to which an increase in metabolic activity is desired. The cells are
incubated at a
moderate growth temperature, for 5 to 10 days. To enhance the selection
process, the plates can
be stored for another 5 to 10 days at a low temperature. The low temperature
sometimes can
allow strains that have gained or increased an activity to continue growing
while other strains are
inhibited for growth at the low temperature. Following the initial selection
and secondary growth at
low temperature, the plates can be replica plated on higher or lower
concentrations of the selection
substance to further select for the desired activity.
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A native, heterologous or mutagenized polynucleotide can be introduced into a
nucleic acid
reagent for introduction into a host organism, thereby generating an
engineered microorganism.
Standard recombinant DNA techniques (restriction enzyme digests, ligation, and
the like) can be
used by the artisan to combine the mutagenized nucleic acid of interest into a
suitable nucleic acid
reagent capable of (i) being stably maintained by selection in the host
organism, or (ii) being
integrating into the genome of the host organism. As noted above, sometimes
nucleic acid
reagents comprise two replication origins to allow the same nucleic acid
reagent to be manipulated
in bacterial before final introduction of the final product into the host
organism (e.g., yeast or fungus
for example). Standard molecular biology and recombinant DNA methods are known
(e.g.,
described in Maniatis, T., E. F. Fritsch and J. Sambrook (1982) Molecular
Cloning: a Laboratory
Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
Nucleic acid reagents can be introduced into microorganisms using various
techniques. Non-
limiting examples of methods used to introduce heterologous nucleic acids into
various organisms
include; transformation, transfection, transduction, electroporation,
ultrasound-mediated
transformation, particle bombardment and the like. In some instances the
addition of carrier
molecules (e.g., bis-benzimdazolyl compounds, for example, see US Patent
5595899) can
increase the uptake of DNA in cells typically though to be difficult to
transform by conventional
methods. Conventional methods of transformation are known (e.g., described in
Maniatis, T., E. F.
Fritsch and J. Sambrook (1982) Molecular Cloning: a Laboratory Manual; Cold
Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.).
Culture, Production and Process Methods
Engineered microorganisms often are cultured under conditions that optimize
yield of a target
molecule (e.g., six-carbon target molecule). Non-limiting examples of such
target molecules are
adipic acid and 6-hydroxyhexanoic acid. Culture conditions often optimize
activity of one or more
of the following activities: 6-oxohexanoic acid dehydrogenase activity, omega
oxo fatty acid
dehydrogenase activity, 6-hydroxyhexanoic acid dehydrogenase activity, omega
hydroxyl fatty acid
dehydrogenase activity, glucose-6-phosphate dehydrogenase activity, hexanoate
synthase activity,
lipase activity, fatty acid synthase activity, acetyl CoA carboxylase
activity, monooxygenase
activity, monooxygenase reductase activity, fatty alcohol oxidase, acyl-CoA
ligase, acyl-CoA
oxidase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and/or acetyl-
CoA C-
acyltransferase activities. In general, non-limiting examples of conditions
that may be optimized
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include the type and amount of carbon source, the type and amount of nitrogen
source, the
carbon-to-nitrogen ratio, the oxygen level, growth temperature, pH, length of
the biomass
production phase, length of target product accumulation phase, and time of
cell harvest.
Culture media generally contain a suitable carbon source. Carbon sources
useful for culturing
microorganisms and/or fermentation processes sometimes are referred to as
feedstocks. The term
"feedstock" as used herein refers to a composition containing a carbon source
that is provided to
an organism, which is used by the organism to produce energy and metabolic
products useful for
growth. A feedstock may be a natural substance, a "man-made substance," a
purified or isolated
substance, a mixture of purified substances, a mixture of unpurified
substances or combinations
thereof. A feedstock often is prepared by and/or provided to an organism by a
person, and a
feedstock often is formulated prior to administration to the organism. A
carbon source may
include, but is not limited to including, one or more of the following
substances: monosaccharides
(e.g., also referred to as "saccharides," which include 6-carbon sugars (e.g.,
glucose, fructose), 5-
carbon sugars (e.g., xylose and other pentoses) and the like), disaccharides
(e.g., lactose,
sucrose), oligosaccharides (e.g., glycans, homopolymers of a monosaccharide),
polysaccharides
(e.g., starch, cellulose, heteropolymers of monosaccharides or mixtures
thereof), sugar alcohols
(e.g., glycerol), and renewable feedstocks (e.g., cheese whey permeate,
cornsteep liquor, sugar
beet molasses, barley malt).
A carbon source also may include a metabolic product that can be used directly
as a metabolic
substrate in an engineered pathway described herein, or indirectly via
conversion to a different
molecule using engineered or native biosynthetic pathways in an engineered
microorganism. In
some embodiments, a carbon source may include glycerol backbones generated by
the action of
an engineered pathway including at least a lipase activity. In certain
embodiments, metabolic
pathways can be preferentially biased towards production of a desired product
by increasing the
levels of one or more activities in one or more metabolic pathways having
and/or generating at
least one common metabolic and/or synthetic substrate. In some embodiments, a
metabolic
byproduct (e.g., glycerol) of an engineered activity (e.g., lipase activity)
can be used in one or more
metabolic pathways selected from gluconeogenesis, pentose phosphate pathway,
glycolysis, fatty
acid synthesis, beta oxidation, and omega oxidation, to generate a carbon
source that can be
converted to adipic acid.
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Carbon sources also can be selected from one or more of the following non-
limiting examples:
paraffin (e.g., saturated paraffin, unsaturated paraffin, substituted
paraffin, linear paraffin, branched
paraffin, or combinations thereof); alkanes (e.g., hexane), alkenes or
alkynes, each of which may
be linear, branched, saturated, unsaturated, substituted or combinations
thereof (described in
greater detail below); linear or branched alcohols (e.g., hexanol); saturated
and/or unsaturated
fatty acids (e.g., each fatty acid is about 1 carbon to about 60 carbons with
0 to 10 unsaturations,
including free fatty acids, mixed fatty acids, single fatty acid, purified
fatty acids (e.g., single fatty
acid or mixture of fatty acids) fatty acid distillates, soap stocks, the like
and combinations thereof);
esters of fatty acids; monoglycerides; diglycerides; triglycerides; and
phospholipids. Non-limiting
commercial sources of products for preparing feedstocks include plants or
plant products (e.g.,
vegetable oils (e.g., almond oil, canola oil, cocoa butter, coconut oil, corn
oil, cottonseed oil,
flaxseed oil, grape seed oil, illipe, olive oil, palm oil, palm olein, palm
kernel oil, safflower oil,
peanut oil, soybean oil, sesame oil, shea nut oil, sunflower oil walnut oil,
oleic acid, the like and
combinations thereof), vegetable oil products and purified fatty acids (e.g.,
myristoleic acid,
palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid,
linoleic acid, linoelaidic acid,
a-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid and
docosahexaenoic acid),
and animal fats (e.g., beef tallow, butterfat, lard, cod liver oil)). A carbon
source may include a
petroleum product and/or a petroleum distillate (e.g., diesel, fuel oils,
gasoline, kerosene, paraffin
wax, paraffin oil, petrochemicals).
The term "paraffin" as used herein refers to the common name for alkane
hydrocarbons,
independent of the source (e.g., plant derived, petroleum derived, chemically
synthesized,
fermented by a microorganism), or carbon chain length. A carbon source
sometimes comprises a
paraffin, and in some embodiments, a paraffin is predominant in a carbon
source (e.g., about 75%,
80%, 85%, 90% or 95% paraffin). A paraffin sometimes is saturated (e.g., fully
saturated),
sometimes includes one or more unsaturations (e.g., about 1, 2, 3, 4, 5, 6, 7,
8, 9, 10
unsaturations) and sometimes is substituted with one or more non-hydrogen
substituents. Non-
limiting examples of non-hydrogen substituents include halo, acetyl, =0, =N-
CN, =N-OR, =NR, OR,
NR2, SR, SO2R, SO2NR2, NRSO2R, NRCONR2, NRCOOR, NRCOR, CN, COOR, CONR2, 00CR,
COR, and NO2, where each R is independently H, C1-C8 alkyl, C2-C8 heteroalkyl,
C1-C8 acyl, C2-
C8 heteroacyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8
heteroalkynyl, C6-C10
aryl, or C5-C10 heteroaryl, and each R is optionally substituted with halo,
=0, =N-CN, =N-OR',
=NR', OR', NR'2, SR', 502R', SO2NR'2, NR'502R', NR'CONR'2, NR'COOR', NR'COR',
CN, COOR',
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CONR'2, 00CR', COR', and NO2, where each R' is independently H, C1-C8 alkyl,
C2-C8
heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl or C5-C10 heteroaryl.
In some embodiments a feedstock is selected according to the genotype and/or
phenotype of the
engineered microorganism to be cultured. For example, a feedstock rich in 12-
carbon fatty acids,
12-carbon dicarboxylic acids or 12-carbon paraffins, or a mixture of 10-carbon
and 12-carbon
compounds can be useful for culturing yeast strains harboring an alteration
that partially blocks
beta oxidation by disrupting PDX4 activity, as described herein. Non-limiting
examples of carbon
sources having 10 and/or 12 carbons include fats (e.g., coconut oil, palm
kernel oil), paraffins (e.g.,
alkanes, alkenes, or alkynes) having 10 or 12 carbons, (e.g., decane, dodecane
(also referred to
as adakane12, bihexyl, dihexyl and duodecane), alkene and alkyne derivatives),
fatty acids
(decanoic acid, dodecanoic acid), fatty alcohols (decanol, dodecanol), the
like, non-toxic
substituted derivatives or combinations thereof.
A carbon source sometimes comprises an alkyl, alkenyl or alkynyl compound or
molecule (e.g., a
compound that includes an alkyl, alkenyl or alkynyl moiety (e.g., alkane,
alkene, alkyne)). In
certain embodiments, an alkyl, alkenyl or alkynyl molecule, or combination
thereof, is predominant
in a carbon source (e.g., about 75%, 80%, 85%, 90% or 95% of such molecules).
As used herein,
the terms "alkyl," "alkenyl" and "alkynyl" include straight-chain (referred to
herein as "linear"),
branched-chain (referred to herein as "non-linear"), cyclic monovalent
hydrocarbyl radicals, and
combinations of these, which contain only C and H atoms when they are
unsubstituted. Non-
limiting examples of alkyl moieties include methyl, ethyl, isobutyl,
cyclohexyl, cyclopentylethyl, 2-
propenyl, 3-butynyl, and the like. An alkyl that contains only C and H atoms
and is unsubstituted
sometimes is referred to as "saturated." An alkenyl or alkynyl generally is
"unsaturated" as it
contains one or more double bonds or triple bonds, respectively. An alkenyl
can include any
number of double bonds, such as 1, 2, 3, 4 or 5 double bonds, for example. An
alkynyl can include
any number of triple bonds, such as 1, 2, 3, 4 or 5 triple bonds, for example.
Alkyl, alkenyl and
alkynyl molecules sometimes contain between about 2 to about 60 carbon atoms
(C). For
example, an alkyl, alkenyl and alkynyl molecule can include about 1 carbon
atom, about 2 carbon
atoms, about 3 carbon atoms, about 4 carbon atoms, about 5 carbon atoms, about
6 carbon
atoms, about 7 carbon atoms, about 8 carbon atoms, about 9 carbon atoms, about
10 carbon
atoms, about 12 carbon atoms, about 14 carbon atoms, about 16 carbon atoms,
about 18 carbon
atoms, about 20 carbon atoms, about 22 carbon atoms, about 24 carbon atoms,
about 26 carbon
atoms, about 28 carbon atoms, about 30 carbon atoms, about 32 carbon atoms,
about 34 carbon
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atoms, about 36 carbon atoms, about 38 carbon atoms, about 40 carbon atoms,
about 42 carbon
atoms, about 44 carbon atoms, about 46 carbon atoms, about 48 carbon atoms,
about 50 carbon
atoms, about 52 carbon atoms, about 54 carbon atoms, about 56 carbon atoms,
about 58 carbon
atoms or about 60 carbon atoms. In some embodiments, paraffins can have a mean
number of
carbon atoms of between about 8 to about 18 carbon atoms (e.g., about 8 carbon
atoms, about 9
carbon atoms, about 10 carbon atoms, about 11 carbon atoms, about 12 carbon
atoms, about 13
carbon atoms, about 14 carbon atoms, about 15 carbon atoms, about 16 carbon
atoms, about 17
carbon atoms and about 18 carbon atoms). A single group can include more than
one type of
multiple bond, or more than one multiple bond. Such groups are included within
the definition of
the term "alkenyl" when they contain at least one carbon-carbon double bond,
and are included
within the term "alkynyl" when they contain at least one carbon-carbon triple
bond. Alkyl, alkenyl
and alkynyl molecules include molecules that comprise an alkyl, alkenyl and/or
alkynyl moiety, and
include molecules that consist of an alkyl, alkenyl or alkynyl moiety (i.e.,
alkane, alkene and alkyne
molecules).
Alkyl, alkenyl and alkynyl substituents sometimes contain 1-20C (alkyl) or 2-
20C (alkenyl or
alkynyl). They can contain about 8-14C or about 10-14C in some embodiments. A
single group
can include more than one type of multiple bond, or more than one multiple
bond. Such groups
are included within the definition of the term "alkenyl" when they contain at
least one carbon-
carbon double bond, and are included within the term "alkynyl" when they
contain at least one
carbon-carbon triple bond.
Alkyl, alkenyl and alkynyl groups or compounds sometimes are substituted to
the extent that such
substitution can be synthesized and can exist. Typical substituents include,
but are not limited to,
halo, acetyl, =0, =N-CN, =N-OR, =NR, OR, NR2, SR, 502R, 502NR2, NRSO2R,
NRCONR2,
NRCOOR, NRCOR, CN, COOR, CONR2, 00CR, COR, and NO2, where each R is
independently
H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C2-C8
alkenyl, C2-C8
heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C11 aryl, or C5-C11
heteroaryl, and each R
is optionally substituted with halo, =0, =N-CN, =N-OR', =NR', OR', NR'2, SR',
502R', SO2NR'2,
NR'502R', NR'CONR'2, NR'COOR', NR'COR', CN, COOR', CONR'2, 00CR', COR', and
NO2,
where each R' is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl,
C2-C8 heteroacyl,
C6-C10 aryl or C5-C10 heteroaryl. Alkyl, alkenyl and alkynyl groups can also
be substituted by
C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl or C5-C10 heteroaryl, each of which
can be substituted
by the substituents that are appropriate for the particular group.
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"Acetylene" or "acetyl" substituents are 2-10C alkynyl groups that are
optionally substituted, and
are of the formula -CEC-Ri, where Ri is H or C1-C8 alkyl, C2-C8 heteroalkyl,
C2-C8 alkenyl, C2-C8
heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8
heteroacyl, C6-C10 aryl,
C5-C10 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl, and each Ri
group is optionally
substituted with one or more substituents selected from halo, =0, =N-CN, =N-
OR', =NR', OR',
NR'2, SR', SO2R', SO2NR'2, NR'502R', NR'CONR'2, NR'COOR', NR'COR', CN, COOR',
CONR'2,
00CR', COR', and NO2, where each R' is independently H, C1-C6 alkyl, C2-C6
heteroalkyl, C1-C6
acyl, C2-C6 heteroacyl, C6-C10 aryl, C5-C10 heteroaryl, C7-12 arylalkyl, or C6-
12 heteroarylalkyl,
each of which is optionally substituted with one or more groups selected from
halo, C1-C4 alkyl,
C1-C4 heteroalkyl, C1-C6 acyl, C1-C6 heteroacyl, hydroxy, amino, and =0; and
where two R' can
be linked to form a 3-7 membered ring optionally containing up to three
heteroatoms selected from
N, 0 and S. In some embodiments, Ri of -CEC-Ri is H or Me.
A carbon source sometimes comprises a heteroalkyl, heteroalkenyl and/or
heteroalkynyl molecule
or compound (e.g., comprises heteroalkyl, heteroalkenyl and/or heteroalkynyl
moiety (e.g.,
heteroalkane, heteroalkene or heteroalkyne)). "Heteroalkyl", "heteroalkenyl",
and "heteroalkynyl"
and the like are defined similarly to the corresponding hydrocarbyl (alkyl,
alkenyl and alkynyl)
groups, but the `hetero' terms refer to groups that contain one to three 0, S
or N heteroatoms or
combinations thereof within the backbone; thus at least one carbon atom of a
corresponding alkyl,
alkenyl, or alkynyl group is replaced by one of the specified heteroatoms to
form a heteroalkyl,
heteroalkenyl, or heteroalkynyl group. The typical and sizes for heteroforms
of alkyl, alkenyl and
alkynyl groups are generally the same as for the corresponding hydrocarbyl
groups, and the
substituents that may be present on the heteroforms are the same as those
described above for
the hydrocarbyl groups. For reasons of chemical stability, it is also
understood that, unless
otherwise specified, such groups do not include more than two contiguous
heteroatoms except
where an oxo group is present on N or S as in a nitro or sulfonyl group.
The term "alkyl" as used herein includes cycloalkyl and cycloalkylalkyl groups
and compounds, the
term "cycloalkyl" may be used herein to describe a carbocyclic non-aromatic
compound or group
that is connected via a ring carbon atom, and "cycloalkylalkyl" may be used to
describe a
carbocyclic non-aromatic compound or group that is connected to a molecule
through an alkyl
linker. Similarly, "heterocycly1" may be used to describe a non-aromatic
cyclic group that contains
at least one heteroatom as a ring member and that is connected to the molecule
via a ring atom,
which may be C or N; and "heterocyclylalkyl" may be used to describe such a
group that is
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connected to another molecule through a linker. The sizes and substituents
that are suitable for
the cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl groups
are the same as those
described above for alkyl groups. As used herein, these terms also include
rings that contain a
double bond or two, as long as the ring is not aromatic.
A carbon source sometimes comprises an acyl compound or moiety (e.g., compound
comprising
an acyl moiety). As used herein, "acyl" encompasses groups comprising an
alkyl, alkenyl, alkynyl,
aryl or arylalkyl radical attached at one of the two available valence
positions of a carbonyl carbon
atom, and heteroacyl refers to the corresponding groups where at least one
carbon other than the
carbonyl carbon has been replaced by a heteroatom chosen from N, 0 and S. Thus
heteroacyl
includes, for example, -C(=0)OR and ¨C(=0)NR2 as well as ¨C(=0)-heteroaryl.
Acyl and heteroacyl groups are bonded to any group or molecule to which they
are attached
through the open valence of the carbonyl carbon atom. Typically, they are C1-
C8 acyl groups,
which include formyl, acetyl, pivaloyl, and benzoyl, and C2-C8 heteroacyl
groups, which include
methoxyacetyl, ethoxycarbonyl, and 4-pyridinoyl. The hydrocarbyl groups, aryl
groups, and
heteroforms of such groups that comprise an acyl or heteroacyl group can be
substituted with the
substituents described herein as generally suitable substituents for each of
the corresponding
component of the acyl or heteroacyl group.
A carbon source sometimes comprises one or more aromatic moieties and/or
heteroaromatic
moieties. "Aromatic" moiety or "aryl" moiety refers to a monocyclic or fused
bicyclic moiety having
the well-known characteristics of aromaticity; examples include phenyl and
naphthyl. Similarly,
"heteroaromatic" and "heteroaryl" refer to such monocyclic or fused bicyclic
ring systems which
contain as ring members one or more heteroatoms selected from 0, S and N. The
inclusion of a
heteroatom permits aromaticity in 5 membered rings as well as 6 membered
rings. Typical
heteroaromatic systems include monocyclic C5-C6 aromatic groups such as
pyridyl, pyrimidyl,
pyrazinyl, thienyl, furanyl, pyrrolyl, pyrazolyl, thiazolyl, oxazolyl, and
imidazolyl and the fused
bicyclic moieties formed by fusing one of these monocyclic groups with a
phenyl ring or with any of
the heteroaromatic monocyclic groups to form a C8-C10 bicyclic group such as
indolyl,
benzimidazolyl, indazolyl, benzotriazolyl, isoquinolyl, quinolyl,
benzothiazolyl, benzofuranyl,
pyrazolopyridyl, quinazolinyl, quinoxalinyl, cinnolinyl, and the like. Any
monocyclic or fused ring
bicyclic system which has the characteristics of aromaticity in terms of
electron distribution
throughout the ring system is included in this definition. It also includes
bicyclic groups where at
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least the ring which is directly attached to the remainder of the molecule has
the characteristics of
aromaticity. Typically, the ring systems contain 5-12 ring member atoms. The
monocyclic
heteroaryls sometimes contain 5-6 ring members, and the bicyclic heteroaryls
sometimes contain
8-10 ring members.
Aryl and heteroaryl moieties may be substituted with a variety of substituents
including C1-C8 alkyl,
C2-C8 alkenyl, C2-C8 alkynyl, C5-C12 aryl, C1-C8 acyl, and heteroforms of
these, each of which
can itself be further substituted; other substituents for aryl and heteroaryl
moieties include halo,
OR, NR2, SR, SO2R, SO2NR2, NRSO2R, NRCONR2, NRCOOR, NRCOR, CN, COOR, CONR2,
00CR, COR, and NO2, where each R is independently H, C1-C8 alkyl, C2-C8
heteroalkyl, C2-C8
alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C10 aryl,
C5-C10
heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl, and each R is
optionally substituted as
described above for alkyl groups. The substituent groups on an aryl or
heteroaryl group may be
further substituted with the groups described herein as suitable for each type
of such substituents
or for each component of the substituent. Thus, for example, an arylalkyl
substituent may be
substituted on the aryl portion with substituents typical for aryl groups, and
it may be further
substituted on the alkyl portion with substituents as typical or suitable for
alkyl groups.
Similarly, "arylalkyl" and "heteroarylalkyl" refer to aromatic and
heteroaromatic ring systems, which
are stand-alone molecules (e.g., benzene or substituted benzene, pyridine or
substituted pyridine),
or which are bonded to an attachment point through a linking group such as an
alkylene, including
substituted or unsubstituted, saturated or unsaturated, cyclic or acyclic
linkers. A linker often is C1-
C8 alkyl or a hetero form thereof. These linkers also may include a carbonyl
group, thus making
them able to provide substituents as an acyl or heteroacyl moiety. An aryl or
heteroaryl ring in an
arylalkyl or heteroarylalkyl group may be substituted with the same
substituents described above
for aryl groups. An arylalkyl group sometimes includes a phenyl ring
optionally substituted with the
groups defined above for aryl groups and a C1-C4 alkylene that is
unsubstituted or is substituted
with one or two C1-C4 alkyl groups or heteroalkyl groups, where the alkyl or
heteroalkyl groups
can optionally cyclize to form a ring such as cyclopropane, dioxolane, or
oxacyclopentane.
Similarly, a heteroarylalkyl group often includes a C5-C6 monocyclic
heteroaryl group optionally
substituted with one or more of the groups described above as substituents
typical on aryl groups
and a C1-C4 alkylene that is unsubstituted. A heteroarylalkyl group sometimes
is substituted with
one or two C1-C4 alkyl groups or heteroalkyl groups, or includes an optionally
substituted phenyl
ring or C5-C6 monocyclic heteroaryl and a C1-C4 heteroalkylene that is
unsubstituted or is
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substituted with one or two C1-C4 alkyl or heteroalkyl groups, where the alkyl
or heteroalkyl groups
can optionally cyclize to form a ring such as cyclopropane, dioxolane, or
oxacyclopentane.
Where an arylalkyl or heteroarylalkyl group is described as optionally
substituted, the substituents
may be on the alkyl or heteroalkyl portion or on the aryl or heteroaryl
portion of the group. The
substituents optionally present on the alkyl or heteroalkyl portion sometimes
are the same as those
described above for alkyl groups, and the substituents optionally present on
the aryl or heteroaryl
portion often are the same as those described above for aryl groups generally.
"Arylalkyl" groups as used herein are hydrocarbyl groups if they are
unsubstituted, and are
described by the total number of carbon atoms in the ring and alkylene or
similar linker. Thus a
benzyl group is a C7-arylalkyl group, and phenylethyl is a C8-arylalkyl.
"Heteroarylalkyl" as described above refers to a moiety comprising an aryl
group that is attached
through a linking group, and differs from "arylalkyl" in that at least one
ring atom of the aryl moiety
or one atom in the linking group is a heteroatom selected from N, 0 and S. The
heteroarylalkyl
groups are described herein according to the total number of atoms in the ring
and linker
combined, and they include aryl groups linked through a heteroalkyl linker;
heteroaryl groups linked
through a hydrocarbyl linker such as an alkylene; and heteroaryl groups linked
through a
heteroalkyl linker. Thus, for example, C7-heteroarylalkyl includes
pyridylmethyl, phenoxy, and N-
pyrrolylmethoxy.
"Alkylene" as used herein refers to a divalent hydrocarbyl group. Because an
alkylene is divalent,
it can link two other groups together. An alkylene often is referred to as
¨(CH2)n- where n can be 1-
20, 1-10, 1-8, or 1-4, though where specified, an alkylene can also be
substituted by other groups,
and can be of other lengths, and the open valences need not be at opposite
ends of a chain. Thus
¨CH(Me)- and ¨C(Me)2- may also be referred to as alkylenes, as can a cyclic
group such as
cyclopropan-1,1-diyl. Where an alkylene group is substituted, the substituents
include those
typically present on alkyl groups as described herein.
In some embodiments, a feedstock includes a mixture of carbon sources, where
each carbon
source in the feedstock is selected based on the genotype of the engineered
microorganism. In
certain embodiments, a mixed carbon source feedstock includes one or more
carbon sources
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selected from sugars, cellulose, fatty acids, triacylglycerides, paraffins,
the like and combinations
thereof.
Nitrogen may be supplied from an inorganic (e.g., (NH
4)
2S04) or
organic source
(e.g., urea or glutamate). In addition to appropriate carbon and nitrogen
sources, culture media
also can contain suitable minerals, salts, cofactors, buffers, vitamins, metal
ions (e.g., Mn
+2,
Co
+2, Zn
+2, Mg
+2) and other components suitable for culture of
microorganisms.
Engineered microorganisms sometimes are cultured in complex media (e.g., yeast
extract-
peptone-dextrose broth (YPD)). In some embodiments, engineered microorganisms
are cultured in
a defined minimal media that lacks a component necessary for growth and
thereby forces selection
of a desired expression cassette (e.g., Yeast Nitrogen Base (DIFCO
Laboratories, Detroit, Mich.)).
Culture media in some embodiments are common commercially prepared media, such
as Yeast
Nitrogen Base (DIFCO Laboratories, Detroit, Mich.). Other defined or synthetic
growth media may
also be used and the appropriate medium for growth of the particular
microorganism are known. A
variety of host organisms can be selected for the production of engineered
microorganisms. Non-
limiting examples include yeast (e.g., Candida tropicalis (e.g., ATCC20336,
ATCC20913,
ATCC20962), Candida viswanithii, Yarrowia lipolytica (e.g., ATCC20228)) and
filamentous fungi
(e.g., Aspergillus nidulans (e.g., ATCC38164) and Aspergillus parasiticus
(e.g., ATCC 24690)). In
specific embodiments, yeast are cultured in YPD media (10 g/L Bacto Yeast
Extract, 20 g/L Bacto
Peptone, and 20 g/L Dextrose). Filamentous fungi, in particular embodiments,
are grown in CM
(Complete Medium) containing 10 g/L Dextrose, 2 g/L Bacto Peptone, 1g/L Bacto
Yeast Extract, 1
g/L Casamino acids, 50 mL /L 20X Nitrate Salts (120 g/L NaNO3, 10.4 g/L KCI,
10.4 g/L MgSO4=7
H20 ), 1 mL/L 1000X Trace Elements (22 g/L ZnSO4=7 H20, 11 g/L H3B03, 5 g/L
MnC12=7 H20, 5
g/L FeSO4=7 H20, 1.7 g/L CoC12=6 H20, 1.6 g/L CuSO4=5 H20, 1.5 g/L Na2Mo04.2
H20, and 50 g/L
Na4EDTA), and 1 mL/L Vitamin Solution (100 mg each of Biotin, pyridoxine,
thiamine, riboflavin, p-
aminobenzoic acid, and nicotinic acid in 100 mL water).
A suitable pH range for the fermentation often is between about pH 4.0 to
about pH 8.0, where a
pH in the range of about pH 5.5 to about pH 7.0 sometimes is utilized for
initial culture conditions.
Depending on the host organism, culturing may be conducted under aerobic or
anaerobic
conditions, where microaerobic conditions sometimes are maintained. A two-
stage process may
be utilized, where one stage promotes microorganism proliferation and another
state promotes
production of target molecule. In a two-stage process, the first stage may be
conducted under
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aerobic conditions (e.g., introduction of air and/or oxygen) and the second
stage may be conducted
under anaerobic conditions (e.g., air or oxygen are not introduced to the
culture conditions). In
some embodiments, the first stage may be conducted under anaerobic conditions
and the second
stage may be conducted under aerobic conditions. In certain embodiments, a two-
stage process
may include two more organisms, where one organism generates an intermediate
product (e.g.,
hexanoic acid produced by Megasphera spp.) in one stage and another organism
processes the
intermediate product into a target product (e.g., adipic acid) in another
stage, for example.
A variety of fermentation processes may be applied for commercial biological
production of a target
product. In some embodiments, commercial production of a target product from a
recombinant
microbial host is conducted using a batch, fed-batch or continuous
fermentation process, for
example.
A batch fermentation process often is a closed system where the media
composition is fixed at the
beginning of the process and not subject to further additions beyond those
required for
maintenance of pH and oxygen level during the process. At the beginning of the
culturing process
the media is inoculated with the desired organism and growth or metabolic
activity is permitted to
occur without adding additional sources (i.e., carbon and nitrogen sources) to
the medium. In
batch processes the metabolite and biomass compositions of the system change
constantly up to
the time the culture is terminated. In a typical batch process, cells proceed
through a static lag
phase to a high-growth log phase and finally to a stationary phase, wherein
the growth rate is
diminished or halted. Left untreated, cells in the stationary phase will
eventually die.
A variation of the standard batch process is the fed-batch process, where the
carbon source is
continually added to the fermentor over the course of the fermentation
process. Fed-batch
processes are useful when catabolite repression is apt to inhibit the
metabolism of the cells or
where it is desirable to have limited amounts of carbon source in the media at
any one time.
Measurement of the carbon source concentration in fed-batch systems may be
estimated on the
basis of the changes of measurable factors such as pH, dissolved oxygen and
the partial pressure
of waste gases (e.g., CO
2).
Batch and fed-batch culturing methods are known in the art. Examples of such
methods may be
found in Thomas D. Brock in Biotechnology: A Textbook of Industrial
Microbiology, 2
nd ed.,
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(1989) Sinauer Associates Sunderland, Mass. and Deshpande, Mukund V., Appl.
Biochem.
Biotechnol., 36:227 (1992).
In continuous fermentation process a defined media often is continuously added
to a bioreactor
while an equal amount of culture volume is removed simultaneously for product
recovery.
Continuous cultures generally maintain cells in the log phase of growth at a
constant cell density.
Continuous or semi-continuous culture methods permit the modulation of one
factor or any number
of factors that affect cell growth or end product concentration. For example,
an approach may limit
the carbon source and allow all other parameters to moderate metabolism. In
some systems, a
number of factors affecting growth may be altered continuously while the cell
concentration,
measured by media turbidity, is kept constant. Continuous systems often
maintain steady state
growth and thus the cell growth rate often is balanced against cell loss due
to media being drawn
off the culture. Methods of modulating nutrients and growth factors for
continuous culture
processes, as well as techniques for maximizing the rate of product formation,
are known and a
variety of methods are detailed by Brock, supra.
In some embodiments involving fermentation, the fermentation can be carried
out using two or
more microorganisms (e.g., host microorganism, engineered microorganism,
isolated naturally
occurring microorganism, the like and combinations thereof), where a feedstock
is partially or
completely utilized by one or more organisms in the fermentation (e.g., mixed
fermentation), and
the products of cellular respiration or metabolism of one or more organisms
can be further
metabolized by one or more other organisms to produce a desired target product
(e.g., adipic acid,
hexanoic acid). In certain embodiments, each organism can be fermented
independently and the
products of cellular respiration or metabolism purified and contacted with
another organism to
produce a desired target product. In some embodiments, one or more organisms
are partially or
completely blocked in a metabolic pathway (e.g., beta oxidation, omega
oxidation, the like or
combinations thereof), thereby producing a desired product that can be used as
a feedstock for
one or more other organisms. Any suitable combination of microorganisms can be
utilized to carry
out mixed fermentation or sequential fermentation. A non-limiting example of
an organism
combination and feedstock suitable for use in mixed fermentations or
sequential fermentations
where the fermented media from a first organism is used as a feedstock for a
second organism is
the use of long chain dicarboxylic acids as a fermentation media for
Megasphaera elsdenii to
produce hexanoic acid, and Candida tropicalis engineered as described herein
to produce adipic
acid from the hexanoic acid produced by Megasphaera elsdenii. Megasphaera
elsdenii is a
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facultative anaerobe. Without being limited by theory, it is believe that
Megasphaera elsdenii
naturally accumulates hexanoic acid as a result of anaerobic respiration.
Candida tropicalis can
grow aerobically and anaerobically. In some embodiments, the hexanoic acid
produced by
Megasphaera elsdenii can be utilized as a feedstock for Candida tropicalis to
produce adipic acid.
In certain embodiments, the Megasphaera produced hexanoic acid is purified
(e.g., partially,
completely) prior to being used as a feedstock for C. tropicalis.
In various embodiments adipic acid is isolated or purified from the culture
media or extracted from
the engineered microorganisms. In some embodiments, fermentation of feedstocks
by methods
described herein can produce a target product (e.g., adipic acid) at a level
of about 80% or more of
theoretical yield (e.g., 80% or more, 81% or more, 82% or more, 83% or more,
84% or more, 85%
or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91%
or more, 92%
or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98%
or more, or
99% or more of theoretical yield). The term "theoretical yield" as used herein
refers to the amount
of product that could be made from a starting material if the reaction is 100%
complete.
Theoretical yield is based on the stoichiometry of a reaction and ideal
conditions in which starting
material is completely consumed, undesired side reactions do not occur, the
reverse reaction does
not occur, and there are no losses in the work-up procedure. Culture media may
be tested for
target product (e.g., adipic acid) concentration and drawn off when the
concentration reaches a
predetermined level. Detection methods are known in the art, including but not
limited to those set
forth in B Stieglitz and P J Weimer, Novel microbial screen for detection of
1,4-butanediol,
ethylene glycol, and adipic acid, Appl Environ Microbiol. 198. Target product
(e.g., adipic acid)
may be present at a range of levels as described herein.
A target product sometimes is retained within an engineered microorganism
after a culture process
is completed, and in certain embodiments, the target product is secreted out
of the microorganism
into the culture medium. For the latter embodiments, (i) culture media may be
drawn from the
culture system and fresh medium may be supplemented, and/or (ii) target
product may be
extracted from the culture media during or after the culture process is
completed. Engineered
microorganisms may be cultured on or in solid, semi-solid or liquid media. In
some embodiments
media is drained from cells adhering to a plate. In certain embodiments, a
liquid-cell mixture is
centrifuged at a speed sufficient to pellet the cells but not disrupt the
cells and allow extraction of
the media, as known in the art. The cells may then be resuspended in fresh
media. Target
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product may be purified from culture media according to known methods, such as
those described
in U.S. Pat. No. 6,787,669 and U.S. Pat. No. 5,296,639, for example.
In certain embodiments, target product is extracted from the cultured
engineered microorganisms.
The micoorganism cells may be concentrated through centrifugation at a speed
sufficient to shear
the cell membranes. In some embodiments, the cells may be physically disrupted
(e.g., shear
force, sonication) or chemically disrupted (e.g., contacted with detergent or
other lysing agent).
The phases may be separated by centrifugation or other method known in the art
and target
product may be isolated according to known methods.
Commercial grade target product sometimes is provided in substantially pure
form (e.g., 90% pure
or greater, 95% pure or greater, 99% pure or greater or 99.5% pure or
greater). In some
embodiments, target product may be modified into any one of a number of
downstream products.
For example, adipic acid may be polycondensed with hexamethylenediamine to
produce nylon.
Nylon may be further processed into fibers for applications in carpeting,
automobile tire cord and
clothing. Adipic acid is also used for manufacturing plasticizers, lubricant
components and
polyester polyols for polyurethane systems. Food grade adipic acid is used as
a gelling aid,
acidulant, leavening and buffering agent. Adipic acid has two carboxylic acid
(-COOH) groups,
which can yield two kinds of salts. Its derivatives, acyl halides, anhydrides,
esters, amides and
nitriles, are used in making downstream products such as flavoring agents,
internal plasticizers,
pesticides, dyes, textile treatment agents, fungicides, and pharmaceuticals
through further
reactions of substitution, catalytic reduction, metal hydride reduction,
diborane reduction, keto
formation with organometallic reagents, electrophile bonding at oxygen, and
condensation.
Target product may be provided within cultured microbes containing target
product, and cultured
microbes may be supplied fresh or frozen in a liquid media or dried. Fresh or
frozen microbes may
be contained in appropriate moisture-proof containers that may also be
temperature controlled as
necessary. Target product sometimes is provided in culture medium that is
substantially cell-free.
In some embodiments target product or modified target product purified from
microbes is provided,
and target product sometimes is provided in substantially pure form. In
certain embodiments
crystallized or powdered target product is provided. Crystalline adipic acid
is a white powder with a
melting point of 360 F and may be transported in a variety of containers
including one ton cartons,
drums, 50 pound bags and the like.
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In certain embodiments, a target product (e.g., adipic acid, 6-hydroxyhexanoic
acid) is produced
with a yield of about 0.30 grams of target product, or greater, per gram of
glucose added during a
fermentation process (e.g., about 0.31 grams of target product per gram of
glucose added, or
greater; about 0.32 grams of target product per gram of glucose added, or
greater; about 0.33
grams of target product per gram of glucose added, or greater; about 0.34
grams of target product
per gram of glucose added, or greater; about 0.35 grams of target product per
gram of glucose
added, or greater; about 0.36 grams of target product per gram of glucose
added, or greater; about
0.37 grams of target product per gram of glucose added, or greater; about 0.38
grams of target
product per gram of glucose added, or greater; about 0.39 grams of target
product per gram of
glucose added, or greater; about 0.40 grams of target product per gram of
glucose added, or
greater; about 0.41 grams of target product per gram of glucose added, or
greater; 0.42 grams of
target product per gram of glucose added, or greater; 0.43 grams of target
product per gram of
glucose added, or greater; 0.44 grams of target product per gram of glucose
added, or greater;
0.45 grams of target product per gram of glucose added, or greater; 0.46 grams
of target product
per gram of glucose added, or greater; 0.47 grams of target product per gram
of glucose added, or
greater; 0.48 grams of target product per gram of glucose added, or greater;
0.49 grams of target
product per gram of glucose added, or greater; 0.50 grams of target product
per gram of glucose
added, or greater; 0.51 grams of target product per gram of glucose added, or
greater; 0.52 grams
of target product per gram of glucose added, or greater; 0.53 grams of target
product per gram of
glucose added, or greater; 0.54 grams of target product per gram of glucose
added, or greater;
0.55 grams of target product per gram of glucose added, or greater; 0.56 grams
of target product
per gram of glucose added, or greater; 0.57 grams of target product per gram
of glucose added, or
greater; 0.58 grams of target product per gram of glucose added, or greater;
0.59 grams of target
product per gram of glucose added, or greater; 0.60 grams of target product
per gram of glucose
added, or greater; 0.61 grams of target product per gram of glucose added, or
greater; 0.62 grams
of target product per gram of glucose added, or greater; 0.63 grams of target
product per gram of
glucose added, or greater; 0.64 grams of target product per gram of glucose
added, or greater;
0.65 grams of target product per gram of glucose added, or greater; 0.66 grams
of target product
per gram of glucose added, or greater; 0.67 grams of target product per gram
of glucose added, or
greater; 0.68 grams of target product per gram of glucose added, or greater;
0.69 or 0.70 grams of
target product per gram of glucose added or greater).
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Examples
The examples set forth below illustrate certain embodiments and do not limit
the technology.
Certain examples set forth below utilize standard recombinant DNA and other
biotechnology
protocols known in the art. Many such techniques are described in detail in
Maniatis, T., E. F.
Fritsch and J. Sambrook (1982) Molecular Cloning: a Laboratory Manual; Cold
Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. DNA mutagenesis can be accomplished using
the
Stratagene (San Diego, CA) "QuickChange" kit according to the manufacturer's
instructions.
Example 1: Cloning Hexanoate Synthase ("HexS') Subunit Genes
Total RNA from Aspergillus parasiticus was prepared using the RiboPure TM
(Ambion, Austin, TX)
kit for yeast. The genes encoding the two subunits of hexanoate synthase
(referred to as "hexA"
and "hex B" were isolated from this total RNA using the 2-step RT-PCR method
with Superscript III
reverse transcriptase (Life Technologies, Carlsbad, CA) and the fragments were
gel purified. The
primers used for each RT-PCR reaction are as follows:
HexA Aspergillus parasiticus primers
SP_HexA_Apar_1_1149 ATGGTCATCCAAGGGAAGAGATTGGCCGCCTCCTCTATTCAGC
ASP_HexA_Apar_1_1149 GTAGGCGTCACAGGAAAGACTGCGTACCA
SP HexA_Apar_941_2270 TATCACCAATGCTGGATGTAAAGAAGTCGCG
ASP_HexA_Apar_941_2270 AATTGGGCTAGGAAACCGGGGATGC
SP HexA_Apar_2067_3016 CGGTCTAATGACGGCGCATGATATCATAGCCGAAACGGTCGAG
ASP_HexA_Apar_2067_3016 ACTTGGCTGGAGTCCATCCCTTCGGCA
SP_HexA_Apar_2812_4181 CTGCCCGAGTTTGAAGTATCTCAACTTACCGCCGACGCCATG
ASP_HexA_Apar_2812_4181 TGAGACGCGCTGCGCAGGGC
SP HexA_Apar_3975_5016 CGAGGTGATCGAGACGCAGATGC
ASP_HexA_Apar_3975_5016 TTATGAAGCACCAGACATCAGCCCCAGC
HexB Aspergillus parasiticus primers
SP_HexB_Apar_1_1166 ATGGGTTCCGTTAGTAGGGAACATGAGTCAATC
ASP_HexB_Apar_1_1166 GTTCCTTGTGTGAGCTCCTGAATAAGACTGCATG
SP HexB_Apar_962_2042 CCATCAAAATCCCCCTCTATCACACGGGCACTGGGAGCAAC
ASP_HexB_Apar_962_2042 CCCACGCCTTGCGCATCTATAATCAGG
SP_HexB_Apar_1837_3527 TGTCCGAATATTCTCCTCGTTGTAGGTAGTGGATT
ASP_HexB_Apar_1837_3527 GCAGTAGTCGATAGGTACACATCCTTGGGGGTTCCATGACTGC
SP HexB_Apar_3322_4460 AGAGGATCAAGGCATTATACATGAGTCTGTGGAACTTGGGCTTTCC
ASP_HexB_Apar_3322_4460 TTCCCCGTCCTCCATGGCCTTATGC
SP HexB_Apar_4256_5667 GGCCTTTGCGCGATACGCTGGTCTCTCGGGTCCCAT
ASP_HexB_Apar_4256_5667 TCACGCCATTTGTTGAAGCAGGGAATG
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Each of the fragments was inserted separately into the plasmid pCRBlunt II
(Life Technologies,
Carlsbad, CA) such that there were four hexA plasmids, each with a different
hexA gene fragment,
and five hex B plasmids each with a different hexB gene fragment. Each hexA
and hexB fragment
was sequence verified, after which the fragments were PCR cloned from each
plasmid. Overlap
PCR was then used to create the full length hexA and hexB genes. The hexA gene
was inserted
into the vector p425GPD which has a LEU2 selectable marker and a
glyceraldehyde 3-phosphate
dehydrogenase promoter (American Type Culture Collection) and the hexB full
length gene was
inserted into p426GPD which has a URA3 selectable marker and a glyceraldehyde
3-phosphate
dehydrogenase promoter (American Type Culture Collection).
Example 2: Transformation of Saccharomyces cerevisiae with HexA and HexE3
Genes
Saccharomyces cereviseae cells (strain BY4742, ATCC Accession Number 201389)
were grown in
standard YPD (10g Yeast Extract, 20g Bacto-Peptone, 20g Glucose, 1L total)
media at about 30
degrees Celsius for about 3 days. The plasmids containing the hexA and hexB
genes were co-
transformed into the Saccharomyces cerevisiae. Transformation was accomplished
using the
Zymo kit (Catalog number T2001; Zymo Research Corp., Orange, CA 92867) using
lug plasmid
DNA and cultured on SC drop out media with glucose (minus uracil and minus
leucine) (20g
glucose; 2.21g SC (-URA,-LEU) dry mix, 6.7g Yeast Nitrogen Base, 1L total) for
2-3 days at about
30 C.
SC(-URA) mix contains:
0.4g Adenine hemisulfate
3.5g Arginine
1g Glutamic Acid
0.433g Histidine
0.4g Myo-lnositol
5.2g lsoleucine
0.9g Lysine
1.5g Methionine
0.8g Phenylalanine
1.1g Serine
1.2g Threonine
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0.8g Tryptophan
0.2g Tyrosine
1.2g Valine
When needed:
0.263g Leucine
0.2g Uracil
Co-transformants were selected and established as liquid cultures in YPD media
under standard
conditions.
Example 3: Production of Synthetic HexA and HexE3 Genes
Synthetic hexaonoate synthase subunit genes were designed for use in Candida
tropicalis. This
organism uses an alternate genetic code in which the codon "CTG" encodes
serine instead of
leucine. Therefore, all "CTG" codons were replaced with the codon "TTG" to
ensure that these
genes, when translated by C. tropicalis, would generate polypeptides with
amino acid sequences
identical to the wild type polypeptides found in A. parasiticus. Due to the
large size of each
subunit, each was synthesized as four fragments, and each fragment was
inserted into the vector
pUC57. PCR was used to clone each fragment, and overlap extension PCR was then
used to
generate each full length gene.
The sequence of the synthetic gene for each hexanoate synthase subunit is set
forth below. The
synthetic gene encoding the hexA subunit is referred to as hexA-AGC
("Alternate Genetic Code")
and the synthetic gene encoding the hexB subunit is referred to as hexB-AGC.
HEXA-AGC for Candida Tropicalis SEQ ID NO: 35
ATGGTCATCCAAGGGAAGAGATTGGCCGCCTCCTCTATTCAGCTTCTCGCAAGCTC
GTTAGACGCGAAGAAGCTTTGTTATGAGTATGACGAGAGGCAAGCCCCAGGTGTAA
CCCAAATCACCGAGGAGGCGCCTACAGAGCAACCGCCTCTCTCTACCCCTCCCTCG
CTACCCCAAACGCCCAATATTTCGCCTATAAGTGCTTCAAAGATCGTGATCGACGA
TGTGGCGCTATCTCGAGTGCAAATTGTTCAGGCTCTTGTTGCCAGAAAGTTGAAGA
CGGCAATTGCTCAGCTTCCTACATCAAAGTCAATCAAAGAGTTGTCGGGTGGTCGG
TCTTCTTTGCAGAACGAGCTCGTGGGGGATATACACAACGAGTTCAGCTCCATCCC
GGATGCACCAGAGCAGATCTTGTTGCGGGACTTTGGCGACGCCAACCCAACAGTGC
AATTGGGGAAAACGTCCTCCGCGGCAGTTGCCAAACTAATCTCGTCCAAGATGCCT
AGTGACTTCAACGCCAACGCTATTCGAGCCCACCTAGCAAACAAGTGGGGTCTAGG
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ACCCTTGCGACAAACAGCGGTGTTGCTCTACGCCATTGCGTCAGAACCCCCATCGC
GTTTAGCTTCATCGAGCGCAGCGGAAGAGTACTGGGACAACGTGTCATCCATGTAC
GCCGAATCGTGTGGCATCACCCTCCGCCCGAGACAAGACACTATGAATGAAGATGC
TATGGCATCGTCGGCGATTGATCCGGCTGTGGTAGCCGAGTTTTCCAAGGGGCACC
GTAGGCTCGGAGTTCAACAGTTCCAAGCGCTAGCAGAATACTTACAAATTGATTTG
TCGGGGTCTCAAGCCTCTCAGTCGGATGCTTTGGTGGCGGAACTTCAGCAGAAAGT
CGATCTCTGGACGGCCGAAATGACCCCCGAGTTTCTCGCCGGGATATCACCAATGT
TGGATGTAAAGAAGTCGCGACGCTATGGCTCGTGGTGGAACATGGCACGGCAGGAT
GTCTTGGCCTTCTATCGCCGTCCTTCCTACAGTGAATTCGTGGACGACGCCTTGGC
CTTCAAAGTTTTTCTCAATCGTCTCTGTAACCGAGCTGATGAGGCCCTCCTCAACA
TGGTACGCAGTCTTTCCTGTGACGCCTACTTCAAGCAAGGTTCTTTGCCCGGATAT
CATGCCGCCTCGCGACTCCTTGAGCAGGCCATCACATCCACAGTGGCGGATTGCCC
GAAGGCACGCCTCATTCTCCCGGCGGTGGGCCCCCACACCACCATTACAAAGGACG
GCACGATTGAATACGCGGAGGCACCGCGCCAGGGAGTGAGTGGTCCCACTGCGTAC
ATCCAGTCTCTCCGCCAAGGCGCATCTTTCATTGGTCTCAAGTCAGCCGACGTCGA
TACTCAGAGCAACTTGACCGACGCTTTGCTTGACGCCATGTGCTTAGCACTCCATA
ATGGAATCTCGTTTGTTGGTAAAACCTTTTTGGTGACGGGAGCGGGTCAGGGGTCA
ATAGGAGCGGGAGTGGTGCGTCTATTGTTAGAGGGAGGAGCCCGAGTATTGGTGAC
GACGAGCAGGGAGCCGGCGACGACATCCAGATACTTCCAGCAGATGTACGATAATC
ACGGTGCGAAGTTCTCCGAGTTGCGGGTAGTTCCTTGCAATCTAGCCAGCGCCCAA
GATTGCGAAGGGTTGATCCGGCACGTCTACGATCCCCGTGGGCTAAATTGGGATTT
GGATGCCATCCTTCCCTTCGCTGCCGCGTCCGACTACAGCACCGAGATGCATGACA
TTCGGGGACAGAGCGAGTTGGGCCACCGGCTAATGTTGGTCAATGTCTTCCGCGTG
TTGGGGCATATCGTCCACTGTAAACGAGATGCCGGGGTTGACTGCCATCCGACGCA
GGTGTTGTTGCCATTGTCGCCAAATCACGGCATCTTCGGTGGCGATGGGATGTATC
CGGAGTCAAAGCTAGCCCTTGAGAGCTTGTTCCATCGCATCCGATCAGAGTCTTGG
TCAGACCAGTTATCTATATGCGGCGTTCGTATCGGTTGGACCCGGTCGACCGGTCT
AATGACGGCGCATGATATCATAGCCGAAACGGTCGAGGAACACGGAATACGCACAT
TTTCCGTGGCCGAGATGGCACTCAACATAGCCATGTTGTTAACCCCCGACTTTGTG
GCCCATTGTGAAGATGGACCTTTGGATGCCGATTTCACCGGCAGCTTGGGAACATT
GGGTAGCATCCCCGGTTTCCTAGCCCAATTGCACCAGAAAGTCCAGTTGGCAGCCG
AGGTGATCCGTGCCGTGCAGGCCGAGGATGAGCATGAGAGATTCTTGTCTCCGGGA
ACAAAACCTACCTTGCAAGCACCCGTGGCCCCAATGCACCCCCGCAGTAGCCTTCG
TGTAGGCTATCCCCGTCTCCCCGATTATGAGCAAGAGATTCGCCCGTTGTCCCCAC
GGTTGGAAAGGTTGCAAGATCCGGCCAATGCTGTGGTGGTGGTCGGGTACTCGGAG
TTGGGGCCATGGGGTAGCGCGCGATTACGGTGGGAAATAGAGAGCCAGGGCCAGTG
GACTTCAGCCGGTTATGTCGAACTTGCCTGGTTGATGAACCTCATCCGCCACGTCA
ACGATGAATCCTACGTCGGCTGGGTGGATACTCAGACCGGAAAGCCAGTGCGGGAT
GGCGAGATCCAGGCATTGTACGGGGACCACATTGACAACCACACCGGTATCCGTCC
TATCCAGTCCACCTCGTACAACCCAGAGCGCATGGAGGTCTTGCAGGAGGTCGCTG
TCGAGGAGGATTTGCCCGAGTTTGAAGTATCTCAACTTACCGCCGACGCCATGCGT
CTCCGCCATGGAGCTAACGTTTCCATCCGCCCCAGTGGAAATCCCGACGCATGCCA
CGTGAAGCTTAAACGAGGCGCTGTTATCCTTGTTCCCAAGACAGTTCCCTTTGTTT
GGGGATCGTGTGCCGGTGAGTTGCCGAAGGGATGGACTCCAGCCAAGTACGGCATC
CCTGAGAACCTAATTCATCAGGTCGACCCCGTCACGCTCTATACAATTTGCTGCGT
GGCGGAGGCATTTTACAGTGCCGGTATAACTCACCCTCTTGAGGTCTTTCGACACA
TTCACCTCTCGGAACTAGGCAACTTTATCGGATCCTCCATGGGTGGGCCGACGAAG
ACTCGTCAGCTCTACCGAGATGTCTACTTCGACCATGAGATTCCGTCGGATGTTTT
GCAAGACACTTATCTCAACACACCTGCTGCCTGGGTTAATATGCTACTCCTTGGCT
GCACGGGGCCGATCAAAACTCCCGTCGGCGCATGTGCCACCGGGGTCGAGTCGATC
GATTCCGGCTACGAGTCAATCATGGCGGGCAAGACAAAGATGTGTCTTGTGGGTGG
CTACGACGATTTGCAGGAGGAGGCATCGTATGGATTCGCACAACTTAAGGCCACGG
TCAACGTTGAAGAGGAGATCGCCTGCGGTCGACAGCCCTCGGAGATGTCGCGCCCC
ATGGCTGAGAGTCGTGCTGGCTTTGTCGAGGCGCATGGCTGCGGTGTACAGTTGTT
GTGTCGAGGTGACATCGCCTTGCAAATGGGTCTTCCTATCTATGCGGTCATTGCCA
GCTCAGCCATGGCCGCCGACAAGATCGGTTCCTCGGTGCCAGCACCGGGCCAGGGC
ATTCTAAGCTTCTCCCGTGAGCGCGCTCGATCCAGTATGATATCCGTCACGTCGCG
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CCCGAGTAGCCGTAGCAGCACATCATCTGAAGTCTCGGACAAATCATCCTTGACCT
CAATCACCTCAATCAGCAATCCCGCTCCTCGTGCACAACGCGCCCGATCCACCACT
GATATGGCTCCGTTGCGAGCAGCGCTTGCGACTTGGGGGTTGACTATCGACGACTT
GGATGTGGCCTCATTGCACGGCACCTCGACGCGCGGTAACGATCTCAATGAGCCCG
AGGTGATCGAGACGCAGATGCGCCATTTAGGTCGCACTCCTGGCCGCCCCTTGTGG
GCCATCTGCCAAAAGTCAGTGACGGGACACCCTAAAGCCCCAGCGGCCGCATGGAT
GCTCAATGGATGCTTGCAAGTATTGGACTCGGGGTTGGTGCCGGGCAACCGCAATC
TTGACACGTTGGACGAGGCCTTGCGCAGCGCGTCTCATCTCTGCTTCCCTACGCGC
ACCGTGCAGCTACGTGAGGTCAAGGCATTCTTGTTGACCTCATTTGGCTTCGGACA
GAAGGGGGGCCAAGTCGTCGGCGTTGCCCCCAAGTACTTCTTTGCTACGCTCCCCC
GCCCCGAGGTTGAGGGCTACTATCGCAAGGTGAGGGTTCGAACCGAGGCGGGTGAT
CGCGCCTACGCCGCGGCGGTCATGTCGCAGGCGGTGGTGAAGATCCAGACGCAAAA
CCCGTACGACGAGCCGGATGCCCCCCGCATTTTTCTCGATCCCTTGGCACGTATCT
CCCAGGATCCGTCGACGGGCCAGTATCGGTTTCGTTCCGATGCCACTCCCGCCCTC
GATGATGATGCTTTGCCACCTCCCGGCGAACCCACCGAGCTAGTGAAGGGCATCTC
CTCCGCCTGGATCGAGGAGAAGGTGCGACCGCATATGTCTCCCGGCGGCACGGTGG
GCGTGGACTTGGTTCCTCTCGCCTCCTTCGACGCATACAAGAATGCCATCTTTGTT
GAGCGCAATTATACGGTAAGGGAGCGCGATTGGGCTGAAAAGAGTGCGGATGTGCG
CGCGGCCTATGCCAGTCGGTGGTGTGCAAAAGAGGCGGTGTTCAAATGTCTCCAGA
CACATTCACAGGGCGCGGGGGCAGCCATGAAAGAGATTGAGATCGAGCATGGAGGT
AACGGCGCACCGAAAGTCAAGCTCCGGGGTGCTGCGCAAACAGCGGCGCGGCAACG
AGGATTGGAAGGAGTGCAATTGAGCATCAGCTATGGCGACGATGCGGTGATAGCGG
TGGCGTTGGGGTTGATGTCTGGTGCTTCATAA
HEXB-AGC for Candida Tropicalis SEQ ID NO: 36
ATGGGTTCCGTTAGTAGGGAACATGAGTCAATCCCCATCCAGGCCGCCCAGAGAGG
CGCTGCCCGGATCTGCGCTGCTTTTGGAGGTCAAGGGTCTAACAATTTGGACGTGT
TAAAAGGTCTATTGGAGTTATACAAGCGGTATGGCCCAGATTTGGATGAGCTACTA
GACGTGGCATCCAACACGCTTTCGCAGTTGGCATCTTCCCCTGCTGCAATAGACGT
CCACGAACCCTGGGGTTTCGACCTCCGACAATGGTTGACCACACCGGAGGTTGCTC
CTAGCAAAGAAATTCTTGCCTTGCCACCACGAAGCTTTCCCTTAAATACGTTACTT
AGCTTGGCGCTCTATTGTGCAACTTGTCGAGAGCTTGAACTTGATCCTGGGCAATT
TCGATCCCTCCTTCATAGTTCCACGGGGCATTCCCAAGGCATATTGGCGGCGGTGG
CCATCACCCAAGCCGAGAGCTGGCCAACCTTTTATGACGCCTGCAGGACGGTGCTC
CAGATCTCTTTCTGGATTGGACTCGAGGCTTACCTCTTCACTCCATCCTCCGCCGC
CTCGGATGCCATGATCCAAGATTGCATCGAACATGGCGAGGGCCTTCTTTCCTCAA
TGCTAAGTGTCTCCGGGCTCTCCCGCTCCCAAGTTGAGCGAGTAATTGAGCACGTC
AATAAAGGGCTCGGAGAATGCAACCGATGGGTTCACTTGGCCTTGGTTAACTCCCA
CGAAAAGTTCGTCTTAGCGGGACCACCTCAATCCTTATGGGCCGTTTGTCTTCATG
TCCGACGGATCAGAGCAGACAATGACCTCGACCAGTCGCGTATCTTGTTCCGCAAC
CGAAAGCCTATAGTGGATATATTATTTCTTCCCATATCCGCACCATTTCACACACC
GTACTTGGACGGTGTTCAAGATCGCGTTATCGAGGCTTTGAGCTCTGCTTCGTTGG
CTCTCCATTCCATCAAAATCCCCCTCTATCACACGGGCACTGGGAGCAACCTACAA
GAACTACAACCACATCAGCTAATCCCGACTCTTATCCGCGCCATTACCGTGGACCA
ATTGGACTGGCCGTTGGTTTGCCGGGGCTTGAACGCAACGCACGTGTTGGACTTTG
GACCTGGACAAACATGCAGTCTTATTCAGGAGCTCACACAAGGAACAGGTGTATCA
GTGATCCAGTTGACTACTCAATCGGGACCAAAACCCGTTGGAGGCCATTTGGCGGC
AGTGAACTGGGAGGCCGAGTTTGGCTTACGACTTCATGCCAATGTCCACGGTGCAG
CTAAATTGCACAACCGTATGACAACATTGCTTGGGAAGCCTCCTGTGATGGTAGCC
GGAATGACACCTACTACGGTGCGCTGGGACTTTGTCGCTGCCGTTGCTCAAGCTGG
ATACCACGTCGAATTGGCTGGTGGTGGCTACCACGCAGAGCGCCAGTTCGAGGCCG
AGATTCGGCGCTTGGCAACTGCCATCCCAGCAGATCATGGCATCACCTGCAATCTC
CTCTACGCCAAGCCTACGACTTTTTCCTGGCAGATCTCTGTCATCAAGGATTTGGT
GCGCCAGGGAGTTCCCGTGGAAGGAATCACCATCGGCGCCGGCATCCCTTCTCCGG
AGGTCGTCCAAGAATGTGTACAGTCCATCGGACTCAAGCACATCTCATTCAAGCCT
GGGTCTTTCGAAGCCATTCACCAAGTCATACAGATCGCGCGTACCCATCCTAACTT
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TTTGATCGGGTTGCAATGGACCGCAGGACGAGGGGGAGGACATCATTCCTGGGAAG
ACTTCCATGGACCTATTTTGGCAACCTACGCTCAAATCCGATCATGTCCGAATATT
CTCCTCGTTGTAGGTAGTGGATTCGGTGGAGGCCCGGACACGTTTCCCTACCTCAC
GGGCCAATGGGCCCAGGCCTTTGGCTATCCATGCATGCCCTTCGACGGAGTGTTGC
TCGGCAGTCGCATGATGGTGGCTCGGGAAGCCCATACGTCAGCCCAGGCAAAACGC
TTGATTATAGATGCGCAAGGCGTGGGAGATGCAGATTGGCACAAGTCTTTCGATGA
GCCTACCGGCGGCGTAGTGACGGTCAACTCGGAATTCGGTCAACCTATCCACGTTC
TAGCTACTCGCGGAGTGATGTTGTGGAAAGAACTCGACAACCGGGTCTTTTCAATC
AAAGACACTTCTAAGCGCTTAGAATATTTGCGCAACCACCGGCAAGAAATTGTGAG
CCGTCTTAACGCAGACTTTGCCCGTCCCTGGTTTGCCGTTGACGGACACGGACAGA
ATGTGGAGTTGGAGGACATGACCTACCTCGAGGTTCTCCGCCGTTTGTGCGATCTC
ACGTATGTTTCCCACCAGAAGCGATGGGTAGATCCATCATATCGAATATTATTGTT
GGACTTCGTTCATTTGCTTCGAGAACGATTCCAATGCGCTATTGACAACCCCGGCG
AATATCCACTCGACATCATCGTCCGGGTGGAAGAGAGCTTGAAGGATAAAGCATAC
CGCACGCTTTATCCAGAAGATGTCTCTCTTCTAATGCATTTGTTCAGCCGACGTGA
CATCAAGCCCGTACCATTCATCCCCAGGTTGGATGAGCGTTTTGAGACCTGGTTTA
AAAAAGACTCATTGTGGCAATCCGAAGATGTGGAGGCGGTAATTGGACAGGACGTC
CAGCGAATCTTCATCATTCAAGGGCCTATGGCCGTTCAGTACTCAATATCCGACGA
TGAGTCTGTTAAAGACATTTTACACAATATTTGTAATCATTACGTGGAGGCTCTAC
AGGCTGATTCAAGAGAAACTTCTATCGGCGATGTACACTCGATCACGCAAAAACCT
CTCAGCGCGTTTCCTGGGCTCAAAGTGACGACAAATAGGGTCCAAGGGCTCTATAA
GTTCGAGAAAGTAGGAGCAGTCCCCGAAATGGACGTTCTTTTTGAGCATATTGTCG
GATTGTCGAAGTCATGGGCTCGGACATGTTTGATGAGTAAATCGGTCTTTAGGGAC
GGTTCTCGTTTGCATAACCCCATTCGCGCCGCACTCCAGCTCCAGCGCGGCGACAC
CATCGAGGTGCTTTTAACAGCAGACTCGGAAATTCGCAAGATTCGACTTATTTCAC
CCACGGGGGATGGTGGATCCACTTCTAAGGTCGTATTAGAGATAGTCTCTAACGAC
GGACAAAGAGTTTTCGCCACCTTGGCCCCTAACATCCCACTCAGCCCCGAGCCCAG
CGTCGTCTTTTGCTTCAAGGTCGACCAGAAGCCGAATGAGTGGACCCTTGAGGAGG
ATGCGTCTGGCCGGGCAGAGAGGATCAAGGCATTATACATGAGTTTGTGGAACTTG
GGCTTTCCGAACAAGGCCTCTGTTTTGGGTCTTAATTCGCAATTCACGGGAGAAGA
ATTGATGATCACAACGGACAAGATTCGTGATTTCGAAAGGGTATTGCGGCAAACCA
GTCCTCTTCAGTTGCAGTCATGGAACCCCCAAGGATGTGTACCTATCGACTACTGC
GTGGTCATCGCCTGGTCTGCTCTTACCAAGCCTTTGATGGTCTCCTCTTTGAAATG
CGACCTCTTGGATTTGCTCCACAGCGCTATAAGCTTCCACTATGCTCCATCTGTCA
AACCATTGCGGGTGGGCGATATTGTCAAAACCTCATCCCGTATCCTAGCGGTCTCG
GTGAGACCTAGGGGAACTATGTTGACGGTGTCGGCGGACATTCAGCGCCAGGGACA
ACATGTAGTCACTGTCAAATCAGATTTCTTTCTCGGAGGCCCCGTTTTGGCATGTG
AAACCCCTTTCGAACTCACTGAGGAGCCTGAAATGGTTGTCCATGTCGACTCTGAA
GTGCGCCGTGCTATTTTACACAGCCGCAAGTGGCTCATGCGAGAAGATCGCGCGCT
AGATTTGCTAGGGAGGCAGCTCCTCTTCAGATTAAAGAGCGAAAAATTGTTCAGGC
CAGACGGCCAGCTAGCATTGTTACAGGTAACAGGTTCCGTGTTCAGCTACAGCCCC
GATGGGTCAACGACAGCATTCGGTCGCGTATACTTCGAAAGCGAGTCTTGTACAGG
GAACGTGGTGATGGACTTCTTGCACCGCTACGGTGCACCTCGGGCGCAGTTGTTGG
AGTTGCAACATCCCGGGTGGACGGGCACCTCTACTGTGGCAGTAAGAGGTCCTCGA
CGCAGCCAATCCTACGCACGCGTCTCCCTCGATCATAATCCCATCCATGTTTGTCC
GGCCTTTGCGCGATACGCTGGTCTCTCGGGTCCCATTGTCCATGGGATGGAAACCT
CTGCCATGATGCGCAGAATTGCCGAATGGGCCATCGGAGATGCAGACCGGTCTCGG
TTCCGGAGCTGGCATATCACCTTGCAAGCACCCGTCCACCCCAACGACCCTTTGCG
GGTGGAGTTGCAGCATAAGGCCATGGAGGACGGGGAAATGGTTTTGAAAGTACAAG
CATTTAACGAAAGGACGGAAGAACGCGTAGCGGAGGCAGATGCCCATGTTGAGCAG
GAAACTACGGCTTACGTCTTCTGTGGCCAGGGCAGTCAACGACAGGGGATGGGAAT
GGACTTGTACGTCAACTGTCCGGAGGCTAAAGCGTTGTGGGCTCGCGCCGACAAGC
ATTTGTGGGAGAAATATGGGTTCTCCATCTTGCACATTGTGCAAAACAACCCTCCA
GCCCTCACTGTTCACTTTGGCAGCCAGCGAGGGCGCCGTATTCGTGCCAACTATTT
GCGCATGATGGGACAGCCACCGATAGATGGTAGACATCCGCCCATATTGAAGGGAT
TGACGCGGAATTCGACCTCGTACACCTTCTCCTATTCCCAGGGGTTGTTGATGTCC
ACCCAGTTCGCCCAGCCCGCATTGGCGTTGATGGAAATGGCTCAGTTCGAATGGCT
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CAAAGCCCAGGGAGTCGTTCAGAAGGGTGCGCGGTTCGCGGGACATTCGTTGGGAG
AATATGCCGCCCTTGGAGCTTGTGCTTCCTTCCTCTCATTTGAAGATCTCATATCT
CTCATCTTTTATCGGGGCTTGAAGATGCAGAATGCGTTGCCGCGCGATGCCAACGG
CCACACCGACTATGGAATGTTGGCTGCCGATCCATCGCGGATAGGAAAAGGTTTCG
AGGAAGCGAGTTTGAAATGTCTTGTCCATATCATTCAACAGGAGACCGGCTGGTTC
GTGGAAGTCGTCAACTACAACATCAACTCGCAGCAATACGTCTGTGCAGGCCATTT
CCGAGCCCTTTGGATGTTGGGTAAGATATGCGATGACCTTTCATGCCACCCTCAAC
CGGAGACTGTTGAAGGCCAAGAGCTACGGGCCATGGTCTGGAAGCATGTCCCGACG
GTGGAGCAGGTGCCCCGCGAGGATCGCATGGAACGAGGTCGAGCGACCATTCCGTT
GCCGGGGATCGATATCCCATACCATTCGACCATGTTACGAGGGGAGATTGAGCCTT
ATCGTGAATATTTGTCTGAACGTATCAAGGTGGGGGATGTGAAGCCGTGCGAATTG
GTGGGACGCTGGATCCCTAATGTTGTTGGCCAGCCTTTCTCCGTCGATAAGTCTTA
CGTTCAGTTGGTGCACGGCATCACAGGTAGTCCTCGGCTTCATTCCTTGCTTCAAC
AAATGGCGTGA
Example 4: Transformation of C. tropicalis with the Synthetic Hexanoate
Synthase Subunit Genes
Candida tropicalis cells (ATCC number 20962) and cultured under standard
conditions in YPD
medium at 30 degrees Celsius. The synthetic genes encoding hexA and hexB are
amplified using
standard PCR amplification techniques. A linear DNA construct comprising, from
5' to 3', the TEF
(transcription elongation factor) promoter, the hexA-AGC gene, the TEF
promoter, the hexB-AGC
gene, and the URA3 marker. Each end of this construct is designed to contain a
mini-URA-Blaster
for integration of the construct into C. tropicalis genomic DNA (Alani E, Cao
L, Kleckner N. A
method for gene disruption that allows repeated use of URA3 selection in the
construction of
multiply disrupted yeast strains. Genetics. 1987 Aug;116 (4):541-545).
The construct is amplified using standard techniques. Transformation of C.
tropicalis cells with this
linear construct is accomplished using standard electroporation techniques
such as those set forth
in U.S. Patent No. 5,648,247 or 5,204,252. Transformants are selected by
plating and growing the
transformed cells on SC-URA media as described above in which only
transformants will survive.
To remove the URA cassette, the confirmed strain is then replated onto SC
complete media
containing 5-Fluoroorotic Acid (5-F0A) and confirmed for the loss of the URA
cassette.
Example 5: Assay of Cytochrome P450 with Activity on Six Carbon Chains in C.
tropicalis
Cultures of C. tropicalis are cultured in YPD media to late log phase and then
exposed to hexane
exposed to various concentrations of hexane up to about 0.1 percent (v/v)
induce the expression of
the cytochrome p450 gene having activity specific for six carbon substrates.
After about 2 hours
exposure to the hexane solution, cells were harvested and RNA isolated using
techniques
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described above. The specifically induced gene may be detected by Northern
blotting and/or
quantitative RT-PCR.
Cells to be analyzed for cytochrome P450 activity are grown under standard
conditions and
harvested for the production of microsomes. Microsomes were prepared by lysing
cells in Tris-
buffered sucrose (10mM Tris-HCI pH 7.5, 1mM EDTA, 0.25M sucrose). Differential
centrifugation
is performed first at 25,000xg then at 100,000xg to pellet cell debris then
microsomes, respectively.
The microsome pellet is resuspended in 0.1M phosphate buffer (pH 7.5), 1mM
EDTA to a final
concentration of approximately 10mg protein/mL.
A reaction mixture containing approximately 0.3mg microsomes, 0.1mM sodium
hexanoate, 0.7mM
NADPH, 50mM Tris-HCI pH 7.5 in 1mL is initiated by the addition of NADPH and
incubated at
37 C for 10 minutes. The reaction is terminated by addition of 0.25mL 5M HCI
and 0.25mL
2.5ug/mL 10-hydroxydecanoic acid is added as an internal standard (3.3 nmol).
The mixture is
extracted with 4.5mL diethyl ether under NaCI-saturated conditions. The
organic phase is
transferred to a new tube and evaporated to dryness. The residue is dissolved
in acetonitrile
containing 10mM 3-bromomethy1-7-methoxy-1,4-benzoxazin-2-one (BrMB) and 0.1mL
of 15mg/mL
18-crown-6 in acetonitril saturated with K2CO3. The solution is incubated at
40 C for 30 minutes
before addition of 0.05mL 2% acetic acid. The fluorescently labeled omega-
hydroxy fatty acids are
resolved via HPLC with detection at 430nm and excitation at 355nm.
Example 6: Examples of Polynucleotide Regulators
Provided in the tables hereafter are non-limiting examples of regulator
polynucleotides that can be
utilized in embodiments herein. Such polynucleotides may be utilized in native
form or may be
modified for use herein. Examples of regulatory polynucleotides include those
that are regulated
by oxygen levels in a system (e.g., upregulated or downregulated by relatively
high oxygen levels
or relatively low oxygen levels).
Regulated Yeast Promoters ¨ Up-regulated by oxygen
ORF name Gene Relative Relative Ratio
name mRNA level mRNA level
(Aerobic) (Anaerobic)
YPL275W 4389 30 219.5
YPL276W 2368 30 118.4
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ORF name Gene Relative Relative Ratio
name mRNA level mRNA level
(Aerobic) (Anaerobic)
YDR256C CTA1 2076 30 103.8
YHR096C HXT5 1846 30 72.4
YDL218W 1189 30 59.4
YCR010C 1489 30 48.8
YOR161C 599 30 29.9
YPL200W 589 30 29.5
YGR110W 1497 30 27
YNL237W YTP1 505 30 25.2
YBR116C 458 30 22.9
Y0R348C PUT4 451 30 22.6
YBR117C TKL2 418 30 20.9
YLL052C 635 30 20
YNL195C 1578 30 19.4
YPR193C 697 30 15.7
YDL222C 301 30 15
YNL335W 294 30 14.6
YPL036W PMA2 487 30 12.8
YML122C 206 30 10.3
YGRO67C 236 30 10.2
YPR192W 204 30 10.2
YNL014W 828 30 9.8
YFLO61W 256 30 9.1
YNR056C 163 30 8.1
YOR186W 153 30 7.6
YDR222W 196 30 6.5
Y0R338W 240 30 6.3
YPR200C 113 30 5.7
YMR018W 778 30 5.2
Y0R364W 123 30 5.1
YNL234W 93 30 4.7
YNR064C 85 30 4.2
YGR213C RTA1 104 30 4
YCL064C CHA1 80 30 4
YOL154W 302 30 3.9
YPR150W 79 30 3.9
YPR196W MAL63 30 30 3.6
YDR420W HKR1 221 30 3.5
YJL216C 115 30 3.5
YNL270C ALP1 67 30 3.3
YHL016C DUR3 224 30 3.2
YOL131W 230 30 3
YOR077W RTS2 210 30 3
YDR536W STL1 55 30 2.7
YNL150W 78 30 2.6
YHR212C 149 30 2.4
YJL108C 106 30 2.4
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ORF name Gene Relative Relative Ratio
name mRNA level mRNA level
(Aerobic) (Anaerobic)
YGRO69W 49 30 2.4
YDR106W 60 30 2.3
YNR034W SOL1 197 30 2.2
YEL073C 104 30 2.1
YOL141W 81 30 1.8
Regulated Yeast Promoters - Down-regulated by oxygen
Relative Relative
Gene mRNA level mRNA level
ORF name name (Aerobic) (Anaerobic) Ratio
YJR047C ANB1 30 4901 231.1
YMR319C FET4 30 1159 58
YPR194C 30 982 49.1
YIR019C STA1 30 981 22.8
YHL042W 30 608 12
YHR210C 30 552 27.6
YHR079B SAE3 30 401 2.7
YGL162W STO1 30 371 9.6
YHL044W 30 334 16.7
YOL015W 30 320 6.1
YCLX07W 30 292 4.2
YIL013C PDR11 30 266 10.6
YDR046C 30 263 13.2
YBRO4OW FIG1 30 257 12.8
YLR040C 30 234 2.9
Y0R255W 30 231 11.6
YOL014W 30 229 11.4
YAR028W 30 212 7.5
YER089C 30 201 6.2
YFLO12W 30 193 9.7
YDR539W 30 187 3.4
YHL043W 30 179 8.9
YJR162C 30 173 6
YMR165C SMP2 30 147 3.5
YER106W 30 145 7.3
YDR541C 30 140 7
YCRX07W 30 138 3.3
YHR048W 30 137 6.9
YCL021W 30 136 6.8
YOL160W 30 136 6.8
YCRX08W 30 132 6.6
YMR057C 30 109 5.5
YDR540C 30 83 4.2
Y0R378W 30 78 3.9
YBRO85W AAC3 45 1281 28.3
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Relative Relative
Gene mRNA level mRNA level
ORF name name (Aerobic) (Anaerobic) Ratio
YER188W 47 746 15.8
YLL065W GIN11 50 175 3.5
YDL241W 58 645 11.1
YBR238C 59 274 4.6
YCR048W ARE1 60 527 8.7
YOL165C 60 306 5.1
YNR075W 60 251 4.2
YJL213W 60 250 4.2
YPL265W DIPS 61 772 12.7
YDL093W PMTS 62 353 5.7
YKR034W DAL80 63 345 5.4
YKR053C 66 1268 19.3
YJR147W 68 281 4.1
Known and putative DNA binding motifs
Regulator Known Consensus Motif
Abf1 TCRNNNNNNACG
Cbf1 RTCACRTG
Ga14 CGGNNNNNNNNNNNCCG
Gcn4 TGACTCA
Gcr1 CTTCC
Hap2 CCAATNA
Hap3 CCAATNA
Hap4 CCAATNA
Hsf1 GAANNTTCNNGAA
I no2 ATGTGAAA
Mata(A1) TGATGTANNT
Mcm1 CCNNNWWRGG
M ig 1 WWWWSYGGGG
Pho4 CACGTG
Rap1 RMACCCANNCAYY
Reb1 CGGGTRR
Ste 12 TGAAACA
Swi4 CACGAAA
Swi6 CACGAAA
Yap1 TTACTAA
Putative DNA
Binding
Motifs
Regulator Best Motif (scored by E- Best Motif (scored by
value) Hypergeometric)
Abf1 TYCGT--R-ARTGAYA TYCGT--R-ARTGAYA
Ace2 RRRAARARAA-A-RARAA GTGTGTGTGTGTGTG
Ad r1 A-AG-GAGAGAG-GGCAG YTSTYSTT-TTGYTWTT
Arg80 T--CCW-TTTKTTTC GCATGACCATCCACG
Arg81 AAAAARARAAAAR MA GSGAYARMGGAMAAAAA
Aro80 YKYTYTTYTT----KY TRCCGAGRYW-SSSGCGS
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Regulator Known Consensus Motif
Ash1 CGTCCGGCGC CGTCCGGCGC
Azf1 GAAAAAGMAAAAAAA AARWTSGARG-A--CSAA
Bas 1 TTTTYYTTYTTKY-TY-T CS-CCAATGK--CS
Cad 1 CATKYTTTTTTKYTY GCT-ACTAAT
Cbf1 CAC GTGACYA CAC GTGACYA
Cha4 CA---ACACASA-A CAYAMRTGY-C
Cin5 none none
Crz 1 GG-A-A--AR-ARGGC- TSGYGRGASA
Cu p9 TTTKYTKTTY-YTTTKTY K-C-C---SCGCTACKGC
DaI81 WTTKTTTTTYTTTTT-T SR-GGCMCGGC-SSG
Da182 TTKTTTTYTTC TACYACA-CACAWGA
Dig 1 AAA--RAA-GARRAA-AR CCYTG-AYTTCW-CTTC
Dot6 GTG MAK-MG RA-G-G GTG MAK-MG RA-G-G
Fh11 -TTWACAYCCRTACAY-Y -TTWACAYCCRTACAY-Y
Fkh 1 TTT-CTTTKYTT-YTTTT AAW-RTAAAYARG
Fkh2 AAARA-RAAA-AAAR-AA GG-AAWA-GTAAACAA
Fzf1 CACACACACACACACAC SASTKCWCTCKTCGT
Gal4 TTGCTTGAACGSATGCCA TTGCTTGAACGSATGCCA
Ga 14 (Gal) YCTTTTTTTTYTTYYKG CGGM---CW-Y--CCCG
Gat1 none none
Gat3 RRSCCGMCGMGRCGCGCS RGARGTSACGCAKRTTCT
Gcn4 AAA-ARAR-RAAAARRAR TGAGTCAY
Gcr1 GGAAGCTGAAACGYMWRR GGAAGCTGAAACGYMWRR
Gcr2 GGAGAGGCATGATGGGGG AGGTGATGGAGTGCTCAG
GIn3 CT-CCTTTCT GKCTRR-RGGAGA-GM
Grf 10 GAAARRAAAAAAMRMARA -GGGSG-T-SYGT-CGA
Gts1 G-GCCRS--TM AG-AWGTTTTTGWCAAMA
Haa 1 none none
Ha19 TTTTTTYTTTTY-KTTTT KCKSGCAGGCWTTKYTCT
Hap2 YTTCTTTTYT-Y-C-KT- G-CCSART-GC
Hap3 T-SYKCTTTTCYTTY SGCGMGGG--CC-GACCG
Hap4 STT-YTTTY-TTYTYYYY YCT-ATTSG-C-GS
Hap5 YK-TTTWYYTC T-TTSMTT-YTTTCCK-C
Hir1 AAAA-A-AARAR-AG CCACKTKSGSCCT-S
Hir2 WAAAAAAGAAAA-AAAAR CRSGCYWGKGC
Hms1 AAA-GG-ARAM -AARAAGC-GGGCAC-C
Hsf1 TYTTCYAGAA--TTCY TYTTCYAGAA--TTCY
Ime4 CACACACACACACACACA CACACACACACACACACA
I no2 TTTYCACATGC SCKKCGCKSTSSTTYAA
I no4 G--GCATGTGAAAA G--GCATGTGAAAA
Ixr1 GAAAA-AAAAAAAARA-A CTTTTTTTYYTSGCC
Leu3 GAAAAARAARAA-AA GCCGGTMMCGSYC--
Mac1 YTTKT--TTTTTYTYTTT A--TTTTTYTTKYGC
Mal 13 GCAG-GCAGG AAAC-TTTATA-ATACA
Ma133 none none
Mata 1 GCCC-C CAAT-TCT-CK
Mbp1 TTTYTYKTTT-YYTTTTT G-RR-A-ACGCGT-R
Mcm1 TTTCC-AAW-RGGAAA TTTCC-AAW-RGGAAA
Met31 YTTYYTTYTTTTYTYTTC
Met4 MTTTTTYTYTYTTC
Mig 1 TATACA-AGMKRTATATG
Mot3 TMTTT-TY-CTT-TTTWK
Msn 1 KT--TTWTTATTCC-C
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Regulator Known Consensus Motif
Msn2 AC CACC
Msn4 R--AAAA-RA-AARAAAT
Mss11 TTTTTTTTCWCTTTKYC
Ndd 1 TTTY-YTKTTTY-YTTYT
Nrg 1 TTY--TTYTT-YTTTYYY
Pdr1 T-YGTGKRYGT-YG
Phd 1 TTYYYTTTTTYTTTTYTT
Pho4 GAMAAAAAARAAAAR
Put3 CYCGGGAAGCSAMM-CCG
Rap1 GRTGYAYGGRTGY
Rcs 1 KMAARAAAAARAAR
Reb1 RTTACCCGS
Rfx1 AYGRAAAARARAAAARAA
Rg m1 GGAKSCC-TTTY-GMRTA
Rgt1 CCCTCC
Rim101 GCGCCGC
Rim 1 TTTTC-KTTTYTTTTTC
Rme 1 ARAAGMAGAAARRAA
Roxl YTTTTCTTTTY-TTTTT
Rph1 ARRARAAAGG-
Rtg 1 YST-YK-TYTT-CTCCCM
Rtg 3 GARA-AAAAR-RAARAAA
Sfl 1 CY--GGSSA-C
Sfp 1 CACACACACACACAYA
Sip4 CTTYTWTTKTTKTSA
Skn7 YTTYYYTYTTTYTYYTTT
Sko1 none
Smp1 AMAAAAARAARWARA-AA
Sok2 ARAAAARRAAAAAG-RAA
Stb 1 RAARAAAAARCMRSRAAA
Ste 12 TTYTKTYTY-TYYKTTTY
Stp 1 GAAAAMAA-AAAAA-AAA
Stp2 YAA-ARAARAAAAA-AAM
Sum 1 TY-TTTTTTYTTTTT-TK
Swi4 RAARAARAAA-AA-R-AA
Swi5 CACACACACACACACACA
Swi6 RAARRRAAAAA-AAAMAA
Thi2 GCCAGACCTAC
Uga3 GG-GGCT
Yap1 TTYTTYTTYTTTY-YTYT
Yap3 none
Yap5 YKSGCGCGYCKCGKCGGS
Yap6 TTTTYYTTTTYYYYKTT
Yap7 none
Yf1044c TTCTTKTYYTTTT
YjI206c TTYTTTTYTYYTTTYTTT
Zap1 TTGCTTGAACGGATGCCA
Zms1 MG-MCAAAAATAAAAS
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Transcriptional repressors
Associated Description(s)
Gene(s)
WHI5 Repressor of G1 transcription that binds to SCB binding factor (SBF)
at SCB target promoters in early G1; phosphorylation of Whi5p by
the CDK, Cln3p/Cdc28p relieves repression and promoter binding by
Whi5; periodically expressed in G1
TUP1 General repressor of transcription, forms complex with Cyc8p,
involved in the establishment of repressive chromatin structure
through interactions with histones H3 and H4, appears to enhance
expression of some genes
ROX1 Heme-dependent repressor of hypoxic genes; contains an HMG
domain that is responsible for DNA bending activity
SFL1 Transcriptional repressor and activator; involved in repression of
flocculation-related genes, and activation of stress responsive
genes; negatively regulated by cAMP-dependent protein kinase A
subunit Tpk2p
RIM101 Transcriptional repressor involved in response to pH and in cell
wall
construction; required for alkaline pH-stimulated haploid invasive
growth and sporulation; activated by proteolytic processing; similar
to A. nidulans PacC
RDR1 Transcriptional repressor involved in the control of multidrug
resistance; negatively regulates expression of the PDR5 gene;
member of the Gal4p family of zinc cluster proteins
SUM1 Transcriptional repressor required for mitotic repression of middle
sporulation-specific genes; also acts as general replication initiation
factor; involved in telomere maintenance, chromatin silencing;
regulated by pachytene checkpoint
XBP1 Transcriptional repressor that binds to promoter sequences of the
cyclin genes, CYS3, and SMF2; expression is induced by stress or
starvation during mitosis, and late in meiosis; member of the
Swi4p/Mbp1p family; potential Cdc28p substrate
NRG2 Transcriptional repressor that mediates glucose repression and
negatively regulates filamentous growth; has similarity to Nrg1p
NRG1 Transcriptional repressor that recruits the Cyc8p-Tup1p complex to
promoters; mediates glucose repression and negatively regulates a
variety of processes including filamentous growth and alkaline pH
response
CUP9 Homeodomain-containing transcriptional repressor of PTR2, which
encodes a major peptide transporter; imported peptides activate
ubiquitin-dependent proteolysis, resulting in degradation of Cup9p
and de-repression of PTR2 transcription
YOX1 Homeodomain-containing transcriptional repressor, binds to Mcm1p
and to early cell cycle boxes (ECBs) in the promoters of cell cycle-
regulated genes expressed in M/G1 phase; expression is cell cycle-
regulated; potential Cdc28p substrate
RFX1 Major transcriptional repressor of DNA-damage-regulated genes,
recruits repressors Tup1p and Cyc8p to their promoters; involved in
DNA damage and replication checkpoint pathway; similar to a family
of mammalian DNA binding RFX1-4 proteins
MIG3 Probable transcriptional repressor involved in response to toxic
agents such as hydroxyurea that inhibit ribonucleotide reductase;
phosphorylation by Snf1p or the Mecip pathway inactivates Mig3p,
allowing induction of damage response genes
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Associated Description(s)
Gene(s)
RGM1 Putative transcriptional repressor with proline-rich zinc fingers;
overproduction impairs cell growth
YHP1 One of two homeobox transcriptional repressors (see also Yoxl p),
that bind to Mcmlp and to early cell cycle box (ECB) elements of
cell cycle regulated genes, thereby restricting ECB-mediated
transcription to the M/G1 interval
HOS4 Subunit of the Set3 complex, which is a meiotic-specific repressor
of
sporulation specific genes that contains deacetylase activity;
potential Cdc28p substrate
CAF20 Phosphoprotein of the mRNA cap-binding complex involved in
translational control, repressor of cap-dependent translation
initiation, competes with elF4G for binding to elF4E
SAP1 Putative ATPase of the AAA family, interacts with the Sinlp
transcriptional repressor in the two-hybrid system
SET3 Defining member of the SET3 histone deacetylase complex which is
a meiosis-specific repressor of sporulation genes; necessary for
efficient transcription by RNAPII; one of two yeast proteins that
contains both SET and PHD domains
RPH1 JmjC domain-containing histone demethylase which can specifically
demethylate H3K36 tri- and dimethyl modification states;
transcriptional repressor of PH R1; Rphlp phosphorylation during
DNA damage is under control of the MEC1-RAD53 pathway
YMR181C Protein of unknown function; mRNA transcribed as part of a
bicistronic transcript with a predicted transcriptional repressor
RGM1/YMR182C; mRNA is destroyed by nonsense-mediated decay
(NMD); YMR181C is not an essential gene
YLR345W Similar to 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase
enzymes responsible for the metabolism of fructoso-2,6-
bisphosphate; mRNA expression is repressed by the Rfx1p-Tupl p-
Ssn6p repressor complex; YLR345W is not an essential gene
MCM1 Transcription factor involved in cell-type-specific transcription
and
pheromone response; plays a central role in the formation of both
repressor and activator complexes
PHR1 DNA photolyase involved in photoreactivation, repairs pyrimidine
dimers in the presence of visible light; induced by DNA damage;
regulated by transcriptional repressor Rphlp
H052 Histone deacetylase required for gene activation via specific
deacetylation of lysines in H3 and H4 histone tails; subunit of the
Set3 complex, a meiotic-specific repressor of sporulation specific
genes that contains deacetylase activity
RGT1 Glucose-responsive transcription factor that regulates expression of
several glucose transporter (HXT) genes in response to glucose;
binds to promoters and acts both as a transcriptional activator and
repressor
SRB7 Subunit of the RNA polymerase 11 mediator complex; associates with
core polymerase subunits to form the RNA polymerase 11
holoenzyme; essential for transcriptional regulation; target of the
global repressor Tupl p
GAL11 Subunit of the RNA polymerase 11 mediator complex; associates with
core polymerase subunits to form the RNA polymerase 11
holoenzyme; affects transcription by acting as target of activators
and repressors
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Transcriptional activators
Associated Description(s)
Gene(s)
SKT5 Activator of Chs3p (chitin synthase 111), recruits Chs3p to the bud
neck
via interaction with Bni4p; has similarity to Shc1p, which activates
Chs3p during sporulation
MSA1 Activator of G1-specific transcription factors, MBF and SBF, that
regulates both the timing of G1-specific gene transcription, and cell
cycle initiation; potential Cdc28p substrate
AMA1 Activator of meiotic anaphase promoting complex (APC/C); Cdc2Op
family member; required for initiation of spore wall assembly; required
for C1b1p degradation during meiosis
STB5 Activator of multidrug resistance genes, forms a heterodimer with
Pdr1p; contains a Zn(I1)2Cys6 zinc finger domain that interacts with a
PDRE (pleotropic drug resistance element) in vitro; binds Sin3p in a
two-hybrid assay
RRD2 Activator of the phosphotyrosyl phosphatase activity of
PP2A,peptidyl-
prolylcis/trans-isomerase; regulates G1 phase progression, the
osmoresponse, microtubule dynamics; subunit of the Tap42p-Pph21p-
Rrd2p complex
BLM10 Proteasome activator subunit; found in association with core
particles,
with and without the 19S regulatory particle; required for resistance to
bleomycin, may be involved in protecting against oxidative damage;
similar to mammalian PA200
SHC1 Sporulation-specific activator of Chs3p (chitin synthase 111),
required for
the synthesis of the chitosan layer of ascospores; has similarity to
Skt5p, which activates Chs3p during vegetative growth;
transcriptionally induced at alkaline pH
NDD1 Transcriptional activator essential for nuclear division; localized
to the
nucleus; essential component of the mechanism that activates the
expression of a set of late-S-phase-specific genes
IMP2' Transcriptional activator involved in maintenance of ion homeostasis
and protection against DNA damage caused by bleomycin and other
oxidants, contains a C-terminal leucine-rich repeat
LYS14 Transcriptional activator involved in regulation of genes of the
lysine
biosynthesis pathway; requires 2-aminoadipate semialdehyde as co-
inducer
MSN1 Transcriptional activator involved in regulation of invertase and
glucoamylase expression, invasive growth and pseudohyphal
differentiation, iron uptake, chromium accumulation, and response to
osmotic stress; localizes to the nucleus
HAA1 Transcriptional activator involved in the transcription of TP02,
YR02,
and other genes putatively encoding membrane stress proteins;
involved in adaptation to weak acid stress
UGA3 Transcriptional activator necessary for gamma-aminobutyrate (GABA)-
dependent induction of GABA genes (such as UGA1, UGA2, UGA4);
zinc-finger transcription factor of the Zn(2)-Cys(6) binuclear cluster
domain type; localized to the nucleus
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Associated Description(s)
Gene(s)
GCR1 Transcriptional activator of genes involved in glycolysis; DNA-
binding
protein that interacts and functions with the transcriptional activator
Gcr2p
GCR2 Transcriptional activator of genes involved in glycolysis; interacts
and
functions with the DNA-binding protein Gcrl p
GAT1 Transcriptional activator of genes involved in nitrogen catabolite
repression; contains a GATA-1-type zinc finger DNA-binding motif;
activity and localization regulated by nitrogen limitation and Ure2p
GLN3 Transcriptional activator of genes regulated by nitrogen catabolite
repression (NCR), localization and activity regulated by quality of
nitrogen source
PUT3 Transcriptional activator of proline utilization genes,
constitutively binds
PUT1 and PUT2 promoter sequences and undergoes a conformational
change to form the active state; has a Zn(2)-Cys(6) binuclear cluster
domain
ARR1 Transcriptional activator of the basic leucine zipper (bZIP) family,
required for transcription of genes involved in resistance to arsenic
compounds
PDR3 Transcriptional activator of the pleiotropic drug resistance
network,
regulates expression of ATP-binding cassette (ABC) transporters
through binding to cis-acting sites known as PDREs (PDR responsive
elements)
MSN4 Transcriptional activator related to Msn2p; activated in stress
conditions, which results in translocation from the cytoplasm to the
nucleus; binds DNA at stress response elements of responsive genes,
inducing gene expression
MSN2 Transcriptional activator related to Msn4p; activated in stress
conditions, which results in translocation from the cytoplasm to the
nucleus; binds DNA at stress response elements of responsive genes,
inducing gene expression
PHD1 Transcriptional activator that enhances pseudohyphal growth;
regulates expression of FLO11, an adhesin required for pseudohyphal
filament formation; similar to StuA, an A. nidulans developmental
regulator; potential Cdc28p substrate
FHL1 Transcriptional activator with similarity to DNA-binding domain of
Drosophila forkhead but unable to bind DNA in vitro; required for rRNA
processing; isolated as a suppressor of splicing factor prp4
VHR1 Transcriptional activator, required for the vitamin H-responsive
element
(VHRE) mediated induction of VHT1 (Vitamin H transporter) and B105
(biotin biosynthesis intermediate transporter) in response to low biotin
concentrations
CDC20 Cell-cycle regulated activator of anaphase-promoting
complex/cyclosome (APC/C), which is required for
metaphase/anaphase transition; directs ubiquitination of mitotic cyclins,
Pdsl p, and other anaphase inhibitors; potential Cdc28p substrate
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Associated Description(s)
Gene(s)
CDH1 Cell-cycle regulated activator of the anaphase-promoting
complex/cyclosome (APC/C), which directs ubiquitination of cyclins
resulting in mitotic exit; targets the APC/C to specific substrates
including Cdc20p, Ase1p, Cin8p and Fin1p
AFT2 Iron-regulated transcriptional activator; activates genes involved
in
intracellular iron use and required for iron homeostasis and resistance
to oxidative stress; similar to Aft1p
MET4 Leucine-zipper transcriptional activator, responsible for the
regulation
of the sulfur amino acid pathway, requires different combinations of the
auxiliary factors Cbf1p, Met28p, Met31p and Met32p
CBS2 Mitochondrial translational activator of the COB mRNA; interacts
with
translating ribosomes, acts on the COB mRNA 5'-untranslated leader
CBS1 Mitochondrial translational activator of the COB mRNA; membrane
protein that interacts with translating ribosomes, acts on the COB
mRNA 5'-untranslated leader
CBP6 Mitochondrial translational activator of the COB mRNA;
phosphorylated
PET111 Mitochondrial translational activator specific for the COX2 mRNA;
located in the mitochondrial inner membrane
PET494 Mitochondrial translational activator specific for the COX3 mRNA,
acts
together with Pet54p and Pet122p; located in the mitochondrial inner
membrane
PET122 Mitochondrial translational activator specific for the COX3 mRNA,
acts
together with Pet54p and Pet494p; located in the mitochondrial inner
membrane
RRD1 Peptidyl-prolyl cis/trans-isomerase, activator of the phosphotyrosyl
phosphatase activity of PP2A; involved in G1 phase progression,
microtubule dynamics, bud morphogenesis and DNA repair; subunit of
the Tap42p-Sit4p-Rrd1p complex
YPR196W Putative maltose activator
POG1 Putative transcriptional activator that promotes recovery from
pheromone induced arrest; inhibits both alpha-factor induced G1 arrest
and repression of CLN1 and CLN2 via SCB/MCB promoter elements;
potential Cdc28p substrate; SBF regulated
MSA2 Putative transcriptional activator, that interacts with G1-specific
transcription factor, MBF and G1-specific promoters; ortholog of
Msa2p, an MBF and SBF activator that regulates G1-specific
transcription and cell cycle initiation
PET309 Specific translational activator for the COX1 mRNA, also influences
stability of intron-containing COX1 primary transcripts; localizes to the
mitochondrial inner membrane; contains seven pentatricopeptide
repeats (PPRs)
TEA1 Ty1 enhancer activator required for full levels of Ty enhancer-
mediated
transcription; C6 zinc cluster DNA-binding protein
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Associated Description(s)
Gene(s)
PIP2 Autoregulatory oleate-specific transcriptional activator of
peroxisome
proliferation, contains Zn(2)-Cys(6) cluster domain, forms heterodimer
with Oaf1p, binds oleate response elements (OREs), activates beta-
oxidation genes
CHA4 DNA binding transcriptional activator, mediates serine/threonine
activation of the catabolic L-serine (L-threonine) deaminase (CHA1);
Zinc-finger protein with Zn[2]-Cys[6] fungal-type binuclear cluster
domain
SFL1 Transcriptional repressor and activator; involved in repression of
flocculation-related genes, and activation of stress responsive genes;
negatively regulated by cAMP-dependent protein kinase A subunit
Tpk2p
RDS2 Zinc cluster transcriptional activator involved in conferring
resistance to
ketoconazole
CAT8 Zinc cluster transcriptional activator necessary for derepression of
a
variety of genes under non-fermentative growth conditions, active after
diauxic shift, binds carbon source responsive elements
AR080 Zinc finger transcriptional activator of the Zn2Cys6 family;
activates
transcription of aromatic amino acid catabolic genes in the presence of
aromatic amino acids
SIP4 C6 zinc cluster transcriptional activator that binds to the carbon
source-
responsive element (CSRE) of gluconeogenic genes; involved in the
positive regulation of gluconeogenesis; regulated by Snf1p protein
kinase; localized to the nucleus
SPT10 Putative histone acetylase, sequence-specific activator of histone
genes, binds specifically and highly cooperatively to pairs of UAS
elements in core histone promoters, functions at or near the TATA box
MET28 Basic leucine zipper (bZIP) transcriptional activator in the Cbf1p-
Met4p-Met28p complex, participates in the regulation of sulfur
metabolism
GCN4 Basic leucine zipper (bZIP) transcriptional activator of amino acid
biosynthetic genes in response to amino acid starvation; expression is
tightly regulated at both the transcriptional and translational levels
CAD1 AP-1-like basic leucine zipper (bZIP) transcriptional activator
involved
in stress responses, iron metabolism, and pleiotropic drug resistance;
controls a set of genes involved in stabilizing proteins; binds
consensus sequence TTACTAA
IN02 Component of the heteromeric Ino2p/Ino4p basic helix-loop-helix
transcription activator that binds inositol/choline-responsive elements
(ICREs), required for derepression of phospholipid biosynthetic genes
in response to inositol depletion
THI2 Zinc finger protein of the Zn(I1)2Cys6 type, probable
transcriptional
activator of thiamine biosynthetic genes
5WI4 DNA binding component of the SBF complex (Swi4p-Swi6p), a
transcriptional activator that in concert with MBF (Mbp1-Swi6p)
regulates late G1-specific transcription of targets including cyclins and
genes required for DNA synthesis and repair
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Associated Description(s)
Gene(s)
HAP5 Subunit of the heme-activated, glucose-repressed Hap2/3/4/5 CCAAT-
binding complex, a transcriptional activator and global regulator of
respiratory gene expression; required for assembly and DNA binding
activity of the complex
HAP3 Subunit of the heme-activated, glucose-repressed Hap2p/3p/4p/5p
CCAAT-binding complex, a transcriptional activator and global
regulator of respiratory gene expression; contains sequences
contributing to both complex assembly and DNA binding
HAP2 Subunit of the heme-activated, glucose-repressed Hap2p/3p/4p/5p
CCAAT-binding complex, a transcriptional activator and global
regulator of respiratory gene expression; contains sequences sufficient
for both complex assembly and DNA binding
HAP4 Subunit of the heme-activated, glucose-repressed Hap2p/3p/4p/5p
CCAAT-binding complex, a transcriptional activator and global
regulator of respiratory gene expression; provides the principal
activation function of the complex
YML037C Putative protein of unknown function with some characteristics of a
transcriptional activator; may be a target of Dbf2p-Mob1p kinase; GFP-
fusion protein co-localizes with clathrin-coated vesicles; YML037C is
not an essential gene
TRA1 Subunit of SAGA and NuA4 histone acetyltransferase complexes;
interacts with acidic activators (e.g., Gal4p) which leads to transcription
activation; similar to human TRRAP, which is a cofactor for c-Myc
mediated oncogenic transformation
YLL054C Putative protein of unknown function with similarity to Pip2p, an
oleate-
specific transcriptional activator of peroxisome proliferation; YLL054C
is not an essential gene
RTG2 Sensor of mitochondrial dysfunction; regulates the subcellular
location
of Rtg1p and Rtg3p, transcriptional activators of the retrograde (RTG)
and TOR pathways; Rtg2p is inhibited by the phosphorylated form of
Mks1p
YBRO12C Dubious open reading frame, unlikely to encode a functional
protein;
expression induced by iron-regulated transcriptional activator Aft2p
JEN1 Lactate transporter, required for uptake of lactate and pyruvate;
phosphorylated; expression is derepressed by transcriptional activator
Cat8p during respiratory growth, and repressed in the presence of
glucose, fructose, and mannose
MRP1 Mitochondrial ribosomal protein of the small subunit; MRP1 exhibits
genetic interactions with PET122, encoding a COX3-specific
translational activator, and with PET123, encoding a small subunit
mitochondrial ribosomal protein
MRP17 Mitochondrial ribosomal protein of the small subunit; MRP17 exhibits
genetic interactions with PET122, encoding a COX3-specific
translational activator
TPI 1 Triose phosphate isomerase, abundant glycolytic enzyme; mRNA half-
life is regulated by iron availability; transcription is controlled by
activators Reb1p, Gcr1p, and Rap1p through binding sites in the 5'
non-coding region
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Associated Description(s)
Gene(s)
PKH3 Protein kinase with similarity to mammalian phosphoinositide-
dependent kinase 1 (PDK1) and yeast Pkh1p and Pkh2p, two
redundant upstream activators of Pkc1p; identified as a multicopy
suppressor of a pkh1 pkh2 double mutant
YGL079W Putative protein of unknown function; green fluorescent protein
(GFP)-
fusion protein localizes to the endosome; identified as a transcriptional
activator in a high-throughput yeast one-hybrid assay
TFB1 Subunit of TFIIH and nucleotide excision repair factor 3 complexes,
required for nucleotide excision repair, target for transcriptional
activators
PET123 Mitochondrial ribosomal protein of the small subunit; PET123
exhibits
genetic interactions with PET122, which encodes a COX3 mRNA-
specific translational activator
MHR1 Protein involved in homologous recombination in mitochondria and in
transcription regulation in nucleus; binds to activation domains of acidic
activators; required for recombination-dependent mtDNA partitioning
MCM1 Transcription factor involved in cell-type-specific transcription
and
pheromone response; plays a central role in the formation of both
repressor and activator complexes
EGD1 Subunit betel of the nascent polypeptide-associated complex (NAC)
involved in protein targeting, associated with cytoplasmic ribosomes;
enhances DNA binding of the Gal4p activator; homolog of human
BTF3b
STE5 Pheromone-response scaffold protein; binds Ste11p, Ste7p, and
Fus3p kinases, forming a MAPK cascade complex that interacts with
the plasma membrane and Ste4p-Ste18p; allosteric activator of Fus3p
that facilitates Ste7p-mediated activation
RGT1 Glucose-responsive transcription factor that regulates expression of
several glucose transporter (HXT) genes in response to glucose; binds
to promoters and acts both as a transcriptional activator and repressor
TYE7 Serine-rich protein that contains a basic-helix-loop-helix (bHLH)
DNA
binding motif; binds E-boxes of glycolytic genes and contributes to their
activation; may function as a transcriptional activator in Ty1-mediated
gene expression
VMA13 Subunit H of the eight-subunit V1 peripheral membrane domain of the
vacuolar H+-ATPase (V-ATPase), an electrogenic proton pump found
throughout the endomembrane system; serves as an activator or a
structural stabilizer of the V-ATPase
GAL11 Subunit of the RNA polymerase II mediator complex; associates with
core polymerase subunits to form the RNA polymerase II holoenzyme;
affects transcription by acting as target of activators and repressors
VAC14 Protein involved in regulated synthesis of PtdIns(3,5)P(2), in
control of
trafficking of some proteins to the vacuole lumen via the MVB, and in
maintenance of vacuole size and acidity; interacts with Fig4p; activator
of Fab1p
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Example 7: Cloning of HEXA and HEXB genes
Aspergillus parasiticus (ATCC 24690) cultures were grown in malt extract broth
media (15 g/L malt
extract broth, Difco) with shaking at 25 C for 3 days. A. parasiticus pellets
were transferred to a
1.5mL tube to provide a volume of pellets equal to approximately 500uL. The
mycelia were frozen
in a dry ice ethanol bath, transferred to a mortar and pestle, and ground into
a fine powder. The
powder was placed in a 1.5mL tube with approximately 500uL 0.7mm Zirconia
beads, and total
RNA was prepared using a Ribopure Plant Kit (Ambion), according to
manufacturer's
recommendations.
First strand synthesis of cDNA was performed with gene-specific primers
oAA0031 (for HEXA) and
oAA0041 (for HEXB) in a reaction containing 0.2uL of gene-specific primer
(10uM), 300ng total
RNA, 1.0uL dNTP (10mM), and sterile water to bring the volume to 13uL. The
total RNA/primer
mixtures were heated at 65 C for 5 minutes then cooled on ice for 5 minutes
before the addition of
4uL 5X First strand buffer, 1uL 0.1M DTT, 1uL H20, and 1uL Superscript III RT
(Invitrogen). First
strand synthesis reactions were incubated at 55 C for 1 hour, followed by
inactivation of the
enzyme at 70 C for 15 minutes and cooling of the reactions to 4 C. The primers
utilized for
isolation of HEXA and HEXB genes were configured to independently amplify the
HEXA and HEXB
genes in fragments, having fragment lengths in the range of between about 1.0
kilobases (kb) to
about 1.6kb, with approximately 200bp of overlapping sequence between the
fragments. The
sequences are shown in the tables below.
Oligonucleotides for cloning of HEXA DNA fragments
HEXA PCR
product
Oligos Sequence
sequence
(bp)
oAA0022 ATGGTCATCCAAGGGAAGAGATTGGCCGCCTCCTCTATTCAGC
1-1149
1149
oAA0023 GTAGGCGTCACAGGAAAGACTGCGTACCA
oAA0024 TATCACCAATGCTGGATGTAAAGAAGTCGCG
941-2270
1330
oAA0025 AATTGGGCTAGGAAACCGGGGATGC
OAA0026 CGGTCTAATGACGGCGCATGATATCATAGCCGAAACGGTCGAG
2067-3016
950
oAA0027 ACTTGGCTGGAGTCCATCCCTTCGGCA
OAA0028 CTGCCCGAGTTTGAAGTATCTCAACTTACCGCCGACGCCATG
2812-4181
1370
oAA0029 TGAGACGCGCTGCGCAGGGC
OAA0030 CGAGGTGATCGAGACGCAGATGC
3975-5016
1042
oAA0031 TTATGAAGCACCAGACATCAGCCCCAGC
gtactagtaaaaaaATGGTCATCCAAGGGAAGAGATTGGCCGCCT
oAA0046
CCTCTATTCAGC 1-5016
5041
oAA0047 gtcccgggctaTTATGAAGCACCAGACATCAGCCCCAGC
tacccgggctattagtgatggtggtgatggtgTGAAGCACCAGAC
oAA0051 1-5016
5062
ATCAGCCCCAGC
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Oligonucleotides for cloning of HEXB DNA fragments
HEXB PCR
product
Oligos Sequence
sequence
(bp)
oAA0032 ATGGGTTCCGTTAGTAGGGAACATGAGTCAATC
1-1166
1166
oAA0033 GTTCCTTGTGTGAGCTCCTGAATAAGACTGCATG
OAA0034 CCATCAAAATCCCCCTCTATCACACGGGCACTGGGAGCAAC
962-2042
1081
oAA0035 CCCACGCCTTGCGCATCTATAATCAGG
oAA0036 TGTCCGAATATTCTCCTCGTTGTAGGTAGTGGATT
1837-3527
1691
oAA0037 GCAGTAGTCGATAGGTACACATCCTTGGGGGTTCCATGACTGC
AGAGGATCAAGGCATTATACATGAGTCTGTGGAACTTGGGCTTTC
oAA0038 c 3323-4461
1139
oAA0039 TTCCCCGTCCTCCATGGCCTTATGC
oAA0040 GGCCTTTGCGCGATACGCTGGTCTCTCGGGTCCCAT
4256-5667
1412
oAA0041 TCACGCCATTTGTTGAAGCAGGGAATG
gtactagtaaaaaaATGGGTTCCGTTAGTAGGGAACATGAGTCAA
oAA0048 TC 1-5667
5694
oAA0049 gtgtttaaacctaTCACGCCATTTGTTGAAGCAGGGAATG
ggtttaaacctatcagtgatggtggtgatggtgCGCCATTTGTTG
1-5667
5714
oAA0111 AAGCAGGGAATGAA
HEXA and HEXB gene fragments were PCR amplified using the cDNA generated above
by the
addition of 5uL 10X Pfu reaction buffer, 1.0uL dNTPs (10mM), 1.0uL Sense and
Antisense Primer
Mix (10uM), 1.0uL Pfu Ultra Fusion HS (Agilent), 2.0uL cDNA, 40uL sterile H20.
Thermocycling
parameters used to amplify the HEXA and HEXB genes were 94 C for 5 minutes, 40
cycles of
94 C 30 seconds, 62 C 40 seconds, 72 C 4 minutes, followed by 72 C 10 minutes
and a 4 C hold.
PCR products of the correct size were gel purified and ligated into pCR-Blunt
II-TOPO (Invitrogen),
transformed into competent TOP10 E. coli cells (Invitrogen) and clones
containing PCR inserts
were sequenced to confirm correct DNA sequence.
DNA fragments of HEXA and HEXB were PCR amplified using the sequence-confirmed
fragments
in pCR-Bluntll as template in order to produce overlapping DNA fragments
covering the entire
sequence of both HEXA and HEXB. The overlapping DNA fragments for each gene
were
combined in a 50uL overlap extension PCR reaction containing each DNA fragment
at 0.2nM,
sense and antisense primers at 0.2uM each, 1X Pfu reaction buffer, 1.0uL Pfu
Ultra Fusion HS
polymerase, and 0.2mM dNTPs. Unique restriction sites were incorporated into
the sense and
antisense primers to allow for cloning the HEXA and HEXB genes into p425GPD
and p426GPD
respectively. For HEXA the restriction sites were Spel / Smal and for HEXB the
restriction sites
were Spel / Pmel. Ligation of the HEXA and HEXB genes into p425GPD and p426GPD
resulted in
plasmids pAA020 and pAA021 respectively. Variants of the HEXA and HEXB genes
that
incorporated C-terminal 6xHis tags were constructed by using an antisense
primer encoding a
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6xHis sequence. Ligation of the HEXA-6xHis and HEXB-6xHis genes into p425GPD
and
p426GPD resulted in plasmids pAA031 and pAA032, respectively. Vectors pAA020,
pAA021,
pAA031 and pAA032 were used to demonstrate protein expression in S.
cerevisiae, as shown in
Figures 11 and 12.
Example 8: Cloning of STCJ and STCK genes
Total RNA was prepared from Aspergillus nidulans (ATCC 38163), as described in
Example 1.
First strand synthesis of cDNA was performed with gene-specific primers
oAA0008 (for STCJ) and
oAA0021 (for STCK) in a reaction containing 0.2uL of gene-specific primer
(10uM), 300ng total
RNA, 1.0uL dNTP (10mM), and sterile water to bring the volume to 13uL. The
total RNA/primer
mixtures were heated at 65 C for 5 minutes then cooled on ice for 5 minutes
before the addition of
4uL 5X First strand buffer, 1uL 0.1M DTT, 1uL H20, and 1uL Superscript III RT
(Invitrogen). First
strand synthesis reactions were incubated at 55 C for 1 hour, followed by
inactivation of the
enzyme at 70 C for 15 minutes and cooling of the reactions to 4 C. Primers
design strategies
substantially similar to those described herein were used to amplify the STCJ
and STCK genes in
fragments in the range of between about 1.1kb to about 1.6kb, with
approximately 200bp of
overlapping sequence between the fragments. The primers used to amplify the
STCJ and STCK
genes are shown in the tables below.
Oligonucleotides for cloning of STCJ DNA fragments
STCJ PCR
product
Oligos Sequence
sequence (bp)
oAA0001 ATGACCCAAAAGACTATACAGCAGGTCCCAAGA
1-1290
1290
oAA0002 TATGGTGCATCGAATGTTGTTTGCCTGG
oAA0009 AAAATGCGTGAGCACTTTGTCCAGCGC
1021-2506 1486
oAA0004 CGACGTAATTGACGTTGTCAACATGCCG
OAA0005 CATCTCGGGTTCCCATCACTCCCTGAGTATGAC
2284-3424 1141
oAA0006 GACAAAGAAGCTGGACACCGCAGCCTTGGGATTCCACGAAC
oAA0007 GATCTGCCTTGTCGGTGGCTATGACGACCTTCAGCCTGAGGAGTCA
TTAACGGATGATAGAGGCCAACGGCCAAAGACACCACTTGCGTACA 3234-4680 1447
oAA0008 C
cacacaactagtaaaaaaATGACCCAAAAGACTATACAGCAGGTCC
oAA0126 CAAGA
1-4680
4710
tgtgtgcccgggTTAACGGATGATAGAGGCCAACGGCCAAAGACAC
OAA0127 CACTTGCGTACAC
tacccgggctattagtgatggtggtgatggtgACGGATGATAGAGG
1-4680
4730
oAA0154 CCAAC
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Oligonucleotides for cloning of STCK DNA fragments
STCK PCR
product
Oligos Sequence
sequence (bp)
oAA0012 ATGACTCCATCACCGTTTCTCGATGCTGT
CACATGGGTAGCATCGTTCATTGCCCAACACAAAGCGGGCCAGTTA 1-1110
1110
oAA0013 ACTC
oAA0014 GTCGAGCTAAGAGTGACTGATGCCATTGGC
901-2510 1610
oAA0015 CGTAATTCAGCTTCTGAACCTGAGCCCAGG
OAA0016 CTTTGCCCGGCCGTGGTTCGC
2301-3555 1255
oAA0017 CCCCCAAGCTCGACAACGGGC
oAA0018 TTCTCAAAATGCACCGGACTGATTACTTGGA
3350-4682 1333
oAA0019 CCCATTCCTCTCTCCTGCGTGCCCTGGCCGGTAAAGACGTAT
CCCTCCTTCGATGGACTTGTCCGGGCAAACGACCGGTTGCGAATGG
oAA0020 AGAT 4477-
5745 1268
oAA0021 CTACCTATTCTCTTCAACCCGCCGTAACAGC
cacacaactagtaaaaaaATGACTCCATCACCGTTTCTCGATGCTG
oAA0128 T 1-5745
5775
oAA0129 tgtgtgcccgggCTACCTATTCTCTTCAACCCGCCGTAACAGC
tgtgtgcccgggctatcagtgatggtggtgatggtgCCTATTCTCT
1-5745
5796
oAA0170 TCAAC
STCJ and STCK fragments were amplified using the cDNA prepared above in PCR
reactions
containing 5uL 10X Pfu reaction buffer, 1.0uL dNTPs (10mM), 1.0uL Sense and
Antisense Primer
Mix (10uM), 1.0uL Pfu Ultra Fusion HS (Agilent), 2.0uL cDNA, 40uL sterile H20.
Thermocycling
parameters used were 94 C for 5 minutes, 40 cycles of 94 C 30 seconds, 62 C 40
seconds, 72 C
4 minutes, followed by 72 C 10 minutes and a 4 C hold. PCR products of the
correct size were gel
purified and ligated into pCR-Blunt II-TOPO (Invitrogen), transformed into
competent TOP10 E. coli
cells (Invitrogen). PCR inserts were sequenced to confirm the correct DNA
sequence. DNA
fragments of STCJ and STCK were PCR amplified using the sequence-confirmed
fragments in
pCR-Bluntll as template in order to produce overlapping DNA fragments covering
the entire
sequence of both STCJ and STCK. The overlapping DNA fragments for each gene
were
combined in a 50uL overlap extension PCR reaction containing each DNA fragment
at 0.2nM,
sense and antisense primers at 0.2uM each, 1X Pfu reaction buffer, 1.0uL Pfu
Ultra Fusion HS
polymerase, and 0.2mM dNTPs.
Sense and antisense primers were designed to incorporate unique restriction
sites for cloning the
STCJ and STCK genes into p425GPD and p426GPD respectively. For STCJ the
restriction sites
were Spel / Xmal and for STCK the restriction sites were Spel / Smal. Ligation
of the STCJ and
STCK genes into p425GPD and p426GPD resulted in plasmids pAA040 and pAA042
respectively.
Variants of the STCJ and STCK genes that incorporated C-terminal 6xHis tags
were constructed
by using an antisense primer encoding a 6xHis sequence. Ligation of the STCJ-
6xHis and STCK-
6xHis genes into p425GPD and p426GPD resulted in plasmids pAA041 and pAA043.
Vectors
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pAA040, pAA0421 pAA042 and pAA043 were used to demonstrate protein expression
in S.
cerevisiae, as shown in Figures 11 and 12.
Example 9: Design and cloning of HEXA and HEXB genes for C. tropicalis
alternate genetic code
The HEXA and HEXB genes contain multiple CTG codons, which normally code for
leucine.
However, certain organisms, Candida tropicalis for example, translate CTG as
serine. DNA
sequences for HEXA and HEXB were prepared that replaced all CTG codons with
TTG codons,
which is translated as leucine in C. tropicalis. The TTG codon was chosen due
to it being the most
frequently used leucine codon in C. tropicalis. The alternate genetic code
(AGC) HEXA and HEXB
genes were synthesized as equal size fragments with 200bp overlaps and ligated
into pUC57
vector (Integrated DNA Technologies). DNA fragments of AGC-HEXA and AGC-HEXB
were PCR
amplified using the fragments in pUC57 as template in order to produce
overlapping DNA
fragments covering the entire sequence of both AGC-HEXA and AGC-HEXB.
The overlapping DNA fragments for each gene were combined in a 50uL overlap
extension PCR
reaction containing each DNA fragment at 0.2nM, sense and antisense primers at
0.2uM each, 1X
Pfu reaction buffer, 1.0uL Pfu Ultra Fusion HS polymerase, and 0.2mM dNTPs.
Sense and
antisense primers incorporated unique Sapl restriction sites for cloning the
AGC-HEXA and AGC-
HEXB genes into pAA105 resulting in plasmids pAA127 and pAA129 respectively.
Gene variants
of AGC-HEXA and AGC-HEXB that contained C-terminal 6xHis tags were ligated
into pAA105
resulting in plasmids pAA128 and pAA130 respectively. The alternate genetic
code primers used
to alter leucine codons for C. tropicalis expression of HEXA and HEXB are
shown in the tables
below.
Oligonucleotides for cloning of AGC-HEXA DNA fragments
AGC-
PCR product
Oligos Sequence HEXA
(bp)
sequence
oAA0383 cacacagctcttctagaATGGTCATCCAAGGGAAGAG
1-1404
1421
oAA0055 AGTATCGACGTCGGCTGACTTGAGACCA
OAA0056 CCATCACATCCACAGTGGCGG
1205-2609
1405
oAA0057 AACCAGGCAAGTTCGACATAACCGGC
oAA0058 GTAGGCTATCCCCGTCTCCCCGATTATG
2410-3814
1405
oAA0059 TGATTGAGGTCAAGGATGATTTGTCCGAGA
oAA0060 TCTTCCTATCTATGCGGTCATTGCCAGCT
3615-5016
1419
oAA0384 cacacagctcttcctttTTATGAAGCACCAGACATCAAC
cacacagctcttcctttttagtgatggtggtgatggtgTGAAGCAC
oAA0385 1-5016 5071
CAGACATCAACCCCAACG
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Oligonucleotides for cloning of AGC-HEXB DNA fragments
AGC-
PCR product
Oligos Sequence HEXB
(bp)
sequence
oAA0386 cacacagctcttctagaATGGGTTCCGTTAGTAGGGA
1-1566
1583
oAA0064 CAAATCCTTGATGACAGAGATCTGCCAGGA
oAA0065 GCTGGGACTTTGTCGCTGCCGTTGCTCAAGCTGGAT
1367-2933
1567
oAA0066 ACTGCTCCTACTTTCTCGAACTTATAGAGCCCTTG
oAA0067 ATATCCGACGATGAGTCTGT
2734-4299
1566
oAA0068 ATGGACAATGGGACCCGAGA
oAA0069 GGACTTCTTGCACCGCTACG
4101-5667
1584
oAA0387 cacacagctcttcctttTCACGCCATTTGTTGAAGCAAAG
cacacagctcttccttttcagtgatggtggtgatggtgCGCCATTT
1-5667
5692
oAA0388 GTTGAAGCA
Example 10: Transformation of S. cerevisiae procedure
Competent cells of S. cerevisiae strain BY4742 were prepared using the Frozen-
EZ Yeast
Transformation II Kit (Zymo Research) following manufacturer's instructions.
50uL aliquots of
competent cells were stored at -80 C until use. Competent cells were
transformed by the addition
of 0.5-1.0ug of intact plasmid DNA as instructed by the Frozen-EZ Yeast
Transformation II Kit
(Zymo Research). Selection for transformants was performed by plating on
selective media; SC-
URA (for p426-based vectors) or SC-LEU (for p425-based vectors).
Example 11: Transformation of C. tropicalis procedure
5mL YPD start cultures were inoculated with a single colony of C. tropicalis
and incubated
overnight at 30 C, with shaking at about 200rpm. The following day, fresh 25mL
YPD cultures,
containing 0.05% Antifoam B, were inoculated to an initial OD600nm of 0.4 and
the culture incubated
at 30 C, with shaking at about 200rpm until an OD600nm of 1.0-2.0 was reached.
Cells were
pelleted by centrifugation at 1,000 x g, 4 C for 10 minutes. Cells were washed
by resuspending in
10mL sterile water, pelleted, resuspended in 1mL sterile water and transferred
to a 1.5mL
microcentrifuge tube. The cells were then washed in 1mL sterile TE/LiOAC
solution, pH 7.5,
pelleted, resuspended in 0.25mL TE/LiOAC solution and incubated with shaking
at 30 C for 30
minutes.
The cell solution was divided into 50uL aliquots in 1.5mL tubes to which was
added 5-8ug of
linearized DNA and 5uL of carrier DNA (boiled and cooled salmon sperm DNA,
10mg/mL). 300uL
of sterile PEG solution (40% PEG 3500, 1X TE, 1X LiOAC) was added, mixed
thoroughly and
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incubated at 30 C for 60 minutes with gentle mixing every 15 minutes. 40uL of
DMSO was added,
mixed thoroughly and the cell solution was incubated at 42 C for 15 minutes.
Cells were then
pelleted by centrifugation at 1,000 x g 30 seconds, resuspended in 500uL of
YPD media and
incubated at 30 C with shaking at about 200rpm for 2 hours. Cells were then
pelleted by
centrifugation and resuspended in 1mL 1X TE, cells were pelleted again,
resuspended in 0.2mL 1X
TE and plated on selective media. Plates were incubated at 30 C for growth of
transformants.
Example 12: HEXA and HEXB expression in S. cerevisiae
Plasmids pAA031 and pAA032 were transformed into competent BY4742 S.
cerevisiae cells
independently and in combination. Selection for transformants containing
pAA031 was performed
on SC-LEU plates. Selection for transformants containing pAA032 was performed
on SC-URA
plates. Selection for transformants containing both pAA031 and pAA032 was
performed on SC-
URA-LEU plates. Single colonies were used to inoculate 5mL of SC drop out
media and grown
overnight at 30 C, with shaking as described herein. Cells from 3mL of
overnight culture were
harvested by centrifugation at 12,000rpm for 2 minutes. Cell pellets were
incubated at -80 C until
frozen.
Approximately 500uL of cold 0.7mm zirconia beads (Ambion) were added on top of
the frozen cell
pellets. Yeast lysis buffer (50mM Tris pH 8.0, 0.1% Triton X100, 0.5mM EDTA,
1X ProCEASE
protease inhibitors [G Biosciences]) was added to fill the tube leaving as
little air in the tube as
possible, the tubes were placed on ice during manipulations. Cells were broken
using three, 2
minute cycles in a Bead Beater (BioSpec) with 1 minute rests on ice between
cycles. 200uL of
whole cell extract (WCE) was removed to a new tube and the remainder of the
whole cell extract
was centrifuged at 16,000 x g, 4 C for 15 minutes to pellet insoluble debris.
The supernatant was
removed to a new tube as the soluble cell extract (SCE). The protein content
in the soluble cell
extract was determined by Bradford assay (Pierce). A volume of SCE containing
5Oug of protein
(and the same volume WCE) was precipitated by the addition of 4 volumes of
cold 100`)/0 acetone.
After centrifugation at 16,000 x g, and 4 C for 15 minutes, the supernatant
was carefully removed
and the pellet washed with 200uL of cold 80% acetone and centrifuged again.
The supernatant
again was carefully removed and the cell pellets air dried for 5 minutes.
Protein pellets were then resuspended in 1X LDS sample buffer containing 50mM
DTT (Invitrogen)
by incubating at 70 C, with shaking at about 1200rpm. After brief
centrifugation and cooling to
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room temperature, samples (2Oug) were separated by SDS PAGE and transferred to
nitrocellulose
for immunodetection with mouse anti-6xHis antibodies (Abcam). Incubation in
1:5,000 primary
antibody was performed overnight at room temperature, incubation in 1:5,000
donkey anti-mouse
HRP conjugate secondary antibody was performed for 3 hours at room
temperature, and detection
was performed with SuperSignal West Pico chemiluminescent substrate (Pierce).
Multiple clones
displayed soluble expression of both HEXA and HEXB subunits of hexanoate
synthase. As shown
in Figure 11, a substantial portion of the expressed protein fractionated with
the insoluble pellet.
Strains sAA061, sAA140, sAA141, sAA142 contained 6xHis-tagged HEXA and HEXB
proteins.
Strain sAA048 contained only vectors p425GPD and p426GPD.
Example 13: STCJ and STCK expression in S. cerevisiae
Plasmids pAA041 and pAA043 were cotransformed into competent BY4742 S.
cerevisiae.
Selection for transformants containing both pAA041 and pAA043 was performed on
SC-URA-LEU
plates. Culture growth, cell extract preparation, SDS PAGE, and
immunodetection were performed
as described herein. One clone displayed soluble expression of both STCJ and
STCK subunits.
As shown in Figure 11, a substantial portion of the expressed protein
fractionated with the
insoluble pellet. Strain sAA144 contained 6xHis-tagged STCJ and STCK proteins.
Strain sAA048
contained only vectors p425GPD and p426GPD.
Example 14: HEXA and HEXB expression in C. tropicalis
Plasmids pAA128 and pAA130 were linearized using Clal, and cotransformed into
competent
sAA103 cells (ura3/ura3, pox4::ura3/p0x4::ura3, pox5::ura3/pox5::ura3). The
Clal recognition sites
in the HEXA and HEXB ORF's are blocked due to overlapping dam methylation.
Selection for
transformants containing integrated vector DNA was performed on SC-URA plates.
Confirmation
of vector integration was performed by PCR using HEXA and HEXB specific
primers.
Transformants that were PCR positive for both HEXA and HEXB were selected for
analysis of
target protein expression. Overnight culture growth was performed as described
herein. Fresh
5mL YPD cultures were inoculated from the overnight cultures to an initial
OD600nm of 0.4 and
incubated until the OD600nm reached ¨5-8, at which point the culture was
harvested. Cell extract
preparation, SDS PAGE, and immunodetection were performed as described herein.
Strains
sAA269 and sAA270 contained plasmids pAA128 and pAA130 integrated into the
genome for
expression of 6xHis-tagged HEXA and HEXB proteins. Both strains displayed
soluble expression
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of 6xHis-tagged HEXA and HEXB subunits as shown in Figure 12. 6xHis tagged
HEXA and HEXB
expressed in strains sAA269 and sAA270 are indicated with arrows. 6xHis tagged
STCJ and
STCK from strain sAA144 were included as a positive control. Strain sAA103 is
the parent strain
for sAA269 and sAA270 and does not contain integrated vectors for the
expression of 6xHis-
tagged HEXA and HEXB.
Example 15: Procedure for recycling of the URA3 marker
C. tropicalis has a limited number of selectable marker, as compared to S.
cerevisiae, therefore,
the URA3 marker is "recycled" to allow multiple rounds of selection using
URA3. To reutilize the
URA3 marker for subsequent engineering of C. tropicalis, a single colony
having the Ura+
phenotype was inoculated into 3 mL YPD and grown overnight at 30 C with
shaking. The
overnight culture was then harvested by centrifugation and resuspended in 1 mL
YNB+YE (6.7 g/L
Yeast Nitrogen Broth, 3g/L Yeast Extract). The resuspended cells were then
serially diluted in
YNB+YE and 100 uL aliquots plated on YPD plates (incubation overnight at 30 C)
to determine
titer of the original suspension. Additionally, triplicate 100 uL aliquots of
the undiluted suspension
were plated on SC Dextrose (Bacto Agar 20g/L, Uracil 0.3 g/L, Dextrose 20 g/L,
Yeast Nitrogen
Broth 6.7 g/L, Amino Acid Dropout Mix 2.14 g/L) and 5-F0A.at 3 different
concentrations (0.5, 0.75,
1 mg/mL).
Plates were incubated for at least 5 days at 30 C. Colonies arising on the SC
Dextrose + 5-
FOA plates were resuspended in 50 uL sterile, distilled water and 5 uL
utilized to streak on to YPD
and SC¨URA (SC Dextrose medium without Uracil) plates. Colonies growing only
on YPD and not
on SC¨URA plates were then inoculated into 3 mL YPD and grown overnight at 30
C with
shaking. Overnight cultures were harvested by centrifugation and resuspended
in 1.5 mL YNB
(6.7 g/L Yeast Nitrogen Broth). The resuspended cells were serially diluted in
YNB and 100 uL
aliquots plated on YPD plates and incubation overnight at 30 C to determine
initial titer. 1 mL of
each undiluted cell suspension also was plated on SC¨URA and incubated for up
to 7 days at
C. Colonies on the SC-URA plates are revertants and the isolate with the
lowest reversion
30 frequency (< 10-7) was used for subsequent strain engineering.
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Example 16: Omega oxidation of hexane and hexanoic acid to adipic acid
Starter cultures of strain sAA003 were grown in YPD2.0 (1% yeast extract, 2%
peptone, 2%
dextrose) overnight as described. Starter cultures were used to inoculate
100mL of fresh YPD2.0 to
an initial OD600nm of 0.4 and incubated overnight at 30 C, with shaking at
about 200rpm. The
100mL culture was pelleted by centrifugation at 4,000 x g, 23 C for 10 minutes
and resuspended in
100mL fresh YPDo.i media (1% yeast extract, 1% peptone, 0.1% dextrose). The
culture was
divided into 4 x 25mL cultures to which were added either 1% hexane, 0.05%
hexanoic acid, 1.0%
hexanoic acid, or no other carbon source. Strain sAA003 is completely blocked
in beta-oxidation,
therefore fermentation tested the ability of the beta-oxidation pathway to
oxidize C6 substrates.
Samples were taken at 24, 48, and 72 hours and analyzed by LC-MS (Scripps
Center for Mass
Spectrometry) using published methods for the detection of adipic acid (Cheng
et al., 2000). The
data for the 72 hour time-point, shown in the table below demonstrates that
strain sAA003 was
able to oxidize both hexanoic acid and hexane to adipic acid. The results also
indicate that the 1%
hexanoic acid level was toxic to the cells leading to no production of adipic
acid over background
levels.
Oxidation of hexane and hexanoic acid to adipic acid
Time (h) MEDIA Adipic acid (mg/L)
0 YPDai 0.000
72 YPDai 0.005
72 YPDai + 0.05% Hexanoic Acid 0.406
72 YPDai + 1% Hexanoic Acid 0.003
72 YPDai + 1% Hexane 0.091
Example 17: Identification of P450 alleles induced by exposure to hexane or
hexanoic acid
350 mL cultures grown overnight in YNB-Salts + 2.0% Glucose (6.7 g/L Yeast
Nitrogen Broth, 3.0
g/L Yeast Extract, 3.0 g/L ammonium sulfate, 3.0 g/L monopotassium phosphate,
0.5 g/L sodium
chloride, and 20 g/L dextrose) were inoculated from a 3 mL overnight culture
of YPD (1% Yeast
Extract, 2% Peptone, 2% Dextrose), and used for RNA preparation. Cultures were
harvested by
centrifugation. Each pellet was resuspended in 100 mL of YNB-Salts medium with
no glucose. A
1 mL aliquot was taken for RNA isolation as a time=0 control. To each 100 mL
suspension, a
different inducer was added, 1% glucose, 1% hexane or 0.05% hexanoic acid and
aliquoted as two
50 mL portions into 250 mL baffled flasks and incubated for 2 or 4 hours at 30
C with shaking. At
2 hours and 4 hours, one flask for each inducer was harvested by
centrifugation and resuspended
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in its own spent media in order to collapse culture foam. 1 mL samples were
isolated by
centrifugation of 1 mL of each culture and RNA prepared using the RiboPure-
Yeast Kit, according
to the manufacturer's directions, with an additional extraction of the initial
RNA preparations with 1
volume of Chloroform:lsoamyl Alcohol (24:1) to the aqueous phase after lysis
and extraction with
Phenol:Chloroform:lsoamyl Alcohol (25:24:1).
Each RNA preparation was further purified by precipitation with ethanol and
treatment with DNase
I, again according to manufacturers' recommendations. All RNA preparations
were shown to be
free of contaminating genomic DNA by electrophoresis and by failure to prime a
PCR product of
the URA3 gene. First strand synthesis reactions were completed for each RNA
preparation using
Superscript III Reverse Transcriptase (Invitrogen), as described herein.
Reactions for each sample
consisted of 1 uL oAA0542 (polyT 10 uM), 1 uL dNTP mix (10 mM each), 1
microgram RNA in 13
uL sterile, distilled water. The RNA/primer mix was heated to 65 C for 5
minutes, and on ice for 1
minute. Primers were generated that amplified a substantially unique area of
each cytochrome
P450 and are shown in the table below.
PCR reactions were performed on the 2 hour induced cDNA samples and compared
to the Time =
0 and genomic DNA controls. PCR reactions for each cDNA and primer pair
combination
consisted of 0.5 uL template, sense and antisense primers at 0.4 uM each, 1X
Taq DNA
polymerase Buffer (New England Biolabs), 0.1 uL Taq DNA polymerase, and 0.2mM
dNTPs and
sterile, distilled water to 25 uL. Cycling parameters used were 95 C for 5
minutes, 30 cycles of 95
C 30 seconds, 50 C 40 seconds, 72 C 2 minutes, followed by 72 C 5 minutes
and a 4 C
hold. PCR reactions were electrophoresed on 1.2% agarose gels to identify
differential expression
due to the inducer used. Several P450s displayed increased induction in the
presence of hexane
or hexanoic acid, however the results were not quantitative. Two Cytochrome
P450's, CYP52A15
and CYP52A16, showed induction only in the presence of hexane and hexanoic
acid and not in the
presence of glucose, as shown in Figure 13. The primers used for PCR analysis
of induced
expression are shown in the table below.
Oligonucleotides for identification of P450 DNA fragments
Oligos Sequence P450 P450 sequence
PCR product (bp)
oAA0082 gattactgcagcagtattagtcttc
CYP52Al2 60-249
190
oAA0083 gtcgaaaacttcatcggcaaag
oAA0084 cacgatattatcgccacatacttc
CYP52A13 10-256
247
oAA0085 cgggacgatcgagatcgtggatacg
oAA0086 caggatattatcgccacatacatc
CYP52A14 10-256
247
oAA0087 ctggacgattgagcgcttggatacg
oAA0088 cgtcttctccatcgtttgcccaagag CYP52A15 5-199
195
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oAA0089 ggtccctgacaaagttaccgagtg
oAA0090 cgtcttctccatcgtttgctcaggag
CYP52A16 5-199
195
oAA0091 gatccaacacgacgttaccgagcg
oAA0092 ggtatgtcgttgtgccagtgttg
CYP52A17 26-248
223
oAA0093 cccacgcttgggttcttggagtggtc
oAA0094 ggtatattgttgtgcctgtgttg
CYP52A18 26-248
223
oAA0095 ccgacgcttgggttcttggagctgtc
oAA0096 ggaaggatgaggtggtgcagtac
CYP52A19 1217-1458
242
oAA0097 gtcttgtgacaagtttggaaactc
oAA0098 gaaagaatgaggtggtgcaatac
CYP52A20 1217-1458
242
oAA0099 gtcctgtgacaagctagggaattc
oAA0104 ctatcgtgggatgtgatctgtgtcg
CYP52D2 19-231
213
oAA0105 ctcgaatctcttgacactgaactcg
Example 18: Cloning and Analysis of C. tropicalis Fatty alcohol oxidase (FAO)
alleles
Isolation of fatty alcohol oxidase genes from C. tropicalis
C. tropicalis (ATCC20336) fatty alcohol oxidase genes were isolated by PCR
amplification using
primers generated to amplify the sequence region covering promoter, fatty
alcohol oxidase gene
(FAO) and terminator of the FA01 sequence (GenBank accession number of FA01
AY538780).
The primers used to amplify the fatty alcohol oxidase nucleotide sequences
from C. tropicalis strain
ATCC20336, are showing in the table below.
Oligonucleotides for cloning FAO alleles
Oligo Sequence
oAA0144 AACGACAAGATTAGATTGGTTGAGA
oAA0145 GTCGAGTTTGAAGTGTGTGTCTAAG
oAA0268 AGATCTCATATGGCTCCATTTTTGCCCGACCAGGTCGACTACAAACACGTC
oAA0269 ATCTGGATCCTCATTACTACAACTTGGCTTTGGTCTTCAAGGAGTCTGCCAAACCTAAC
oAA0282 ACATCTGGATCCTCATTACTACAACTTGGCCTTGGTCT
oAA0421 CACACAGCTCTTCTAGAATGGCTCCATTTTTGCCCGACCAGGTCGAC
oAA0422 CACACAGCTCTTCCTTTCTACAACTTGGCTTTGGTCTTCAAGGAGTCTGC
oAA0429 GTCTACTGATTCCCCTTTGTC
oAA0281 TTCTCGTTGTACCCGTCGCA
PCR reactions contained 25uL 2X master mix, 1.5 uL of oAA0144 and oAA0145
(10uM), 3.0uL
genomic DNA, and 19uL sterile H20. Thermocycling parameters used were 98 C for
2 minutes, 35
cycles of 98 C 20 seconds, 52 C 20 seconds, 72 C 1 minute, followed by 72 C 5
minutes and a
4 C hold. PCR products of the correct size were gel purified, ligated into pCR-
Blunt II-TOPO
(Invitrogen) and transformed into competent TOP10 E. coli cells (Invitrogen).
Clones containing
PCR inserts were sequenced to confirm correct DNA sequence. Four FAO alleles
were identified
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from sequence analysis and designated as FAO-13, FAO-17, FAO-18 and FAO-20.
The sequence
of the clone designated FA0-18 had a sequence that was substantially identical
to the sequence of
FA01 from GenBank. The resulting plasmids of the four alleles were designated
pAA083, pAA084,
pAA059 and pAA085, respectively. Sequence identity comparisons of FAO genes
isolated as
described herein are shown in the tables below.
DNA sequence identity
FAO 1 FAO-18 FAO-17 FAO-13 FAO-20 FA02a FA02 b
FA01 100 100 98 96 95 83 82
FAO-18 100 98 96 95 83 82
FAO-17 100 98 98 83 82
FAO-13 100 99 83 83
FAO-20 100 83 83
FA02a 100 96
FA02 b 100
Protein sequence identity
FAO 1 FAO-18 FAO-17 FAO-13 FAO-20 FA02a FA02 b
FA01 100 100 99 98 98 81 80
FAO-18 100 99 98 98 81 80
FAO-17 100 99 99 82 81
FAO-13 100 99 82 81
FAO-20 100 82 81
FA02a 100 97
FA02 b 100
Amino acid differences in FAO alleles
32 75 89 179 185 213 226 352 544 590
FAO 1 EMGL Y T R HS P
FAO-13QT A L Y A K Q A A
FAO-20Q T AMD A K Q A A
Expression of FAO alleles in E. coli
To determine the levels of FAO enzyme activity with respect to various carbon
sources, the four
isolated FAO alleles were further cloned and over-expressed in E. coli. The
FAOs were amplified
using the plasmids mentioned above as DNA template by PCR with primers oAA0268
and
oAA0269 for FAO-13 and FAO-20 and oAA0268 and oAA0282 for FAO-17 and FAO-18,
using
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conditions as described herein. PCR products of the correct size were gel
purified and ligated into
pET11a vector between Ndel and BamHI sites and transformed into BL21 (DE3) E.
coli cells. The
colonies containing corresponding FAOs were confirmed by DNA sequencing.
Unmodified
pET11 a vector also was transformed into BL21 (DE3) cells, as a control. The
resulting strains and
plasmids were designated sAA153 (pET11a), sAA154 (pAA079 containing FA0-13),
sAA155
(pAA080 containing FAO-17), sAA156 (pAA081 containing FAO-18) and sAA157
(pAA082
containing FAO-20), respectively. The strains and plasmids were used for FAO
over-expression in
E. coli. One colony of each strain was transferred into 5 mL of LB medium
containing 100
micrograms/mL ampicillin and grown overnight at 37 C, 200 rpm. The overnight
culture was used
to inoculate a new culture to OD600nm 0.2 in 25 ml LB containing 100
micrograms/ml ampicillin.
Cells were induced at OD600nm 0.8 with 0.3 mM IPTG for 3 hours and harvested
by centrifugation at
4 C 1,050xg for 10 minutes. The cell pellet was stored at -20 C.
Expression of FAOs in C. tropicalis
Two alleles, FA0-13 and FAO-20, were chosen for amplification in C. tropicalis
based on their
substrate specificity profile, as determined from enzyme assays of soluble
cell extracts of E. coli
with over expressed FAOs. DNA fragments containing FA0-13 and FAO-20 were
amplified using
plasmids pAA079 and pAA082 as DNA templates, respectively, by PCR with primers
oAA0421 and
oAA0422. PCR products of the correct sizes were gel purified and ligated into
pCR-Blunt II-TOPO
(Invitrogen), transformed into competent TOP10 E. coli cells (Invitrogen) and
clones containing
FAO inserts were sequenced to confirm correct DNA sequence. Plasmids
containing FAO-13 and
FAO-20 were digested with Sapl and ligated into vector pAA105, which includes
the C. tropicalis
PGK promoter and terminator. The resulting plasmids were confirmed by
restriction digestion and
DNA sequencing and designated as pAA115 (FA0-13) and pAA116 (FA0-20),
respectively.
Plasmids pAA115 and pAA116 were linearized with Spel, transformed into
competent C. tropicalis
Ura- strains sAA002 (SU-2, ATCC20913) and sAA103. The integration of FAO-13
and FAO-20
was confirmed by colony PCR using primers oAA0429 and oAA0281. The resulting
strains were
designated as sAA278 (pAA115 integrated in strain sAA002), sAA280 (pAA116
integrated in
sAA002), sAA282 (pAA115 integrated in sAA103), and sAA284 (pAA116 integrated
in sAA103),
and were used for fatty alcohol oxidase over-expression in C. tropicalis.
One colony of each strain was inoculated into 5 ml YPD and grown overnight as
described herein.
The overnight culture was used to inoculate a new 25 mL YPD culture to about
OD600nm 0.5. FAO
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over-expression was regulated by the PGK promoter/terminator, induced with
glucose in the
medium and expressed constitutively. Strains sAA002 and sAA103 (e.g.,
untransformed starting
strains) were included as negative controls for FAO over-expression. Cells
were harvested at early
log phase (0D600nm = in the range of between about 3 to about 5) by
centrifugation at 4 C for 10
minutes at 1,050 x g. Cell pellets were stored at -20 C.
Cell extract preparation from E. coli
Cell pellets from 25 mL of FAO expressing E. coli cultures were resuspended in
10 mL phosphate-
glycerol buffer containing 50 mM potassium phosphate buffer (pH7.6), 20%
glycerol, 1 mM
Phenylmethylsulfonyl fluoride (PMSF), 2uL Benzonase 25U/uL, 20uL 10mg/mL
lysozyme. The
cells were then lysed by incubation at room temperature for 50 minutes on a
rotating shaker, and
the cell suspension centrifuged for 30 minutes at 4 C using 15,000 x g for.
The supernatant was
aliquoted in 1.5 ml microcentrifuge tubes and stored at -20 C for FAO enzyme
activity assays.
Cell extract preparation from C. tropicalis
Frozen C. tropicalis cell pellets were resuspended in 1.2 ml of phosphate-
glycerol buffer containing
50 mM potassium phosphate buffer (pH7.6), 20% glycerol, 1 mM
Phenylmethylsulfonyl fluoride
(PMSF). Resuspended cells were transferred to 1.5mL screw-cap tubes containing
about 500uL of
zirconia beads on ice. The cells were lysed with a Bead Beater (Biospec) using
2 minute pulses
and 1 minute rest intervals on ice. The process was repeated 3 times. The
whole cell extract was
then transferred to a new 1.5 ml tube and centrifuged at 16,000 x g for 15
minutes at 4 C. The
supernatant was transferred into a new tube and used for FAO enzyme activity
assays.
Protein concentration determination
Protein concentration of the cell extracts was determined using the Bradford
Reagent following
manufacturers' recommendations (Cat# 23238, Thermo scientific).
FAO enzyme activity assay
FAO enzyme activity assays were performed using a modification of Eirich et
al., 2004). The assay
utilizes a two-enzyme coupled reaction (e.g., FAO and horse radish peroxidase
(HRP)) and can be
monitored by spectrophotometry. 1-Dodecanol was used as a standard substrate
for fatty alcohol
oxidase enzymatic activity assays. FAO oxidizes the dodecanol to dodecanal
while reducing
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molecular oxygen to hydrogen peroxide simultaneously. HRP reduces (2,2'-azino-
bis 3-
ethylbenzthiazoline-6-sulfonic acid; ABTS) in the two-enzyme coupled reaction,
where the electron
obtained from oxidizing hydrogen peroxide to ABTS, which can be measured by
spectrometry at
405 nm. The assay was modified using aminotriazole (AT) to prevent the
destruction of H202 by
endogenous catalase, thus eliminating the need for microsomal fractionation.
The final reaction
mixture (1.0 mL) for FAO enzyme assay consisted of 500 pL of 200 mM HEPES
buffer, pH 7.6; 50
pL of a 10 mg/mL ABTS solution in deionized water; 10 pL of 5 mM solution of
dodecanol in
acetone; 40 pL of 1M AT and 5 pL of a 2 mg/mL horseradish peroxidase solution
in 50 mM
potassium phosphate buffer, pH 7.6. Reaction activity was measured by
measuring light
absorbance at 405 nm for 10 minutes at room temperature after adding the
extract. The amount of
extract added to the reaction mixture was varied so that the activity fell
within the range of 0.2 to
1.0 AA405nm/min. The actual amounts of extract used were about 1.69 U/mg for
E. coli expressed
FAO-13, 0.018U/mg for E. coli expressed FAO-17, 0.35U/mg for E. coli expressed
FAO-18 (e.g.,
FA01), 0.47U/mg E. coli expressed FAO-20, 0.036U/mg C. tropicalis (strain
5AA278) expressed
FAO-13, 0.016U/mg C. tropicalis (strain 5AA282) expressed FAO-13, 0.032U/mg C.
tropicalis
(strain 5AA280) expressed FAO-20 and 0.029U/mg C. tropicalis (strain 5AA284)
expressed FAO-
20. FAO activity was reported as activity units/mg of total protein (1 unit =
1 micromole substrate
oxidized/min). An extinction coefficient at 405 nm of 18.4 was used for ABTS
and was equivalent
to 0.5 mM oxidized substrate. The results of the activity assays are shown in
the tables below.
FAO activity (units/mg total protein) on primary alcohols
1- 1- 1- 1- 1- 1- 1-
Hexadecanol
Butanol Pentanol Hexanol Octanol Decanol Dodecanol Tetradecanol
FAO-13 0.01 0.09 1.17 82.67 70.94 100 79.35
58.88
FAO-17 0.72 0.26 1.06 66.23 22.00 100 47.86
60.98
FAO-18 0.07 0.11 0.26 60.56 54.56 100 114.47
50.65
FAO-20 0.07 0.11 0.91 55.96 74.57 100 89.52
42.59
FAO activity (units/mg total protein) on omega hydroxy fatty acids
1- 6-0H-HA 10-OH-DA 12-0H- 16-0H-
Dodecanol DDA HDA
FAO-13 100 4.18 4.14 6.87 8.57
FAO-17 100 1.18 0.00 0.59 0.94
FAO-18 100 0.00 0.00 4.87 2.94
FAO-20 100 0.03 0.04 2.25 7.46
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Example 19: Construction of C. tropicalis shuttle vector pAA061
Vector pAA061 was constructed from a pUC19 backbone to harbor the selectable
marker URA3
from C. tropicalis strain ATCC20336 as well as modifications to allow
insertion of C. tropicalis
promoters and terminators. A 1,507bp DNA fragment containing the promoter,
ORF, and
terminator of URA3 from C. tropicalis ATCC20336 was amplified using primers
oAA0124 and
oAA0125, shown in the table below. The URA3 PCR product was digested with Ndel
/ Mlul and
ligated into the 2,505bp fragment of pUC19 digested with Ndel / BsmBI (an Mlul
compatible
overhang was produced by BsmBI). In order to replace the lac promoter with a
short 21bp linker
sequence, the resulting plasmid was digested with Sphl / Sapl and filled in
with a linker produced
by annealing oligos oAA0173 and oAA0174. The resulting plasmid was named
pAA061, and is
shown in Figure 30.
Oligonucleotides for construction of pAA061
Oligos Sequence PCR product (bp)
oAA0124 cacacacatatgCGACGGGTACAACGAGAATT
1507
oAA0125 cacacaacgcgtAGACGAAGCCGTTCTTCAAG
oAA0173 ATGATCTGCCATGCCGAACTC
21 (linker)
oAA0174 AGCGAGTTCGGCATGGCAGATCATCATG
Example 20: Cloning of C. tropicalis PGK promoter and terminator
Vector pAA105 was constructed from base vector pAA061 to include the
phosphoglycerate kinase
(PGK) promoter and terminator regions from C. tropicalis ATCC20336 with an
intervening multiple
cloning site (MCS) for insertion of open reading frames (ORF's). The PGK
promoter region was
amplified by PCR using primers oAA0347 and oAA0348, shown in the table below.
The 1,029bp
DNA fragment containing the PGK promoter was digested with restriction enzymes
Pstl / Xmal.
The PGK terminator region was amplified by PCR using primers oAA0351 and
oAA0352, also
shown in the table below. The 396bp DNA fragment containing the PGK terminator
was digested
with restriction enzymes Xmal / EcoRl. The 3,728bp Pstl / EcoRI DNA fragment
from pAA061 was
used in a three piece ligation reaction with the PGK promoter and terminator
regions to produce
pAA105. The sequence between the PGK promoter and terminator contains
restriction sites for
incorporating ORF's to be controlled by the functionally linked constitutive
PGK promoter.
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Oligonucleotides for cloning C. tropicalis PGK promoter and terminator
Oligos Sequence PCR product
(bp)
oAA0347 CACACACTGCAGTTGTCCAATGTAATAATTTT
CACACATCTAGACCCGGGCTCTTCTTCTGAATAGGCAATTGATA 1028
oAA0348 AACTTACTTATC
GAGCCCGGGTCTAGATGTGTGCTCTTCCAAAGTACGGTGTTGTT
oAA0351 GACA 396
oAA0352 CACACACATATGAATTCTGTACTGGTAGAGCTAAATT
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Example 21: Cloning of the PDX4 locus
Primers oAA0138 and oAA0141 (shown in the table below) were generated to
amplify the entire
sequence of NCB! accession number M12160 for the YSAPDX4 locus from genomic
DNA
prepared from C. tropicalis strain ATCC20336. The 2,845bp PCR product was
cloned into the
vector, pCR-Blunt1I-TOPO (Invitrogen), sequenced and designated pAA052, and is
shown in
Figure 29.
Oligonucleotides for cloning of PDX4
Oligos Sequence PCR product (bp)
oAA0138 GAG CTCCAATTGTAATATTTCGG G
2845
oAA0141 GTCGACCTAAATTCGCAACTATCAA
Example 22: Cloning of the PDX5 locus
Primers oAA0179 and oAA0182 (shown in the table below) were generated to
amplify the entire
sequence of NCB! accession number M12161 for the YSAPDX5 locus from genomic
DNA
prepared from C. tropicalis strain ATCC20336. The 2,624bp PCR product was
cloned into the
vector, pCR-Blunt1I-TOPO (Invitrogen), sequenced and designated pAA049, and is
shown in
Figure 28.
Oligonucleotides for cloning of PDX5
Oligos Sequence PCR product (bp)
GAATTCACATGGCTAATTTGGCCTCGGTTCCACAACGCACTCAGC
oAA0179 ATTAAAAA 2624
oAA0182 GAG CTCCCCTGCAAACAGG GAAACACTTGTCATCTGATTT
Example 23: Construction of strain sAA105 and sAA106
Functional PDX4 alleles were restored in C. tropicalis strain sAA003
(ATCC20962; ura3/ura3,
pox4::ura3/pox4::ura3, pox5::ura3/pox5::URA3) by transformation of sAA003 with
PDX4 linear
DNA to replace the ura3-disrupted loci with functional alleles. A 2,845bp DNA
fragment was
amplified by PCR using primers oAA0138 and oAA0141 (described in Example 21)
that contained
the PDX4 ORF as well as 531bp upstream and 184bp downstream of the ORF, using
plasmid
pAA052 as template. The purified PCR product was used to transform competent
sAA003 cells
which were plated on YNB-agar plates supplemented with hexadecane vapor as the
carbon source
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(e.g., by placing a filter paper soaked with hexadecane in the lid of the
inverted petri dish) and
incubated at 30 C for 4-5 days. Colonies growing on hexadecane as the sole
carbon source were
restreaked onto YPD-agar and incubated at 30 C. Single colonies were grown in
YPD cultures
and used for the preparation of genomic DNA.
PCR analysis of the genomic DNA prepared from the transformants was performed
with oligos
oAA0138 and oAA0141. An URA3-disrupted PDX4 would produce a PCR product of
5,045bp,
while a functional PDX4 would produce a PCR product of 2,845bp. In strain
sAA105 only one
PCR product was amplified with a size of 2,845bp indicating that both PDX4
alleles had been
functionally restored. In strain sAA106 PCR products of both 2,845bp and
5,045bp were amplified
indicating that one PDX4 allele had been functionally restored while the other
PDX4 allele
remained disrupted by URA3. The resultant strain genotypes were: sAA105
(ura3/ura3,
PDX4/PDX4, pox5::ura3/pox5::URA3) and sAA106 (ura3/ura3, PDX4/pox4::ura3,
pox5::ura3/pox5::URA3).
Example 24: Construction of strain sAA152
Functional PDX5 alleles were restored in C. tropicalis strain sAA103
(ura3/ura3,
pox4::ura3/pox4::ura3, pox5::ura3/pox5::ura3) by transformation of sAA103 with
PmII-linearized
plasmid pAA086 (containing the PDX5 promoter, gene, terminator and a URA3
marker). Selection
of transformants was performed by plating on SC-URA agar plates. Verification
of plasmid
integration was performed by PCR with primers oAA179 and oAA182 (described in
Example 22).
Integration of the plasmid was shown by a PCR product of 2,584bp indicating
the presence of a
functional PDX5 allele. Other PDX5 alleles in sAA152 were disrupted with an
ura3 gene
increasing the PCR product size for nonfunctional alleles to 4,734bp. Genotype
for strain sAA152
is ura3/ura3, pox4::ura3/pox4::ura3, pox5::ura3/pox5::ura3, ura3::P0X5,URA3).
Example 25: Construction of strain sAA232
Functional PDX5 alleles were restored in C. tropicalis strain sAA003 by
transformation of sAA003
with PDX5 linear DNA to replace the URA3-disrupted loci with a functional
allele. A 2,584bp DNA
fragment was amplified by PCR using primers oAA0179 and oAA0182 (described in
Example 22 )
that contained the PDX5 ORF as well as 456bp upstream and 179bp downstream of
the ORF
using plasmid pAA049 as template. The purified PCR product was used to
transform competent
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sAA003 cells which were plated on SC+URA+5F0A plates and incubated at 30 C for
3-4 days.
Colonies were restreaked onto YPD-agar and incubated at 30 C. Single colonies
were grown in
YPD cultures and used for the preparation of genomic DNA. PCR analysis of the
genomic DNA
prepared from the transformants was performed with oligos oAA0179 and oAA0182.
An ura3-
disrupted PDX5 would produce a PCR product of 4,784bp while a functional PDX5
would produce
a PCR product of 2,584bp. In strain sAA232 PCR products of both 2,584bp and
4,784bp were
amplified indicating that one PDX5 allele had been functionally restored while
the other PDX5
allele remained disrupted by ura3. The resultant genotype of strain sAA232 is
ura3/ura3,
pox4::ura3/pox4::ura3, pox5::ura3/P0X5. 5-FOA selection restored the PDX5
allele that had been
disrupted with the functional URA3 leaving the sAA232 strain Lira-.
Example 26: Construction of strain sAA235
Functional PDX5 alleles were restored in C. tropicalis strain sAA003 by
transformation of sAA003
with PDX5 linear DNA to replace the URA3-disrupted loci with a functional
allele. A 2,584bp DNA
fragment was amplified by PCR using primers oAA0179 and oAA0182 (described in
Example 22 )
that contained the PDX5 ORF as well as 456bp upstream and 179bp downstream of
the ORF
using plasmid pAA049 as template. The purified PCR product was used to
transform competent
sAA003 cells which were plated on YNB-agar plates supplemented with dodecane
vapor as the
carbon source (e.g., by placing a filter paper soaked with dodecane in the lid
of the inverted petri
dish) and incubated at 30 C for 4-5 days. Colonies growing on dodecane as the
sole carbon
source were restreaked onto YPD-agar and incubated at 30 C. Single colonies
were grown in
YPD cultures and used for the preparation of genomic DNA. PCR analysis of the
genomic DNA
prepared from the transformants was performed with oligos oAA0179 and oAA0182.
An ura3-
disrupted PDX5 would produce a PCR product of 4,784bp while a functional PDX5
would produce
a PCR product of 2,584bp. In strain sAA235 a PCR product of 2,584bp was
amplified indicating
that both PDX5 alleles had been functionally restored. An unintended
consequence of the
selection strategy (YNB-agar with dodecane) was that the cells reverted back
to an Lira
phenotype. Without being limited by any theory, it is believed the absence of
uracil in the solid
media and the replacement of the only functional URA3 forced the cells to
mutate one of the other
ura3 loci back to a functional allele. Therefore the genotype of the strain
sAA235 is believed to be
URA3/ura3, pox4::ura3/pox4::ura3, PDX5/PDX5. Verification of which of the loci
is the functional
URA3 is underway.
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Example 27: Construction of strains with amplified CPR and CYP52 genes
Strains having an increased number of copies of cytochrome P450 reductase
(CPR) and/or for
cytochrome P450 monooxygenase (CYP52) genes were constructed to determine how
over
expression of CPR and CYP52 affected diacid production.
Cloning and integration of the CPR gene.
A 3,019bp DNA fragment encoding the CPR promoter, ORF, and terminator from C.
tropicalis
ATCC750 was amplified by PCR using primers oAA0171 and oAA0172 (see table
below)
incorporating unique Sapl and Sphl sites. The amplified DNA fragment was cut
with the indicated
restriction enzymes and ligated into plasmid pAA061, shown in Figure 30, to
produce plasmid
pAA067, shown in Figure. 32. Plasmid pAA067 was linearized with Clal and
transformed into C.
tropicalis Lira- strain sAA103 (ura3/ura3, pox4::ura3/pox4::ura3,
pox5::ura3/pox5::ura3).
Transformations were performed with plasmid pAA067 alone and in combination
with plasmids
harboring the CYP52A15 or CYP52A16 genes, described below.
Cloning and integration of CYP52A15 gene.
A 2,842bp DNA fragment encoding the CYP52A15 promoter, ORF, and terminator
from C.
tropicalis ATCC20336 was amplified by PCR using primers oAA0175 and oAA0178
(see table
below) and cloned into pCR-Blunt1I-TOPO for DNA sequence verification. The
cloned CYP52A15
DNA fragment was isolated by restriction digest with Xbal / BamHI (2,742bp)
and ligated into
plasmid pAA061, shown in Figure 30, to produce plasmid pAA077, shown in Figure
33. Plasmid
pAA077 was linearized with Pm11 and transformed into C. tropicalis Lira-
strain sAA103 (ura3/ura3,
pox4::ura3/pox4::ura3, pox5::ura3/pox5::ura3). pAA077 was cotransformed with
plasmid pAA067
harboring the CPR gene.
Cloning and integration of CYP52A16 gene.
A 2,728bp DNA fragment encoding the CYP52A16 promoter, ORF, and terminator
from C.
tropicalis ATCC20336 was amplified by PCR using primers oAA0177 and oAA0178
(see table
below) and cloned into pCR-Blunt1I-TOPO for DNA sequence verification. The
cloned CYP52A16
DNA fragment was amplified with primers oAA0260 and oAA0261(see table below)
which
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incorporated unique Sacl / Xbal restriction sites. The amplified DNA fragment
was digested with
Sacl and Xbal restriction enzymes and ligated into plasmid pAA061 to produce
plasmid pAA078,
shown in Figure 34. Plasmid pAA078 was linearized with Clal and transformed
into C. tropicalis
Lira- strain sAA103 (ura3/ura3, pox4::ura3/pox4::ura3, pox5::ura3/pox5::ura3).
pAA078 was
cotransformed with plasmid pAA067 harboring the CPR gene.
Oligonucleotides for cloning of CPR, CYP52A15 and CYP52A16
Oligos Sequence PCR product (bp)
oAA0171 cacctcgctcttccAGCTGTCATGTCTATTCAATGCTTCGA
3019
oAA0172 cacacagcatgcTAATGTTTATATCGTTGACGGTGAAA
cacaaag cggaagag cAAATTTTGTATTCTCAGTAGGATTT
oAA0175 CATC 2842
oAA0178 cacacagcatgCAAACTTAAGGGTGTTGTAGATATCCC
cacacacccgggATCGACAGTCGATTACGTAATCCATATT
oAA0177 ATTT 2772
oAA0178 cacacagcatgCAAACTTAAGGGTGTTGTAGATATCCC
oAA0260 cacacagagctcACAGTCGATTACGTAATCCAT
2772
oAA0261 cacatctagaGCATGCAAACTTAAGGGTGTTGTA
Preparation of genomic DNA.
Genomic DNA was prepared from transformants for PCR verification and for
Southern blot
analysis. Isolated colonies were inoculated into 3 mL YPD and grown overnight
at 30 C with
shaking. Cells were pelleted by centrifugation. To each pellet, 200 uL
Breaking Buffer (2% Triton
X-100, 1% SDS, 100 mM NaCI, 10 mM Tris pH 8 and, 1 mM EDTA) was added, and the
pellet
resuspended and transferred to a fresh tube containing 200 uL 0.5 mm
Zirconia/Silica Beads. 200
uL Phenol:Chloroform:lsoamyl Alcohol (25:24:1) was added to each tube,
followed by vortexing for
1 minute. Sterile distilled water was added (200 uL) to each tube and the
tubes were centrifuged
at 13000 rpm for 10 minutes. The aqueous layer was ethanol precipitated and
washed with 70%
ethanol. The pellet was resuspended in 100-200 microliters 10 mM Tris, after
drying. Genomic
DNA preparation for southern blot analysis was performed using the same
procedure on 25 mL
cultures for each colony tested.
Characterization of strains with amplified CPR and CYP52 genes.
Verification of integrants was performed by PCR using primers oAA0252 and
oAA0256 (CPR),
oAA0231 and oAA0281 (CYP52A15), and oAA242 and oAA0257 (CYP52A16). The primers
used
for verification are shown in the table below.
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Oligonucleotides for PCR verification of CPR, CYP52A15 and CYP52A16
Oligos Sequence PCR product (bp)
oAA0252 TTAATGCCTTCTCAAGACAA
743
oAA0256 GGTTTTCCCAGTCACGACGT
oAA0231 CCTTGCTAATTTTCTTCTGTATAGC
584
oAA0281 TTCTCGTTGTACCCGTCGCA
oAA0242 CACACAACTTCAGAGTTGCC
974
oAA0257 TCGCCACCTCTGACTTGAGC
Southern blot analysis was used to determine the copy number of the CPR,
CYP52A15 and
CYP52A15 genes. Biotinylated DNA probes were prepared with gene specific
oligonucleotides
using the NEBlot Phototope Kit from New England BioLabs (Catalog #N7550S) on
PCR products
generated from each gene target as specified in the table below. Southern
Hybridizations were
performed using standard methods (e.g., Sambrook, J. and Russell, D. W. (2001)
Molecular
Cloning: A Laboratory Manual, (3rd ed.), pp. 6.33-6.64. Cold Spring Harbor
Laboratory Press).
Detection of hybridized probe was performed using the Phototope-Star Detection
Kit from New
England BioLabs (Catalog #N70205). Copy number was determined by densitometry
of the
resulting bands.
Oligonucleotides for Probe Template PCR of CPR, CYP52A15 and CYP52A16
Oligos Sequence Gene Template
PCR product (bp)
oAA0250 AATTGAACATCAGAAGAGGA
oAA0254 CCTGAAATTTCCAAATGGTGTCTAA CPR pAA067 1313
oAA0227 TTTTTTGTGCGCAAGTACAC CYP52A15
pAA077 905
oAA0235 CAACTTGACGTGAGAAACCT
oAA0239 AGATGCTCGTTTTACACCCT
CYP52A16 pAA078 672
oAA0247 ACACAGCTTTGATGTTCTCT
Example 28: Strain evaluation of partially beta-oxidation blocked strains
Fermentation of methyl laurate feedstock.
5mL starter cultures, in 5P92 media (6.7g/L Difco yeast nitrogen base, 3.0g/L
Difco yeast extract,
3.0g/L ammonium sulfate, 1.0g/L potassium phosphate monobasic, 1.0g/L
potassium phosphate
dibasic, 75g/L dextrose) were incubated overnight at 30 C, with shaking and
used to inoculate
flasks containing 25mL of 5P92 media to an initial OD600nm of about 0.4.
Cultures were incubated
approximately 18 hours at 30 C, with shaking at about 200rpm. Cells were
pelleted by
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centrifugation at 4 C for 10 minutes at 4,000 x g, then resuspended in SP92-D
media (6.7g/L Difco
yeast nitrogen base, 3.0g/L Difco yeast extract, 3.0g/L ammonium sulfate,
1.0g/L potassium
phosphate monobasic, 1.0g/L potassium phosphate dibasic) supplemented with
0.1% dextrose
and 2% methyl laurate. Incubation of the cultures continued at 30 C, with
shaking and samples
were taken for analysis of fatty acids and diacids by gas chromatography (GC).
Sample for GC were prepared by adding 0.8mL of 6.0M HCI to 1mL of whole
culture samples and
the samples were stored at 4 C to await processing. Samples were processed by
incubating in a
60 C water bath for 5 minutes, after which 4.0 mL of MTBE was added to the 1.8
mL acidified
whole culture samples and vortexed for 20 seconds. The phases were allowed to
separate for 10
min at room temperature. 1mL of the MTBE phase was drawn and dried with sodium
sulfate.
Aliquots of the MTBE phase were derivatized with BSTFA reagent (Regis
Technologies Inc.) and
analyzed by GC equipped with a Flame Ionization Detector. The results of the
gas
chromatography are shown in the table below.
Fatty acid and Diacid profile (g/L) in Methyl Laurate fermentation
Strain Time
C12 Acid C12 Diacid C10 Diacid C8 Diacid C6 Diacid
(h)
0 0.00 0.00 0.00 0.00 0.00
sAA105 24 0.05 0.00 0.00 0.07 0.42
48 0.00 0.00 0.00 0.00 0.00
0 0.00 0.00 0.00 0.00 0.00
sAA106 24 2.92 1.29 0.15 0.58 0.37
48 0.04 0.02 0.00 0.00 0.01
0 0.02 0.00 0.00 0.00 0.00
sAA152 24 0.58 0.55 0.07 0.43 0.03
48 0.00 0.03 0.00 0.05 0.58
0 0.00 0.00 0.00 0.00 0.00
sAA003 24 1.96 0.41 0.00 0.00 0.00
48 1.43 0.47 0.00 0.00 0.00
Fermentation of methyl myristate and oleic acid feedstocks.
Fermentations were performed essentially as described for methyl laurate
feedstock except that
2% methyl myristate or 2% oleic acid was substituted for the 2% methyl
laurate. The results of the
gas chromatography are shown in the tables below.
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Fatty acid and Diacid profile (g/L) in Methyl Myristate fermentation
Strain Time
C14 Acid C14 Diacid C12 Diacid C10 Diacid C8 Diacid
C6 Diacid
(h)
0 0.02 0.00 0.00 0.00 0.00 0.00
sAA105 24 0.02 0.00 0.00 0.00 0.00 0.29
48 0.01 0.00 0.00 0.00 0.00 0.03
0 0.01 0.00 0.00 0.00 0.00 0.00
sAA106 24 0.02 0.00 0.00 0.00 0.08 1.71
48 0.01 0.00 0.00 0.00 0.00 0.04
0 0.01 0.00 0.00 0.00 0.00 0.00
sAA232 24 0.01 0.00 0.00 0.00 0.59 0.26
48 0.01 0.00 0.00 0.00 0.35 0.47
0 0.01 0.00 0.00 0.00 0.00 0.00
sAA235 24 0.02 0.00 0.00 0.00 0.25 0.38
48 0.01 0.00 0.00 0.00 0.04 0.66
0 0.02 0.01 0.01 0.00 0.00 0.00
sAA003 24 0.55 0.25 0.00 0.00 0.00 0.00
48 0.49 0.38 0.00 0.00 0.00 0.00
Diacid profile (g/L) in Oleic acid fermentation
Strain Time C14 Diacid C12 Diacid C10 Diacid C8 Diacid C6 Diacid
(h)
sAA105 0 0.00 0.00 0.00 0.00 0.00
24 0.00 0.00 0.00 0.00 0.03
48 0.00 0.00 0.00 0.00 0.01
sAA106 0 0.00 0.00 0.00 0.00 0.00
24 0.00 0.00 0.00 0.00 1.48
48 0.00 0.00 0.00 0.00 0.42
sAA232 0 0.00 0.00 0.00 0.00 0.00
24 0.00 0.00 0.00 0.00 0.09
48 0.00 0.00 0.00 0.00 0.10
sAA235 0 0.00 0.00 0.00 0.00 0.00
24 0.00 0.00 0.00 0.00 0.10
48 0.00 0.00 0.00 0.00 0.11
Fermentations also were performed using coconut oil as a feed stock. Coconut
oil contains a
mixture of fatty acids of different carbon chain lengths. The percent
composition of fatty acids, by
weight, is about 6% capric acid (C10:0, where 0 refers to the number of double
or unsaturated
bonds), about 47% lauric acid (C12:0), about 18% myristic acid (C14:0), about
9% palmitic acid
(C16:0). About 3% stearic acid (C18:0), about 6% oleic acid (C18:1, where 1
refers to the number
of double bonds), and about 2% linoleic acid (omega-6 fatty acid, C18:2). In
some embodiments,
palm kernel oil can be substituted for coconut oil. Palm kernel oil has a
distribution of fatty acids
similar to that of coconut oil. Fermentations and GC were carried out
essentially as described
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herein with the exception of feedstock used. The result of fermentations
performed using coconut
oil as a feedstock are presented below.
Diacid profile (g/L) in Coconut Oil fermentation
Strain Time C14 Diacid C12 Diacid C10 Diacid C8 Diacid C6 Diacid
(h)
sAA105 0 0.00 0.00 0.00 0.00 0.00
24 0.00 0.00 0.00 0.00 0.00
48 0.00 0.00 0.00 0.00 0.00
sAA106 0 0.00 0.00 0.00 0.00 0.00
24 0.00 0.00 0.00 0.00 0.01
48 0.00 0.00 0.00 0.00 0.00
sAA152 0 0.00 0.00 0.00 0.00 0.00
24 0.00 0.00 0.00 0.00 0.43
48 0.00 0.00 0.00 0.00 0.45
sAA232 0 0.00 0.00 0.00 0.00 0.00
24 0.00 0.00 0.00 0.00 0.41
48 0.00 0.00 0.00 0.00 0.58
sAA235 0 0.00 0.00 0.00 0.00 0.00
24 0.00 0.00 0.00 0.00 0.43
48 0.00 0.00 0.00 0.00 0.76
Example 29: Strain evaluation of completely beta-oxidation blocked strains
Fermentations also were performed using methyl myristate as a feed stock.
Fermentations and
GC were carried out essentially as described herein with the exception of
feedstock used. The
result of fermentations performed using coconut oil as a feedstock are
presented below.
C14 Diacid production in strains with amplified CPR, CYP52A15, and/or CYP52A16
Strain 014 diacid, 72h (g/L) CPR A15 A16
sAA003 0.98 2 1 1
sAA318 1.19 3 1 1
sAA239 2.75 3 1 3
sAA319 1.37 7 1 1
sAA238 1.93 7 2 1
Example 30: Nucleic acid and amino acid sequences of novel fatty alcohol
oxidase genes
As noted above, novel fatty alcohol oxidase genes were identified and cloned.
The nucleotide and
amino acid sequences of the novel sequences are presented herein. Nucleotide
and amino acid
sequence identity comparison are shown in Example 18.
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Nucleotide Sequences
FAO-13 (SEQ ID NO:1)
ATGGCTCCATTTTTGCCCGACCAGGTCGACTACAAACACGTCGACACCCTTATGTTATTATGTGACGGGATCATCCACG
A
AACCACCGTCGACCAAATCAAAGACGTTATTGCTCCTGACTTCCCTGCTGACAAGTACGAAGAGTACGTCAGGACATTC
A
CCAAACCCTCCGAAACCCCAGGGTTCAGGGAAACCGTCTACAACACAGTCAACGCAAACACCACGGACGCAATCCACCA
G
TTCATTATCTTGACCAATGTTTTGGCATCCAGGGTCTTGGCTCCAGCTTTGACCAACTCGTTGACGCCTATCAAGGACA
T
GAGCTTGGAAGACCGTGAAAAATTGTTGGCCTCGTGGCGCGACTCCCCAATCGCTGCCAAAAGGAAGTTGTTCAGGTTG
G
TTTCTACGCTTACCTTGGTCACGTTCACGAGATTGGCCAATGAGTTGCATTTGAAAGCCATTCATTATCCAGGAAGAGA
A
GACCGTGAAAAGGCTTATGAAACCCAGGAGATTGACCCTTTTAAGTACCAGTTTTTGGAAAAACCGAAGTTTTACGGCG
C
TGAGTTGTACTTGCCAGATATTGATGTGATCATTATTGGATCTGGTGCCGGTGCTGGTGTTGTGGCCCACACTTTGGCC
A
ACGATGGCTTCAAGAGTTTGGTTTTGGAAAAGGGCAAATACTTTAGCAACTCCGAGTTGAACTTTGATGACAAGGACGG
C
GTTCAAGAATTATACCAAAGTGGAGGTACTTTGACTACAGTCAACCAACAGTTGTTTGTTCTTGCTGGTTCCACTTTTG
G
TGGCGGTACCACTGTCAATTGGTCAGCCTGTCTTAAGACGCCATTCAAGGTGCGTAAGGAATGGTATGATGAGTTTGGT
G
TTGACTTTGCTGCTGATGAAGCATACGATAAAGCGCAGGATTATGTTTGGCAGCAAATGGGAGCTTCTACCGAAGGCAT
C
ACCCACTCTTTGGCTAACGAGATTATTATTGAAGGTGGTAAGAAATTAGGTTACAAGGCCAAGGTATTAGACCAAAACA
G
CGGTGGTCATCCTCAGCACAGATGCGGTTTCTGTTATTTGGGCTGTAAGCACGGTATCAAGCAGGGTTCTGTTAATAAC
T
GGTTTAGAGACGCAGCTGCCCACGGTTCCCAGTTCATGCAACAGGTTAGAGTTTTGCAAATACTTAACAAGAAGGGGAT
C
GCTTACGGTATCTTGTGTGAGGATGTTGTAACCGGCGCCAAGTTCACCATTACTGGCCCCAAAAAGTTTGTTGTTGCTG
C
CGGTGCTTTGAACACTCCATCTGTGTTGGTCAACTCCGGCTTCAAGAACAAGAACATCGGTAAGAACTTAACTTTGCAC
C
CAGTTTCTGTCGTGTTTGGTGATTTTGGCAAAGACGTTCAAGCAGACCACTTCCACAACTCCATCATGACTGCCCTTTG
T
TCAGAAGCCGCTGATTTAGACGGCAAGGGCCATGGATGCAGAATTGAAACCATCTTGAACGCTCCATTCATCCAGGCTT
C
ATTCTTACCATGGAGAGGTAGTAACGAGGCTAGACGAGACTTGTTGCGTTACAACAACATGGTGGCGATGTTGCTCCTT
A
GTCGTGACACCACCAGTGGTTCCGTTTCTGCTCATCCAACCAAACCTGAAGCTTTGGTTGTCGAGTACGACGTGAACAA
G
TTTGACAGAAACTCGATCTTGCAGGCATTGTTGGTCACTGCTGACTTGTTGTATATCCAAGGTGCCAAGAGAATCCTTA
G
TCCACAGGCATGGGTGCCAATTTTTGAATCCGACAAGCCAAAGGATAAGAGATCAATCAAGGACGAGGACTATGTCGAA
T
GGAGAGCCAAGGTTGCCAAGATTCCTTTCGACACCTACGGCTCACCTTATGGTTCGGCACATCAAATGTCTTCTTGCCG
T
ATGTCAGGTAAGGGTCCTAAATACGGTGCTGTTGACACCGATGGTAGATTGTTTGAATGTTCGAATGTTTATGTTGCCG
A
TGCAAGTCTTTTGCCAACTGCAAGCGGTGCCAACCCTATGGTCACCACCATGACTCTTGCCAGACATGTTGCGTTAGGT
T
TGGCAGACTCCTTGAAGACCAAAGCCAAGTTGTAG
FAO-17 (SEQ ID NO:2)
ATGGCTCCATTTTTGCCCGACCAGGTCGACTACAAACACGTCGACACCCTTATGTTATTATGTGACGGGATCATCCACG
A
AACCACCGTGGACGAAATCAAAGACGTCATTGCCCCTGACTTCCCCGCCGACAAATACGAGGAGTACGTCAGGACATTC
A
CCAAACCCTCCGAAACCCCAGGGTTCAGGGAAACCGTCTACAACACCGTCAACGCAAACACCATGGATGCAATCCACCA
G
TTCATTATCTTGACCAATGTTTTGGGATCAAGGGTCTTGGCACCAGCTTTGACCAACTCGTTGACTCCTATCAAGGACA
T
GAGCTTGGAAGACCGTGAAAAGTTGTTAGCCTCGTGGCGTGACTCCCCTATTGCTGCTAAAAGGAAGTTGTTCAGGTTG
G
TTTCTACGCTTACCTTGGTCACGTTCACGAGATTGGCCAATGAGTTGCATTTGAAAGCCATTCATTATCCAGGAAGAGA
A
GACCGTGAAAAGGCTTATGAAACCCAGGAGATTGACCCTTTTAAGTACCAGTTTTTGGAAAAACCGAAGTTTTACGGCG
C
TGAGTTGTACTTGCCAGATATTGATGTGATCATTATTGGATCTGGTGCCGGTGCTGGTGTTGTGGCCCACACTTTGGCC
A
ACGATGGCTTCAAGAGTTTGGTTTTGGAAAAGGGCAAATACTTTAGCAACTCCGAGTTGAACTTTGATGACAAGGACGG
C
GTTCAAGAATTATACCAAAGTGGAGGTACTTTGACTACAGTCAACCAACAGTTGTTTGTTCTTGCTGGTTCCACTTTTG
G
TGGCGGTACCACTGTCAATTGGTCAGCCTGTCTTAAGACGCCATTCAAGGTGCGTAAGGAATGGTATGATGAGTTTGGT
G
TTGACTTTGCTGCTGATGAAGCATACGATAAAGCGCAGGATTATGTTTGGCAGCAAATGGGAGCTTCTACCGAAGGCAT
C
ACCCACTCTTTGGCTAACGAGATTATTATTGAAGGTGGTAAGAAATTAGGTTACAAGGCCAAGGTATTAGACCAAAACA
G
CGGTGGTCATCCTCAGCACAGATGCGGTTTCTGTTATTTGGGTTGTAAGCACGGTATCAAGCAGGGCTCTGTTAATAAC
T
GGTTTAGAGACGCAGCTGCCCACGGTTCTCAGTTCATGCAACAGGTTAGAGTTTTGCAAATCCTTAACAAGAAGGGCAT
C
GCTTATGGTATCTTGTGTGAGGATGTTGTAACCGGTGCCAAGTTCACCATTACTGGCCCCAAAAAGTTTGTTGTTGCCG
C
CGGCGCCTTAAACACTCCATCTGTGTTGGTCAACTCCGGATTCAAGAACAAGAACATCGGTAAGAACTTAACTTTGCAT
C
CAGTTTCTGTCGTGTTTGGTGATTTTGGCAAAGACGTTCAAGCAGACCACTTCCACAACTCCATCATGACTGCCCTTTG
T
TCAGAAGCCGCTGATTTAGACGGCAAGGGCCATGGATGCAGAATTGAAACCATCTTGAACGCTCCATTCATCCAGGCTT
C
ATTCTTACCATGGAGAGGTAGTAACGAGGCTAGACGAGACTTGTTGCGTTACAACAACATGGTGGCGATGTTGCTCCTT
A
GTCGTGACACCACCAGTGGTTCCGTTTCTGCTCATCCAACCAAACCTGAAGCTTTGGTTGTCGAGTACGACGTGAACAA
G
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TTTGACAGAAACTCGATCTTGCAGGCATTGTTGGTCACTGCTGACTTGTTGTATATCCAAGGTGCCAAGAGAATCCTTA
G
TCCACAGGCATGGGTGCCAATTTTTGAATCCGACAAGCCAAAGGATAAGAGATCAATCAAGGACGAGGACTATGTCGAA
T
GGAGAGCCAAGGTTGCCAAGATTCCTTTCGACACCTACGGCTCACCTTATGGTTCGGCACATCAAATGTCTTCTTGCCG
T
ATGTCAGGTAAGGGTCCTAAATACGGTGCTGTTGACACCGATGGTAGATTGTTTGAATGTTCGAATGTTTATGTTGCCG
A
TGCAAGTCTTTTGCCAACTGCAAGCGGTGCCAACCCTATGGTCACCACCATGACTCTTGCAAGACATGTTGCGTTAGGT
T
TGGCAGACTCCTTGAAGACCAAGGCCAAGTTGTAG
FAO-20 (SEQ ID NO:3)
ATGGCTCCATTTTTGCCCGACCAGGTCGACTACAAACACGTCGACACCCTTATGTTATTATGTGACGGGATCATCCACG
A
AACCACCGTCGACCAAATCAAAGACGTTATTGCTCCTGACTTCCCTGCTGACAAGTACGAAGAGTACGTCAGGACATTC
A
CCAAACCCTCCGAAACCCCAGGGTTCAGGGAAACCGTCTACAACACAGTCAACGCAAACACCACGGACGCAATCCACCA
G
TTCATTATCTTGACCAATGTTTTGGCATCCAGGGTCTTGGCTCCAGCTTTGACCAACTCGTTGACGCCTATCAAGGACA
T
GAGCTTGGAAGACCGTGAAAAATTGTTGGCCTCGTGGCGCGACTCCCCAATCGCTGCCAAAAGGAAATTGTTCAGGTTG
G
TTTCCACGCTTACCTTGGTTACTTTCACGAGATTGGCCAATGAGTTGCATTTGAAAGCCATTCACTATCCAGGAAGAGA
A
GACCGTGAAAAGGCTTATGAAACCCAGGAGATTGACCCTTTCAAGTACCAGTTTATGGAAAAGCCAAAGTTTGACGGCG
C
TGAGTTGTACTTGCCAGATATTGATGTTATCATTATTGGATCTGGTGCCGGTGCTGGTGTTGTGGCCCACACTTTGGCC
A
ACGATGGCTTCAAGAGTTTGGTTTTGGAAAAGGGCAAATACTTTAGCAACTCCGAGTTGAACTTTGATGACAAGGACGG
C
GTTCAAGAATTATACCAAAGTGGAGGTACTTTGACTACAGTCAACCAACAGTTGTTTGTTCTTGCTGGTTCCACTTTTG
G
TGGCGGTACCACTGTCAATTGGTCAGCCTGTCTTAAGACGCCATTCAAGGTGCGTAAGGAATGGTATGATGAGTTTGGT
G
TTGACTTTGCTGCTGATGAAGCATACGATAAAGCGCAGGATTATGTTTGGCAGCAAATGGGAGCTTCTACCGAAGGCAT
C
ACCCACTCTTTGGCTAACGAGATTATTATTGAAGGTGGTAAGAAATTAGGTTACAAGGCCAAGGTATTAGACCAAAACA
G
CGGTGGTCATCCTCAGCACAGATGCGGTTTCTGTTATTTGGGCTGTAAGCACGGTATCAAGCAGGGTTCTGTTAATAAC
T
GGTTTAGAGACGCAGCTGCCCACGGTTCCCAGTTCATGCAACAGGTTAGAGTTTTGCAAATACTTAACAAGAAGGGGAT
C
GCTTACGGTATCTTGTGTGAGGATGTTGTAACCGGCGCCAAGTTCACCATTACTGGCCCCAAAAAGTTTGTTGTTGCTG
C
CGGTGCTTTGAACACTCCATCTGTGTTGGTCAACTCCGGCTTCAAGAACAAGAACATCGGTAAGAACTTAACTTTGCAC
C
CAGTTTCTGTCGTGTTTGGTGATTTTGGCAAAGACGTTCAAGCAGACCACTTCCACAACTCCATCATGACTGCCCTTTG
T
TCAGAAGCCGCTGATTTAGACGGCAAGGGCCATGGATGCAGAATTGAAACCATCTTGAACGCTCCATTCATCCAGGCTT
C
ATTCTTACCATGGAGAGGTAGTAACGAGGCTAGACGAGACTTGTTGCGTTACAACAACATGGTGGCGATGTTGCTCCTT
A
GTCGTGACACCACCAGTGGTTCCGTTTCTGCTCATCCAACCAAACCTGAAGCTTTGGTTGTCGAGTACGACGTGAACAA
G
TTTGACAGAAACTCGATCTTGCAGGCATTGTTGGTCACTGCTGACTTGTTGTATATCCAAGGTGCCAAGAGAATCCTTA
G
TCCACAGGCATGGGTGCCAATTTTTGAATCCGACAAGCCAAAGGATAAGAGATCAATCAAGGACGAGGACTATGTCGAA
T
GGAGAGCCAAGGTTGCCAAGATTCCTTTCGACACCTACGGCTCACCTTATGGTTCGGCACATCAAATGTCTTCTTGCCG
T
ATGTCAGGTAAGGGTCCTAAATACGGTGCTGTTGACACCGATGGTAGATTGTTTGAATGTTCGAATGTTTATGTTGCCG
A
TGCAAGTCTTTTGCCAACTGCAAGCGGTGCCAACCCTATGGTCACCACCATGACTCTTGCCAGACATGTTGCGTTAGGT
T
TGGCAGACTCCTTGAAGACCAAAGCCAAGTTGTAG
FAO2A (SEQ ID NO:4)
ATGAATACCTTCTTGCCAGACGTGCTCGAATACAAACACGTCGACACCCTTTTGTTATTGTGTGACGGGATCATCCACG
A
AACCACAGTCGATCAGATCAAGGACGCCATTGCTCCCGACTTCCCTGAGGACCAGTACGAGGAGTATCTCAAGACCTTC
A
CCAAGCCATCTGAGACCCCTGGGTTCAGAGAAGCCGTCTACGACACGATCAACGCCACCCCAACCGATGCCGTGCACAT
G
TGTATTGTCTTGACCACCGCATTGGACTCCAGAATCTTGGCCCCCACGTTGACCAACTCGTTGACGCCTATCAAGGATA
T
GACCTTGAAGGAGCGTGAACAATTGTTGGCCTCTTGGCGTGATTCCCCGATTGCGGCAAAGAGAAGATTGTTCAGATTG
A
TTTCCTCGCTTACCTTGACGACGTTTACGAGATTGGCCAGCGAATTGCACTTGAAAGCCATCCACTACCCTGGCAGAGA
C
TTGCGTGAAAAGGCGTATGAAACCCAGGTGGTTGACCCTTTCAGGTACCTGTTTATGGAGAAACCAAAGTTTGACGGCG
C
CGAATTGTACTTGCCAGATATCGACGTCATCATCATTGGATCAGGCGCCGGTGCTGGTGTCATGGCCCACACTCTCGCC
A
ACGACGGGTTCAAGACCTTGGTTTTGGAAAAGGGAAAGTATTTCAGCAACTCCGAGTTGAACTTTAATGACGCTGATGG
C
GTGAAAGAGTTGTACCAAGGTAAAGGTGCTTTGGCCACCACCAATCAGCAGATGTTTATTCTTGCCGGTTCCACTTTGG
G
CGGTGGTACCACTGTCAACTGGTCTGCTTGCCTTAAAACACCATTTAAAGTGCGTAAGGAGTGGTACGACGAGTTTGGT
C
TTGAATTTGCTGCCGATGAAGCCTACGACAAAGCGCAGGATTATGTTTGGAAACAAATGGGTGCTTCAACAGATGGAAT
C
ACTCACTCCTTGGCCAACGAAGTTGTGGTTGAAGGAGGTAAGAAGTTGGGCTACAAGAGCAAGGAAATTGAGCAGAACA
A
CGGTGGCCACCCTGACCACCCATGTGGTTTCTGTTACTTGGGCTGTAAGTACGGTATTAAACAGGGTTCTGTGAATAAC
T
GGTTTAGAGACGCAGCTGCCCACGGGTCCAAGTTCATGCAACAAGTCAGAGTTGTGCAAATCCTCAACAAGAATGGCGT
C
GCTTATGGTATCTTGTGTGAGGATGTCGAAACCGGAGTCAGGTTCACTATTAGTGGCCCCAAAAAGTTTGTTGTTTCTG
C
TGGTTCTTTGAACACGCCAACTGTGTTGACCAACTCCGGATTCAAGAACAAGCACATTGGTAAGAACTTGACGTTGCAC
C
CAGTTTCCACCGTGTTTGGTGACTTTGGCAGAGACGTGCAAGCCGACCATTTCCACAAATCTATTATGACTTCGCTTTG
T
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TACGAGGTTGCTGACTTGGACGGCAAGGGCCACGGATGCAGAATCGAAACCATCTTGAACGCTCCATTCATCCAAGCTT
C
TTTGTTGCCATGGAGAGGAAGTGACGAGGTCAGAAGAGACTTGTTGCGTTACAACAACATGGTGGCCATGTTGCTTATC
A
CGCGTGATACCACCAGTGGTTCAGTTTCTGCTGACCCAAAGAAGCCCGACGCTTTGATTGTCGACTATGAGATTAACAA
G
TTTGACAAGAATGCCATCTTGCAAGCTTTCTTGATCACTTCCGACATGTTGTACATTGAAGGTGCCAAGAGAATCCTCA
G
TCCACAGCCATGGGTGCCAATCTTTGAGTCGAACAAGCCAAAGGAGCAAAGAACGATCAAGGACAAGGACTATGTTGAG
T
GGAGAGCCAAGGCTGCTAAGATACCTTTCGACACCTACGGTTCTGCATATGGGTCCGCACATCAAATGTCCACCTGTCG
T
ATGTCCGGAAAGGGTCCTAAATACGGTGCTGTTGATACTGATGGTAGATTGTTTGAATGTTCGAATGTCTATGTTGCTG
A
TGCTAGTGTTTTGCCTACTGCCAGCGGTGCCAACCCAATGATATCCACCATGACCTTTGCTAGACAGATTGCGTTAGGT
T
TGGCTGACTCCTTGAAGACCAAACCCAAGTTGTAG
FAO2B (SEQ ID NO:5)
ATGAATACCTTCTTGCCAGACGTGCTCGAATACAAACACGTCGATACCCTTTTGTTATTATGTGACGGGATCATCCACG
A
AACCACAGTCGACCAGATCAGGGACGCCATTGCTCCCGACTTCCCTGAAGACCAGTACGAGGAGTATCTCAAGACCTTC
A
CCAAGCCATCTGAGACCCCTGGGTTCAGAGAAGCCGTCTACGACACGATCAACAGCACCCCAACCGAGGCTGTGCACAT
G
TGTATTGTATTGACCACCGCATTGGACTCGAGAATCTTGGCCCCCACGTTGACCAACTCGTTGACGCCTATCAAGGATA
T
GACCTTGAAAGAGCGTGAACAATTGTTGGCTGCCTGGCGTGATTCCCCGATCGCGGCCAAGAGAAGATTGTTCAGATTG
A
TTTCCTCACTTACCTTGACGACCTTTACGAGATTGGCCAGCGACTTGCACTTGAGAGCCATCCACTACCCTGGCAGAGA
C
TTGCGTGAAAAGGCATATGAAACCCAGGTGGTTGACCCTTTCAGGTACCTGTTTATGGAAAAACCAAAGTTTGACGGCA
C
CGAGTTGTACTTGCCAGATATCGACGTCATCATCATTGGATCCGGTGCCGGTGCTGGTGTCATGGCCCACACTTTAGCC
A
ACGACGGGTACAAGACCTTGGTTTTGGAAAAGGGAAAGTATTTCAGCAACTCCGAGTTGAACTTTAATGATGCCGATGG
T
ATGAAAGAGTTGTACCAAGGTAAATGTGCGTTGACCACCACGAACCAGCAGATGTTTATTCTTGCCGGTTCCACTTTGG
G
CGGTGGTACCACTGTTAACTGGTCTGCTTGTCTTAAAACACCATTTAAAGTGCGTAAGGAGTGGTACGACGAGTTTGGT
C
TTGAATTTGCTGCCGACGAAGCCTACGACAAAGCACAAGACTATGTTTGGAAACAAATGGGCGCTTCTACCGAAGGAAT
C
ACTCACTCTTTGGCGAACGCGGTTGTGGTTGAAGGAGGTAAGAAGTTGGGTTACAAGAGCAAGGAAATCGAGCAGAACA
A
TGGTGGCCATCCTGACCACCCCTGTGGTTTCTGTTACTTGGGCTGTAAGTACGGTATTAAGCAGGGTTCTGTGAATAAC
T
GGTTTAGAGACGCAGCTGCCCACGGGTCCAAGTTCATGCAACAAGTCAGAGTTGTGCAAATCCTCCACAATAAAGGCGT
C
GCTTATGGCATCTTGTGTGAGGATGTCGAGACCGGAGTCAAATTCACTATCAGTGGCCCCAAAAAGTTTGTTGTTTCTG
C
AGGTTCTTTGAACACGCCAACGGTGTTGACCAACTCCGGATTCAAGAACAAACACATCGGTAAGAACTTGACGTTGCAC
C
CAGTTTCGACCGTGTTTGGTGACTTTGGCAGAGACGTGCAAGCCGACCATTTCCACAAATCTATTATGACTTCGCTCTG
T
TACGAAGTCGCTGACTTGGACGGCAAGGGCCACGGATGCAGAATCGAGACCATCTTGAACGCTCCATTCATCCAAGCTT
C
TTTGTTGCCATGGAGAGGAAGCGACGAGGTCAGAAGAGACTTGTTGCGTTACAACAACATGGTGGCCATGTTGCTTATC
A
CCCGTGACACCACCAGTGGTTCAGTTTCTGCTGACCCAAAGAAGCCCGACGCTTTGATTGTCGACTATGACATCAACAA
G
TTTGACAAGAATGCCATCTTGCAAGCTTTCTTGATCACCTCCGACATGTTGTACATCGAAGGTGCCAAGAGAATCCTCA
G
TCCACAGGCATGGGTGCCAATCTTTGAGTCGAACAAGCCAAAGGAGCAAAGAACAATCAAGGACAAGGACTATGTCGAA
T
GGAGAGCCAAGGCTGCCAAGATACCTTTCGACACCTACGGTTCTGCCTATGGGTCCGCACATCAAATGTCCACCTGTCG
T
ATGTCCGGAAAGGGTCCTAAATACGGCGCCGTTGATACCGATGGTAGATTGTTTGAATGTTCGAATGTCTATGTTGCTG
A
TGCTAGTGTTTTGCCTACTGCCAGCGGTGCCAACCCAATGATCTCCACCATGACGTTTGCTAGACAGATTGCGTTAGGT
T
TGGCTGACTCTTTGAAGACCAAACCCAAGTTGTAG
In addition to the novel FAO genes isolated, a sequence substantially
identical to the sequence
used for primer design, described above, also was isolated. The nucleotide
sequence of the gene
is presented below.
FAO-18 (SEQ ID NO: 6)
ATGGCTCCATTTTTGCCCGACCAGGTCGACTACAAACACGTCGACACCCTTATGTTATTATGTGACGGGATCATCCACG
A
AACCACCGTGGACGAAATCAAAGACGTCATTGCCCCTGACTTCCCCGCCGACAAATACGAGGAGTACGTCAGGACATTC
A
CCAAACCCTCCGAAACCCCAGGGTTCAGGGAAACCGTCTACAACACCGTCAACGCAAACACCATGGATGCAATCCACCA
G
TTCATTATCTTGACCAATGTTTTGGGATCAAGGGTCTTGGCACCAGCTTTGACCAACTCGTTGACTCCTATCAAGGACA
T
GAGCTTGGAAGACCGTGAAAAGTTGTTAGCCTCGTGGCGTGACTCCCCTATTGCTGCTAAAAGGAAGTTGTTCAGGTTG
G
TTTCTACGCTTACCTTGGTCACGTTCACGAGATTGGCCAATGAGTTGCATTTGAAAGCCATTCATTATCCAGGAAGAGA
A
GACCGTGAAAAGGCTTATGAAACCCAGGAGATTGACCCTTTTAAGTACCAGTTTTTGGAAAAACCGAAGTTTTACGGCG
C
192
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TGAGTTGTACTTGCCAGATATTGATGTGATCATTATTGGATCTGGGGCCGGTGCTGGTGTCGTGGCCCACACTTTGACC
A
ACGACGGCTTCAAGAGTTTGGTTTTGGAAAAGGGCAGATACTTTAGCAACTCCGAGTTGAACTTTGATGACAAGGACGG
G
GTTCAAGAATTATACCAAAGTGGAGGTACTTTGACCACCGTCAACCAGCAGTTGTTTGTTCTTGCTGGTTCCACTTTTG
G
TGGTGGTACCACTGTCAATTGGTCGGCCTGTCTTAAAACGCCATTCAAGGTGCGTAAGGAATGGTATGATGAGTTTGGC
G
TTGACTTTGCTGCCGATGAAGCCTACGACAAAGCACAGGATTATGTTTGGCAGCAAATGGGAGCTTCTACCGAAGGCAT
C
ACCCACTCTTTGGCTAACGAGATTATTATTGAAGGTGGCAAGAAATTAGGTTACAAGGCCAAGGTATTAGACCAAAACA
G
CGGTGGTCATCCTCATCACAGATGCGGTTTCTGTTATTTGGGTTGTAAGCACGGTATCAAGCAGGGCTCTGTTAATAAC
T
GGTTTAGAGACGCAGCTGCCCACGGTTCTCAGTTCATGCAACAGGTTAGAGTTTTGCAAATCCTTAACAAGAAGGGCAT
C
GCTTATGGTATCTTGTGTGAGGATGTTGTAACCGGTGCCAAGTTCACCATTACTGGCCCCAAAAAGTTTGTTGTTGCCG
C
CGGCGCCTTAAACACTCCATCTGTGTTGGTCAACTCCGGATTCAAGAACAAGAACATCGGTAAGAACTTAACTTTGCAT
C
CAGTTTCTGTCGTGTTTGGTGATTTTGGCAAAGACGTTCAAGCAGATCACTTCCACAACTCCATCATGACTGCTCTTTG
T
TCAGAAGCCGCTGATTTAGACGGCAAGGGTCATGGATGCAGAATTGAAACCATCTTGAACGCTCCATTCATCCAGGCTT
C
ATTCTTACCATGGAGAGGTAGTAACGAGGCTAGACGAGACTTGTTGCGTTACAACAACATGGTGGCCATGTTACTTCTT
A
GTCGTGATACCACCAGTGGTTCCGTTTCGTCCCATCCAACTAAACCTGAAGCATTAGTTGTCGAGTACGACGTGAACAA
G
TTTGACAGAAACTCCATCTTGCAGGCATTGTTGGTCACTGCTGACTTGTTGTACATTCAAGGTGCCAAGAGAATCCTTA
G
TCCCCAACCATGGGTGCCAATTTTTGAATCCGACAAGCCAAAGGATAAGAGATCAATCAAGGACGAGGACTATGTCGAA
T
GGAGAGCCAAGGTTGCCAAGATTCCTTTTGACACCTACGGCTCGCCTTATGGTTCGGCGCATCAAATGTCTTCTTGTCG
T
ATGTCAGGTAAGGGTCCTAAATACGGTGCTGTTGATACCGATGGTAGATTGTTTGAATGTTCGAATGTTTATGTTGCTG
A
CGCTAGTCTTTTGCCAACTGCTAGCGGTGCTAATCCTATGGTCACCACCATGACTCTTGCAAGACATGTTGCGTTAGGT
T
TGGCAGACTCCTTGAAGACCAAGGCCAAGTTGTAG
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H H H H H H 4< F, F, F, F, F, F, 4, 4, H H H H H H 4< 0
0 0 0 0 0 4<
H H H H H H 4< CD CD CD CD CD CD 4< 4 CD CD CD CD CD CD
4< CD CD CD CD CD CD 4<
H H H H H H 4< F, F, F, F, F, F, 4, 0 0 0 0 0 0 4<
CD CD CD CD CD CD 4< F, F, F, 4,
HHHHOO CD CD CD CD CD CD 4< H H H H H H 4< H H H H
H H 4< F<F<H00
H H H H H H 4< CD CD CD CD CD CD 4< F, F, F, F, F, F, 4, F, F,
F, F, F, F, 4, 0 0 0 0 0 0 4<
i<
4 H H H H H H 4< 0 0 0 0 0 0 4< CD CD CD CD CD CD 4<
O 0 0 0 0 0
U U U U H U CD CD CD
CD CD CD 4<
H H H H H H 4<
4< 4
0 0 0 0 0 0 4< 0 0 0 0 0 0 4< 0 0 0 0 0 0 4<
O 0 0 0 0 0 4<
CD CD CD CD CD CD 4< U U U U H U CD CD CD CD U U U
U U H H H H H
H H H H H H 4< H H H H H H 4< 0 0 0 0 0 0 4< F, F, F, F, F, F, 4,
0 0 0 0 0 0 4<
CD CD CD CD CD CD 4< CD CD CD CD CD CD 4< H H H H H H 4< CD
CD CD CD CD CD 4< 0 0 0 0 0 0 4<
CD CD CD CD CD CD 4< H H H H H H 4< H H H H CD CD H H H H H
H 4< CD CD CD CD CD CD 4<
CD CD CD CD CD CD 4< F, F, F, F, F, F, 4, CD CD CD CD CD CD 4<
H H H H H H 4< H H H H H H 4<
H H H H H H 4< H H H H H H 4< CD CD CD CD CD CD 4< CD CD CD CD CD CD
4< H H H H H H 4<
0 0 0 0 0 0 4< H H H H H H 4< HHHHHO H H H H H H 4<
O 0 0 0 0 0 4< F, F, F, F, F, F, 4, F, F, F, F, F, F,
4, 0 0 0 0 0 0 4< H H H H H H 4<
CD CD CD U U CD CD CD CD CD CD CD 4< H H H H H H 4< CD CD CD CD CD
CD 4< 0 0 0 0 CD CD
CD CD CD Pul CD CD CD CD CD CD CD CD 4< H H H H F: U
HHHHHO H H H H H H 4<
0 0 0 0 0 0 4< CD CD CD CD CD CD 4< CD CD CD
CD CD CD 4<
O 0 0 0 0 0 4<CD CD CD CD -Kt; 0 0 0
0 CD CD CD CD CD CD CD CD 4< F, F, F, 4,
0 0 0 0 0 0 4< F, F, F= CD H H 0 0 0 0 0 0 4< FFHHH
O 0 0 0 0 0 4< Fz Fz Fz Fz Fz Fz 4, 0 0 0 0 0 0 4<
Fz Fz Fz Fz Fz Fz 4, 0 0 0 0 0 0 4<
O 0 0 0 0 0 4< CD CD CD CD CD CD 4< H H H H H H 4<
H H H H H H 4< CD CD CD CD CD CD 4<
H H H H H H 4< CD CD CD CD CD CD 4< U U U U H H 4, HHHOHH
CD CD CD CD CD CD 4< 4< CD CD CD CD CD CD 4< 4,
FzFzFz Fz Fz Fz 4,
FzFzFzFzFz Fz 4, * OLD OLD OLD* 4 00000CD*
HHHHOO 0 0 0 0 0 0 4< 0 0 0 0 0 0 4< H H H H H H 4< U U U H H H
H H H H H H 4< H H H H H H 4< Fz Fz Fz Fz Fz Fz 4, 0 0 0 0 0 0 4<
0 0 0 0 0 0 4<
O 0 0 0 0 0 4< * H H H H H H 4< 0 0 0 0 0 0 4<
CD CD CD CD CD CD 4<
O 0 0 0 0 0< 0 0 0 0 0 0 4< H H H H H H 4< H H H H H H 4<
H H H H H H 4< 0 0 0 0 0 0 4< 0 0 0 0 0 0 4, CD CD CD CD CD CD 4<
H H H H H H 4<
i<
H H H H
4 -e. -e.
0 0 0 0 0 0 4< CD CD CD CD CD CD 4<
CD CD CD CD CD CD 4< CD CD CD CD CD CD
4<
H H H H H H 4<
CD CD CD CD CD CD 4< CD CD CD CD CD CD 4< Fz Fz Fz
Fz Fz Fz 4, i< FzFzFz Fz Fz Fz 4,
FzFzFzFzFz Fz 4, OLD OLD OLD* 4 H H H H H H 4<
CD CD CD CD CD CD 4< * U U U H U U HHHHFF
HHHHOO
i<
4 CD 00* H H H H H H 4<
H H H H H H 4< CD CD CD CD CD CD 4< CD CD CD CD
CD 4< H H H H H H4<
CD CD CD CD CD CD 4<
O 0 0 0 0 0 4< H H H H CD CD H H H H H H 4< Fz f, Fz1100
H H H H H H 4<
O 0 0 0 0 0 4< Fz Fz Fz Fz Fz Fz 4, 0 0 0
0 0 0 4< 0 0 0 0 0 0 4< i<
00000CD* OLD OLD OLD 4< 000000* H H H H H H 4<
4
H H H H H H 4< CD CD CD CD CD CD 4< H H H H F: F: 0 0 0 0 0 L9*
0 0 0 0 0 L9*
H H H H H H 4< H H H H H H 4< 0 0 0 0 0 0 4<
CD CD CD CD CD CD 4< 4 Fz Fz Fz Fz Fz Fz 4, Fz Fz
Fz Fz Fz Fz 4, H H H H H H 4<
Cn 0 C--- CO
,¨ICV,-1,¨I ni,Q ,¨ICV,-1,-1 ni,Q ,¨ICV,-1,¨I ni,Q
,¨ICV,-1,¨I ni,Q ,¨ICV,-1,¨I ni,Q
MINN MINN MINN MINN MINN
O00000 000000 000000 000000 000 000
FzFzFzFzFz Fz FzFz FzFzFzFz FzFzFzFz FzFz Fz
FzFzFzFzFz FzFzFz FzFzFz
LO 0 LO 0 LO 0 LO 0
N¨ N¨ (NI (NI CO co 71-
200
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DDDDDD
-1 -1 -1 -1 -1
CVC\ICVC\ICV
UUUUU
UUUUU 04c
CD CD CD CD 0 0 *
CD CD CD CD 0 0 *
H H H H H H
H H H H H H
H
U= UUUU
H H H H H H
UUUUU
CD CD CD CD CD CD*
HH
UUUUU
CD CD CD CD CD CD*
CD CD CD CD CD CD*
H H H H H H
H H H H H H
H H H H H H
CD CD CD CD CD CD*
CD CD CD CD CD CD*
H H H H H H
H H H H H H
CD CD CD CD CD CD*
UUUUU
CD CD CD CD CD CD*
H H H H H H
H H H H H H
CD CD CD CD r= r=
HHHHCD
UUUUU
CD CD CD CD CD CD*
U U H
U U U U U U
0*
H H H H H H
H H H H H H CVC\ICVCVCVN
O000H H
H H U CD CD CD CD CD CD
0*
HHHHHH-x
CD CD CD CD CD CD CD CD CD CD CD CD
HHHHHH HHHHHH-x
HHHHHH-x
O000004c 000000*
O000004c
4*
O00000-x 000000*
O00000* 000000*
4 4 4 4 H 0000uu
uuuufu
H H H H H H
CD CD CD CD r= r=
Cn0C---00 Cn0C---00
rci,Q rci,Q
MINN MINN
O00000 000000
LO O LO
201
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Amino acid sequences
FAO-1 - SEQ ID NO:7
MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDEIKDVIAPDFPADKYEEYVRTFTKPSETPGFRETVYNTVNANTMDAIH
Q
FIILTNVLGSRVLAPALTNSLTPIKDMSLEDREKLLASWRDSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHYPGR
E
DREKAYETQEIDPFKYQFLEKPKFYGAELYLPDIDVIIIGSGAGAGVVAHTLTNDGFKSLVLEKGRYFSNSELNFDDKD
G
VQELYQSGGTLTTVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADEAYDKAQDYVWQQMGASTEG
I
THSLANEIIIEGGKKLGYKAKVLDQNSGGHPHHRCGFCYLGCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKG
I
AYGILCEDVVTGAKFTITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSVVFGDFGKDVQADHFHNSIMTAL
C
SEAADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSGSVSSHPTKPEALVVEYDVN
K
FDRNSILQALLVTADLLYIQGAKRILSPQPWVPIFESDKPKDKRSIKDEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSC
R
MSGKGPKYGAVDTDGRLFECSNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL
FAO-13 - SEQ ID NO:8
MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDQIKDVIAPDFPADKYEEYVRTFTKPSETPGFRETVYNTVNANTTDAIH
Q
FIILTNVLASRVLAPALTNSLTPIKDMSLEDREKLLASWRDSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHYPGR
E
DREKAYETQEIDPFKYQFLEKPKFYGAELYLPDIDVIIIGSGAGAGVVAHTLANDGFKSLVLEKGKYFSNSELNFDDKD
G
VQELYQSGGTLTTVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADEAYDKAQDYVWQQMGASTEG
I
THSLANEIIIEGGKKLGYKAKVLDQNSGGHPQHRCGFCYLGCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKG
I
AYGILCEDVVTGAKFTITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSVVFGDFGKDVQADHFHNSIMTAL
C
SEAADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSGSVSAHPTKPEALVVEYDVN
K
FDRNSILQALLVTADLLYIQGAKRILSPQAWVPIFESDKPKDKRSIKDEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSC
R
MSGKGPKYGAVDTDGRLFECSNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL
FAO-20 - SEQ ID NO:9
MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDQIKDVIAPDFPADKYEEYVRTFTKPSETPGFRETVYNTVNANTTDAIH
Q
FIILTNVLASRVLAPALTNSLTPIKDMSLEDREKLLASWRDSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHYPGR
E
DREKAYETQEIDPFKYQFMEKPKFDGAELYLPDIDVIIIGSGAGAGVVAHTLANDGFKSLVLEKGKYFSNSELNFDDKD
G
VQELYQSGGTLTTVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADEAYDKAQDYVWQQMGASTEG
I
THSLANEIIIEGGKKLGYKAKVLDQNSGGHPQHRCGFCYLGCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKG
I
AYGILCEDVVTGAKFTITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSVVFGDFGKDVQADHFHNSIMTAL
C
SEAADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSGSVSAHPTKPEALVVEYDVN
K
FDRNSILQALLVTADLLYIQGAKRILSPQAWVPIFESDKPKDKRSIKDEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSC
R
MSGKGPKYGAVDTDGRLFECSNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL
FAO-17 - SEQ ID NO: 10
MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDEIKDVIAPDFPADKYEEYVRTFTKPSETPGFRETVYNTVNANTMDAIH
Q
FIILTNVLGSRVLAPALTNSLTPIKDMSLEDREKLLASWRDSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHYPGR
E
DREKAYETQEIDPFKYQFLEKPKFYGAELYLPDIDVIIIGSGAGAGVVAHTLANDGFKSLVLEKGKYFSNSELNFDDKD
G
VQELYQSGGTLTTVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADEAYDKAQDYVWQQMGASTEG
I
THSLANEIIIEGGKKLGYKAKVLDQNSGGHPQHRCGFCYLGCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKG
I
AYGILCEDVVTGAKFTITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSVVFGDFGKDVQADHFHNSIMTAL
C
SEAADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSGSVSAHPTKPEALVVEYDVN
K
FDRNSILQALLVTADLLYIQGAKRILSPQAWVPIFESDKPKDKRSIKDEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSC
R
MSGKGPKYGAVDTDGRLFECSNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAK1
202
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FAO-2a - SEQ ID NO: 11
MNTFLPDVLEYKHVDTLLLLCDGIIHETTVDQIKDAIAPDFPEDQYEEYLKTFTKPSETPGFREAVYDTINATPTDAVH
M
CIVLTTALDSRILAPTLTNSLTPIKDMTLKEREQLLASWRDSPIAAKRRLFRLISSLTLTTFTRLASELHLKAIHYPGR
D
LREKAYETQVVDPFRYSFMEKPKFDGAELYLPDIDVIIIGSGAGAGVMAHTLANDGFKTLVLEKGKYFSNSELNFNDAD
G
VKELYQGKGALATTNQQMFILAGSTLGGGTTVNWSACLKTPFKVRKEWYDEFGLEFAADEAYDKAQDYVWKQMGASTDG
I
THSLANEVVVEGGKKLGYKSKEIEQNNGGHPDHPCGFCYLGCKYGIKQGSVNNWFRDAAAHGSKFMQQVRVVQILNKNG
V
AYGILCEDVETGVRFTISGPKKFVVSAGSLNTPTVLTNSGFKNKHIGKNLTLHPVSTVFGDFGRDVQADHFHKSIMTSL
C
YEVADLDGKGHGCRIETILNAPFIQASLLPWRGSDEVRRDLLRYNNMVAMLLITRDTTSGSVSADPKKPDALIVDYEIN
K
FDKNAILQAFLITSDMLYIEGAKRILSPQPWVPIFESNKPKEQRTIKDKDYVEWRAKAAKIPFDTYGSAYGSAHQMSTC
R
MSGKGPKYGAVDTDGRLFECSNVYVADASVLPTASGANPMISTMTFARQIALGLADSLKTKPKL
FAO-2b - SEQ ID NO:12
MNTFLPDVLEYKHVDTLLLLCDGIIHETTVDQIRDAIAPDFPEDQYEEYLKTFTKPSETPGFREAVYDTINSTPTEAVH
M
CIVLTTALDSRILAPTLTNSLTPIKDMTLKEREQLLAAWRDSPIAAKRRLFRLISSLTLTTFTRLASDLHLRAIHYPGR
D
LREKAYETQVVDPFRYSFMEKPKFDGTELYLPDIDVIIIGSGAGAGVMAHTLANDGYKTLVLEKGKYFSNSELNFNDAD
G
MKELYQGKCALTTTNQQMFILAGSTLGGGTTVNWSACLKTPFKVRKEWYDEFGLEFAADEAYDKAQDYVWKQMGASTEG
I
THSLANAVVVEGGKKLGYKSKEIEQNNGGHPDHPCGFCYLGCKYGIKQGSVNNWFRDAAAHGSKFMQQVRVVQILHNKG
V
AYGILCEDVETGVKFTISGPKKFVVSAGSLNTPTVLTNSGFKNKHIGKNLTLHPVSTVFGDFGRDVQADHFHKSIMTSL
C
YEVADLDGKGHGCRIETILNAPFIQASLLPWRGSDEVRRDLLRYNNMVAMLLITRDTTSGSVSADPKKPDALIVDYDIN
K
FDKNAILQAFLITSDMLYIEGAKRILSPQAWVPIFESNKPKEQRTIKDKDYVEWRAKAAKIPFDTYGSAYGSAHQMSTC
R
MSGKGPKYGAVDTDGRLFECSNVYVADASVLPTASGANPMISTMTFARQIALGLADSLKTKPKL
203
Clustal amino acid sequence alignments
FAO-13
MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDQIKDVIAPDFPADKYEEYVRTFTKPSETP 60
0
FAO-20
MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDQIKDVIAPDFPADKYEEYVRTFTKPSETP 60
w
FAO-1 MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDEIKDVIAPDFPADKYEEYVRTFTKPSETP
60 o
1¨,
w
*******************************:****************************
.6.
oo
oo
FAO-13
GFRETVYNTVNANTTDAIHQFIILTNVLASRVLAPALTNSLTPIKDMSLEDREKLLASWR 120
oo
FAO-20
GFRETVYNTVNANTTDAIHQFIILTNVLASRVLAPALTNSLTPIKDMSLEDREKLLASWR 120
FAO-1 GFRETVYNTVNANTMDAIHQFIILTNVLGSRVLAPALTNSLTPIKDMSLEDREKLLASWR
120
************** *************.*******************************
FAO-13
DSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHYPGREDREKAYETQEIDPFKYQFLE 180
FAO-20
DSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHYPGREDREKAYETQEIDPFKYQFME 180
FAO-1 DSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHYPGREDREKAYETQEIDPFKYQFLE
180
**********************************************************:*
n
0
FAO-13
KPKFYGAELYLPDIDVIIIGSGAGAGVVAHTLANDGFKSLVLEKGKYFSNSELNFDDKDG 240
n)
co
FAO-20
KPKFDGAELYLPDIDVIIIGSGAGAGVVAHTLANDGFKSLVLEKGKYFSNSELNFDDKDG 240
tri
0
w 20 FAO-1
KPKFYGAELYLPDIDVIIIGSGAGAGVVAHTLTNDGFKSLVLEKGRYFSNSELNFDDKDG 240
0
ko
o
tri
.6. ****
***************************:************:**************
n)
0
H
FAO-13
VQELYQSGGTLTTVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADE 300
.4.
1
FAO-2 0
VQELYQSGGTLTTVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADE 300
0
w
1
FAO-1 VQELYQSGGTLTTVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADE
300 n)
************************************************************
tri
FAO-13
AYDKAQDYVWQQMGASTEGITHSLANEIIIEGGKKLGYKAKVLDQNSGGHPQHRCGFCYL 360
FAO-2 0
AYDKAQDYVWQQMGASTEGITHSLANEIIIEGGKKLGYKAKVLDQNSGGHPQHRCGFCYL 360
FAO-1 AYDKAQDYVWQQMGASTEGITHSLANEIIIEGGKKLGYKAKVLDQNSGGHPHHRCGFCYL
360
***************************************************:********
V
FAO-13
GCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKGIAYGILCEDVVTGAKFTITGP 420
n
1-i
FAO-20
GCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKGIAYGILCEDVVTGAKFTITGP 420
FAO-1 GCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKGIAYGILCEDVVTGAKFTITGP
420 cr
w
************************************************************
o
1¨,
w
-CIS
FAO-13
KKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSVVFGDFGKDVQADHFHNSIMTALC 480
col
cA
col
FAO-2 0
KKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSVVFGDFGKDVQADHFHNSIMTALC 480
cA
w
FAO-1 KKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSVVFGDFGKDVQADHFHNSIMTALC
480
************************************************************
0
w
o
1-,
w
-,ii
.6.
m
m
m
FAO-13
SEAADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSG 540
FAO-2 0
SEAADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSG 540
FAO-1
SEAADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSG 540
************************************************************
FAO-13
SVSAHPTKPEALVVEYDVNKFDRNSILQALLVTADLLYIQGAKRILSPQAWVPIFESDKP 600
n
FAO-20
SVSAHPTKPEALVVEYDVNKFDRNSILQALLVTADLLYIQGAKRILSPQAWVPIFESDKP 600
0
FAO-1
SVSSHPTKPEALVVEYDVNKFDRNSILQALLVTADLLYIQGAKRILSPQPWVPIFESDKP 600
n)
0
***:*********************************************.**********
tri
0
0
w
,c,
o
FAO-13
KDKRSIKDEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGRLFEC 660
tri
col
FAO-2 0
KDKRSIKDEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGRLFEC 660
n)
0
FAO-1
KDKRSIKDEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGRLFEC 660
H
IA
I
************************************************************
0
w
,
1.)
FAO-13 SNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL
704 tri
FAO-2 0 SNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL
704
FAO-1 SNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL
704
********************************************
,-o
n
,-i
cp
t..)
t..)
--c-,.5
u4
c.,
u4
c.,
t..)
CA 02850095 2014-03-25
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Example 31: Addition and/or Amplification of Monooxygenase and Monooxygenase
reductase
activities.
Cytochrome P450's often catalyze a monooxygenase reaction, e.g., insertion of
one atom of
oxygen into an organic substrate (RH) while the other oxygen atom is reduced
to water:
RH + 02 + 2H+ + 2e¨ ¨> ROH + H20
The substrates sometimes are of a homogeneous carbon chain length. Enzymes
with
monooxygenase activity sometimes recognize substrates of specific carbon chain
lengths, or a
subgroup of carbon chain lengths with respect to organic substrates of
homogenous carbon chain
length. Addition of novel cytochrome activities (e.g., B. megaterium BM3)
and/or amplification of
certain or all endogenous or heterologous monooxygenase activities (e.g.,
CYP52Al2
polynucleotide, CYP52A13 polynucleotide, CYP52A14 polynucleotide, CYP52A15
polynucleotide,
CYP52A16 polynucleotide, CYP52A17 polynucleotide, CYP52A18 polynucleotide,
CYP52A19
polynucleotide, CYP52A20 polynucleotide, CYP52D2 polynucleotide, BM3
polynucleotide) can
contribute to an overall increase in carbon flux through native and/or
engineered metabolic
pathways, in some embodiments. In certain embodiments, adding a novel
monooxygenase or
increasing certain or all endogenous or heterologous monooxygenase activities
can increase the
flux of substrates of specific carbon chain length or subgroups of substrates
with mixtures of
specific carbon chain lengths. In some embodiments, the selection of a
monooxygenase activity
for amplification in an engineered strain is related to the feedstock utilized
for growth of the
engineered strain, pathways for metabolism of the chosen feedstock and the
desire end product
(e.g., adipic acid).
Strains engineered to utilize plant-based oils for conversion to adipic acid,
would benefit by having
one or more monooxygenase activities with substrate specificity that matches
the fatty acid chain-
length distribution of the oil. For example, the most prevalent fatty acid in
coconut oil is lauric acid
(12 carbons long), therefore, the monooxygenase activity chosen for a coconut
oil-utilizing strain
would have a substrate preference for C12 fatty acids. For strains engineered
to utilize other plant
based oils with different fatty acid chain-length distributions it would be
desirable to amplify a
monooxygenase activity that has a matching substrate preference. In some
embodiments, a
genetic modification that alters monooxygenase activity increases the activity
of one or more
monooxygenase activities with a substrate preference for fatty acids prevalent
in coconut oil. In
206
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certain embodiments, the genetic modification increases the activity of a
monooxygenase activity
with a preference for C12 fatty acids.
Strains engineered to utilize glucose for conversion to adipic acid, would
benefit by choosing a
monooxygenase activity that can utilize the distribution of chain lengths
produced by fatty acid
synthase (FAS). For strains engineered to utilize the long chain fatty acids
produced by FAS, it
often is desirable to add and/or amplify one or more monooxygenase activities
preferring long
chain fatty acids. For strains engineered to utilize a mutant FAS that
produces medium- or short-
chain fatty acids, or a specialized FAS (e.g., hexanoate synthase), it often
is desirable to add
and/or amplify one or more monooxygenase activities preferring medium- or
short-chain fatty acids.
In some embodiments, a genetic modification that alters monooxygenase activity
increases the
activity of one or more monooxygenase activities with a preference for the
long chain fatty acids
produced by fatty acid synthase activity (e.g., FAS). In certain embodiments,
the genetic
modification increases the activity of a monooxygenase activity with a
preference for the medium-
or short-chain fatty acids produced by a mutant or specialized FAS.
As mentioned previously, the enzymes that carry out the monooxygenase activity
are reduced by
the activity of monooxygenase reductase, thereby regenerating the enzyme.
Selection of a CPR
for amplification in an engineered strain depends upon which P450 is
amplified, in some
embodiments. A particular CPR may interact preferentially with one or more
monooxygenase
activities, in some embodiments, but not well with other monooxygenases. A
monooxygenase
reductase from C. tropicalis strain ATCC750, two monooxygenase reductase
activities from C.
tropicalis strain ATCC20336 and a monooxygenase reductase activity from
Bacillus megaterium
are being evaluated for activity with the added and/or amplified
monooxygenases described herein.
Provided in the tables below are nucleotide sequences used to add or amplify
monooxygenase
and monooxygenase reductase activities.
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99131V9V9V333V3V9V33991197/9111319139111931911911999139137/33V3V9V9VV9933991191
1317/37/7/
91113197/337/919391191917/7/3333V9V9VV3V9V397/7/31911397/937/191131191917/19991
9VVV39VVVV9113
97/97/7/97/33397/13917/9911139VVV9V311917/137/137/7/337/31197/7/37/391991397/7/
337/19137/97/97/11197/997/7/
37/7/37/7/31991199137/131193V9V3919911337/7/97/117/13991137/17/7/997/3339VVV37/
7/31193197/913911137/9
97/7/7/97/1991391111V9VIVIVS3V97/7/337/7/317/199117/1317/7/937/99VVV1133137/391
933197/91991117/11311
oo
o
97/9937/33993117/9119337/111V9V1113119117/7/997/331VIV911137/17/7/919997/7/37/3
97/7/11311VIV397/7/97/7/
oo
oo
.re
91191197/33113V3V337/7/9911931937/91917/31391197/7/37/7/97/97/33911197/37/337/9
7/911917/3397/97/397/37/3
o
(.9)
97/7/9913997/7/939937/9911V3V311317/99917/91997/1191113397/7/11111313V3V3997/97
/7/39991113111117/9
,--i
o
19911197/3337/7/39911117/139VVV3IVIVV9V9V3317/9VVV337/919319317/997/337/3993111
13117/197/31937/1
el
33V39991939VV333VVSVV3313V33VSLILSVV3VLLV93VV3V,LSVSVV313999V3999VSVVVSVV3LLSV3
313139 (LINndA0)
0
97/7/9917/9917/7/319317/19931113937/7/37/937/191197/7/3VVV3V3197/33139139199911
9VVVVV917/911319393 LIN 0917 d
,L3VSVVV3V3V,LV3SLL33,L3VV3VVV3,LV3,LV3V,L9,L,LS,LSV339,LS,L,L93,LS,LV,L99,L,LV
,LVVSV,L33,L3VV3VVS,LIveIv 0W0-11-1001A0 91. :ON 0102S
amanbas uoRdposaa ON 01 02S
V lo
SVIVV3917/97/7/31937/17/3999937/9
111911391917/937/9113V3V39911V9V1337/337/3337/197/9137/7/7/933337/997/37/319113
97/1333117/7/997/3119
113V9V11991111937/11993397/7/9337/9111391119V3VV3V99911391317/7/97/7/3319919937
/7/3117/3391137/1
1399917/999113VVV9V137/7/317/3397/9111991V9VVV99337/97/11117/913913917/93393991
7/117/113913317/7/
ci)
V3V9V3137/7/39931317/33137/17/7/391991997/917/7/9VVV9V317/911317/13317/997/7/39
937/7/3333991993997/97/
V337/11137/93V3V9VVVIIV9391117/97/7/393VVV97/7/33319197/7/3337/37/117/97/91193V
VV937/7/911919339VVV
91137/197/9191V9V97/7/911397/97/9111337/11V9V97/7/911919313137/997/919991399911
13V3V317/7/911VVV9
9V9V9V91197/7/33999131V3V3V3337/7/997/33991197/9111919139111331911911999139137/
33V3V9997/7/99
139911911317/37/7/91113197/337/919391191917/7/3333V9VVV93V9V397/7/33911397/937/
191131191917/1999
3997/7/37/VVVV91137/7/997/97/33337/13917/9911339VVV9V311917/137/137/7/337/31197
/7/37/3319117/37/937/7/391
97/997/7/3119V9VVV37/7/3717/31991199137/1911V3V9V3V11139119V9V337/33191117/1319
7/7/393197/7/37/7/311
93937/9339111VVV3937/97/VV97/7/9139917/37/997/7/937/17/937/139911VVV997/937/999
7/7/1133137/391933197/9
1991117/1131197/9937/9397/3117/9119337/111V9V1113119117/7/997/3317/17/91113VIVV
919997/7/37/397/7/113
Lc)
IIVIV397/7/97/7/91191197/331117/37/337/7/9911931937/91917/31391197/7/397/97/97/
33911197/37/337/97/91191V
C\I
3397/97/397/37/397/7/9913997/7/939937/9911337/311317/19937/19999119117/33VVV311
311VVV319997/397/7/99
9117/3111117/919911197/3337/7/3991111393997/7/31VIVV9V933337/9997/337/311919919
17/337/7/399111139
17/1397/331311137/37/997/91717/17/937/3311193331317/31717/1117/911997/3997/9991
3919VVVV13971717/313311 (CZVndA )
0
7/7/9193399111VVV3113991119919993V9V137/7/33191V3V3V3117/3397/7/13939991197/7/9
7/7/917/91117/117/7/ NV 0917d
C\I
337/997/33917/3139919317/97/39717/317/117/3199119117/33911319117/37/199137/117/
3311317/97/337/931391V 0W0-11-1001A0 :ON 01 02S
Lc)
o
o
cs)
SVIV31917/97/7/31937/37/3919937/9
Lc)
111911391917/937/91137/37/39911V9V1337/337/3337/197/7/33VVV933337/97/7/37/31911
3VVV3311197/997/3119
OD
C\I
113V9V11991111937/11993397/7/9137/9111391119V3VV3V99911191317/7/97/7/3319919937
/7/3117/33911311
1399917/999113VVV9V137/7/317/337/7/91119917/9VVV99337/97/11117/913913917/933939
917/117/113913317/7/
V3V9V3137/7/39931317/33137/197/391991997/917/997/7/997/317/911317/13317/997/7/3
9937/7/333399199399V9V
/337/11137/93V3V9VVVIIV9391117/97/7/393VVV97/7/33319197/7/3337/37/117/97/91113V
VV937/7/91191933997/7/
91137/197/9191V9V97/7/911397/97/9111337/11V9V97/7/91191931313V9VV9V991131991119
7/39V3VV911VVV9
9V9V9V91197/7/33999131V3V3V333VVV9V33991197/9111919139111331911911999139137/33V
3V9V97/7/99
/39911911317/37/7/91113197/337/919391191997/7/3333V9V9V93V9V397/7/31911397/917/
191131191917/1999
3997/7/39VVVV91137/7/997/97/33337/13917/9911339VVV9V311917/137/137/7/337/11197/
7/37/3319117/37/917/7/391
97/997/7/3119V9VVV37/7/3717/31991199137/191137/7/97/37/11139119V9V337/33991937/
13197/7/393197/7/37/7/311
93937/93391117/97/393V9VVV97/7/913191V3V9VVV937/37/937/139911VVV997/937/99VVV11
33137/391933197/9
oo
1991117/1131197/9937/33993137/9319137/111V9V1113119117/7/997/3317/17/911137/197
/919997/V3V3VVV113
oo
oo
DIVIV397/7/97/7/91191197/331137/37/337/7/9911931937/91917/31391197/7/37/V9V9V33
911197/37/337/97/91191V
(.9)
3397/97/397/37/397/7/9913997/7/933937/9911V3V311317/19937/19999117/117/33VVV311
31197/7/319997/397/399
9117/3111117/919911197/3337/7/3991111393997/7/31VIVV9V933337/9997/337/311919919
17/337/7/399111139
17/1397/331311137/37/997/91717/17/937/3311193331317/31VVILLV91199137/97/9991391
9VVITT/3971717/313311 (6 I'VndA0)
7/7/9193399311VVV31139991199199937/91937/7/33193V3V3V3117/3397/7/139199911VVV97
/7/917/91117/117/7/ 6V' 0917d
337/997/31917/3139919317/97/33717/317/117/3399119117/33911319117/37/199137/117/
37/11317/97/317/931391V 0W0-111001A0 (ig :ON 01 02S
amanbas uoRdposaa ON 01 02S
el
0
In
0
In
0
es1
Il
0
es1
C/)
c.)
a,
Lc)
c1
1
co
o
1
.i.
H
0
(N
f n
Li)
,--i
(3)
el
o
o
Lc)
V91137/7/97/137/7/93317/37/191999937/7/313113197/917/937/31317/3119313VVV9VVV33
7/3337/1
OD
C \ I
97/9337/137/7/97/1139V3V1V937/937/37/139197/37/37/9117/9313991111917/19311397/7
/9113317/7/3911197/397/3
0
199113391397/7/97/93339939937/7/3117/3311317/1931991399911197/97/7/3999V9V11939
139991V9V9V9933
4
o
V9311191917/339VIV91717/9991VIVISSVV9V917/7/9113V3V3V139191117/31117/19999197/3
397/33999VVV9V
/IV911117/9331119317/991V9V331997/99399193133113937/937/17/7/997/937/139111V9V9
3937/7/997/133919
197/7/333VIVI3V93113137/7/7/93VVV13317/7/39VVV31337/1193391197/97/7/91199933991
933VVIVVV917/919V
3319337/99917/937/913317/11997/997/97/937/1397/7/997/99191V3V337/917/7/9933397/
1397/9111V3VV39111931
/IIV11931939337/337/37/93937/9913911391131937/7/9119397/7/337/97/7/37/111191193
3317/97/9313V3V3VVV
31991197/939331311191937/197/9397/33V3V97/7/99337/391191V9V1193997/7/37/3319191
97/3397/39131197/7/
/9391911997/7/37/7/37/197/3393111997/11199937/919111311137/191197/937/3911937/7
/39997/137/7/3991117/1
97/7/97/393197/917/7/3117/397/7/9913311997/93917/97/7/937/9VVV91193199991913197
/939911191111199993V
oo
o
93991917/9917/93131199331137/131397/997/331V3V931137/13V9V9917/7/37/339917/911V
3V397/7/993113911
oo
oo
.re
91911117/39337/7/97/1317/9913317/17/31311199931V9VVV3391117/7/3933193911917/131
7/9339V9V397/7/991
o
9V9V3339937/9911137/111317/399VVV99991191193393911197/33V3V137/97/9119991133311
1137/917/9111
(.9)
,--i
o
97/3337/9317/13917/939WV3IVIVV9197/3337/997/7/337/3137/17/919133119397/9117/399
9337/7/319911V3V9VV
el
3717/97/37/3317/37/3393993197/93117/97/9717/319197/31197/9317/1137/9339997/9171
7/397/3397/93917/3137/13911 (CindA0)
0
1337/1139331199911131199917/937/7/317/91917/7/97/931VVV9V931393991V3VVV3V3917/9
113VIVVVIDVIVI O 0917d
ISLLVIVVSIVI9993LILVILLSISILL939LILVaILDISDISISIDIVSISIVSSSISDIVIDSILLSVIDIVIVI
DSSIV 01-1-10-11-1001A0 :ON CII 02S
amanbas uoRdposaa ON 01 02S
CA 02850095 2014-03-25
WO 2013/048898
PCT/US2012/056562
A comparison of the amino acid sequence identities of the monooxygenase
activities is presented
in the table below. The black colored boxes indicate regions of 100% identity.
Many of the
monooxygenase activities shown in the table below have a closely related
"sibling" sequence.
Protein Sequence Identity
CYP52Al2 CYP52A17 CYP52A18 CYP52A19 CYP52A20 CYP52A13 CYP52A14 CYP52A15
CYP52A16 CYP52D2
CYP52Al2 62 61 60 60 59 59 55 55
51
CYP52A17 94 73 73 62 62 60 59
54
CYP52A18 74 74 61 61 60 59
54
CYP52A19 95 64 63 59 58
54
CYP52A20 64 63 59 59
55
CYP52A13 96 68 66
53
CYP52A14 68 67
53
CYP52A15 96
50
CYP52A16
49
CYP52D2
214
SU
(/)o)
mm 0
O0 =
0
00 0
X
Z Z ,..<
cAD
00 CD
K.) =
C.A.) DJ
cn
0
ca as. cn cn
0 C CD 0 3 0 0
3 -0 = DJ
0
<
.=
15,F2i9i1ElinGiE'i`c--))P6t)1212i9i15,`(--))P12Pli9rAi9iPl'aiii)i'2i(c-Dr)
=
110 i926t)120015N-3P,2)6t)1222115,1212i2i1116=12)2)212)(161116-)P-2 ,..<
c
C)
P-'') 2i'2irli'2iP122i9 PPii9P1Piiii215,i9ii'2iP,(-)i'2iN-32)PicS)
(T)
o
..,
",,!"0 61H,,H-inno"H6-)H HpopPi-i6-) 06-)6-)PH6-)H ca
O 6-) ni-i 6-)606-)6-)6-)Popo P G- n
ppoHo HpoonP
! o
H p n p H
o n n H H o p H o p H n p H o H H p n p H H Cl)
H 0 H H
oi-i opoponnHHo HHoHHPHHHHHPoP 0 _ 0
r2,C - ) P. 7 1 6 - ), - 6 - ) PA n- 1 , 7 _ ,i1-1- 6
- ) 2P22P2Pi'2i2)(-12i92211Pi'2i215,1i'2ii121 _o
! c
o
=
o
O
cn
,----,-H000nnH 0 H H 0 0 H Hopoi-i nonnonHp G) G) n
H0PHHHH PnooHHooHnonHHHoHoHpoopopoopp
E2i9151201516-)2112)611-161216=1216=116=1
211111Pii916=1P.2)221116=12
i
00000p000n o oopHn oonnHoH pj(c-_2],]=_),
-)P-)1P,2116=1`)Po6t)12)i'2i2212 i' 1
--)ii'2i2)i'-i5,02i-iP6-)6-)6-)-36-)
2'(-= -3Pi'2i116=1-1-11i9iiIIH2)ini'2],12215,1216=12i9in i6=1,1,9]
,iiiip61,yq,ii
i'-''i2V2)i92222i9r6--))PonP,2221i'2i1P2 2')
i'2i2i9G)2HE
!
IiIIH2)2'(--321215,125M-)iPi Pipp5 -'')112 5i2iiiiiiG') 161122 11
!
i= '2'i(t)1111221(=)12112i'2iPiPi15,i'2'i
nIPEIEli'2i15,11i'2i1Ili'2i16=1 02),i
i
PiE2i'2'i6t)12,i161i9Pc-3N--))1Pli'2]
P52PiiELV2)in2 -=',(,-3 121
HnnG)HG)nG'n'''nHnH_,,nHHH6t)12HPi22)16=122)i'2ii'2i6-)161M-)P2)
ni_i oG) 6-1 on (-1pH nH on Pio Pi (-1 6--)H 6-)
!,2],= i1,9],v,-36,-3-=', 2'62)i'2ii'2],iPii'2ii'2iHi6t1i'2]
6)'il'-36-) v)HoHHHHP
ni-iHpoHHonnonoPoo P.J.. 6-)006-
)6,2]_-)), p ,q-).Qc-)P_-))01
i
onnonopcInHG 1 6-)6-)P
pa) 6 h I H 0 0 J P 0 6 H Poo
Q,L4,,,,,, ,,Q,, PoHHHpoalHoHn pa)D.J..06-)P 0
!
np nn G--) 6.16-) G--) ,21 j)HJo (--) G--
)izi j G--) n1 6:1 G--) n ,21 1H Giln n1 , ihip 1 H 6-) 6-)Jo , GIP Glp ilJo
01 il 0 6-)iij
!06= >7:)00`630`67-)) 6,,-)0g,069-6006i0m6P2i 00n00Q0Qp,i065'0o6P 00 0 ,
onnnonH H0i-iH06-)
HoHoHHoonHHopHH6-)HPPHHHHP06-)6-)0PHHo Hpoi-i
H 6-) 6-) n 6-) H H 6--) n 1-] 0 H 6--) n 6--) 6--) P H 6-) P H 6-) 6-) 6-)
n n p p H p o n H n H p H
Z9S9S0/ZIOZS9lIDcl 86881'0/10Z OM
S3-0-17T03 S600S830 VD
CA 02850095 2014-03-25
WO 2013/048898 PCT/US2012/056562
EI -0,D E2i ri ri EY! rp' ri (c-3 H EH,' EH,' 6 6
EY , , H ' cc -3 cr -2 = ,) .., .1 EY , cr -2 ri Et i' S 6 -- ,) EY ,
cr -2
O
H gOOHCD< CD<HO<CD 0000100H Hrt.HC_D<<CDC_DU< HH
E-1 CDO<H E-1 E-1
OH H0E-10E-1E-1 E-1E-1 <<
E-1E-1< 00<<CDUE-100<0<0
O <HOUCD<CD 00 HHH 0 U<OH< HH 0 <<<<HOU <OHOU
< UH<<H<CDOCDO H<H<CDOCDUCDHOCD<< U<CDOHO< CD<HCD<
1
O E-1E¨I<HHUH<CDO E-1<<E-1E-1E-1E-10E-1HUH<E-1 UCD<UOHE-1 HUOUE-1
H gOUHUH<<HHUOUCDOHUHHOHHHO <HOUCD<CD CDU<HH
0 g0E-1<<E-100E-1E-1HE-1000<< E-1(_DE-1E-100
< H < < H < 0 0 0 0 < H <
0 CDHUOUHUE-1< E-1HUHE-1 <00E-1E-1E-1
JEH-11D 6 E H¨ , ' 6 E H¨ , ' ' ) E t , ' ' c D ) ' r - 2 r i r D ' 0 '' '
r - 2 E t , ' ' c D ) ' c D ) u ' r -1 E iu , 'u ' r - 2L) , 'u 6u E
t i9 E t i9 E i` ' N ' al
1
HOOHOUHHOU<OU
<= 00E-1 CDE-1<CD<HOUOUHE-1H<HE-1 OH
OgE-1 UHE-1<<H<E-10<<<0
O00H OHOO<CDUHOU<OH<<H000<00 CDUHOUHOHHHO<<
HE-1E-1000<H<CDE-1E-1E-1HUH .ralE-1E-1(_D<E¨I<E-1E-
1 1 E-11<00E-10E¨I HU
<<
E ¨ 1 0 E ¨ 1 E ¨ 1 < E ¨ 1 < < < E ¨ 1 < < E ¨ 1 E ¨ 1 E ¨ 1 0
< E ¨ 1 < < 0 0 0 E ¨ 1 E ¨ 1 E ¨ 1 0 E-1 < 0 < E ¨ 1 0 0 0 0
CDOH<<HOHOHOU<HHH 0<<<U<CD 0 HH OHOO<CDUHOU
O0 HHUOUHHH<HH<<<HOUCDUHOO HO HUOUOU<CD<UHH 0
< 0 < < UUUUUHHUH
1
O H HOO CD<CDOU<CDOUH<H
Pl< F:paGHCDCDH HCDpi.GHLDHOHUUFHH
HILDH<H H<OHH HHOUUH< OH HH<<<H 000000HHOUU<<
O E-100< 00<<<<H<HHOU HE-1E-10E-1E-100 U<U<<UU<OU<<0
O0 CD<HOOHH<CDUH<<H CDOUHH <00H<H CDOCD<CD<CDOH<CDOU
HU<<HUHH V_DH H g HUH HHOCT_DH<HH
U<UHUOUHOUHOU
<<UU<E-1<<
g <g HUH HE-1 E-1 E-1HUHU 00<<00<<<<E-1<E-1
H
0 CDHOH <PC(1<(__DCD<HH HO H HOH<CD CD<HOOHH<CDUH<<
O HH<HHHH UHHUH<<H OUHHOCDOH <OH CDHUV_DHU<HU
1
<U<OCH 0
1
<<00000HOHO HUOCDO<CD<<CD<<CD CDHOH<<CD<CDOCD<<
<H<HULDHUH<H H<<UHU<HHHH<CD HU<HHHHOHUHUU
OUOU<UHE-1000< 00<0000E-1E-1<<CDE-1 0<<<<CDUE-10E-1E-1 <
O 000H<HHHE-1000<<UE-1<<<CDOE-10<H 0<00000H OHO H
CDUHH<H<H<H<HHHOHOCD<U<<H<<H< OU<OULDHUH<0 H
H<H<O<U00<HOH<OU<OHHOUCD<<CDPC HOU<OHHOUU< 0
<H CDO<CDUCD<OHOCDOHH<HHOU0000<0 CD<OH<HHOHOUCD<
H<CD<HH<HUHH HHHHH<<HHUHHHOH CD<OHHOCDUCDHUOU
E-10E-1000gH<E-1< CDO<H<OUE-100 0E-100 E-100<000<HOE-1<0
O000000HOHO HHOCDOUHOO< <000 CDO<CDUCD<OHO<OH
CDUCDH<OHHHOHHHOOHH<OHHH CDHHH OUHH<OUHUOHOU
O0 HOUE-10E-10E-1<0<<E-100000<0 0<<0 E-100U<H<E-1<<CDO<
V__D H< H 0000O<U<HUO<H0<< HHOO H<CDOCDHOH CD<HH 0
UUHHUUUUUU
O =
<0000<0< H<HOCDCD<H<H <0000 <CD<H00000<UPCH
<HUH HE-1 E-1 00< <HUHE-1E-1E-1 E-1E-1
<00E-100E-1H<CD<U0
CD< HU<<<UCD<OHOUU
O0 <<HOLDH 00000 H<<HOCD<OU HO <<U0C-D<CD<<H<H 0
H
g HH<HH OH H<CDOH <UH<H 0 5CDHP..0 UOU<H(10000<0
E-1g E-10110E-1< <0 H g CDHE-100E-10< g E ¨ 1 g
E ¨ 1 < 0 0 0 < < < < < 0 <
O0 <<CDOH 0<<CDOHH <H<UH (_)R <00 <<HOOH 00000,g
U<HHHOHO<CDOHHU< HHO<H HUHHU OULDHHr_DH0000
UH HHHH UHOOH CDUH< UOUCD<UH<U<< HHOH< <<UH<CDH
O0 CDUH<<HOHH OH<CDCD<OH<OUCDH<HU 0000H
HH
HOHU<OHHOU '4,<H<HCDHHO<H<<CD<HU HUH 000000000<
O<<HE-1000E-10 <00E-1E-1E-1H<OHHOHUHE-1 <HE-1E-10E-100E-100E-1<
UUHHH CD<H0 CD<CD<HHHHOOH HU<< HHH<<H<HHOH<CD
<CD<CD< HHOOH HHHHH <<HHC1 UHHH 00000000rUHH
U< HOH HOHHHUHHH<OH<<H
H<H < HOHUOUHU <CDUH
CDUHOH CD<HHOHHUOUHHHO< CDHO<CD HOH CD<H0 CD<CD<
1
E-1 U CD
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SEQ ID NO Description Sequence
SEQ ID NO:41 cytochrome MT I KEMPQPKT FGELKNL PLLNTDKPVQALMK IADELGE
I FKFEAPGRVTRYLS SQRL I KEACDESR FDKNL S QALKFVRD
P450:NADPH FAGDGLFTSWTHEKNWKKAHNI LL PS
FSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDT I GL CGFNYR
0
P450 FNS FYRDQPHPFI TSMVRASDEAMNKS
QRANPDDPAYDENKRQ FQED I KVMNDLVDKI IADRKASGEQSDDLLTHMLNGKD n.)
r PE TGE PLDDENIRYQ I I TFL IAGHE T T S GLL S
FAS Y FLVKNPHVLQKAAEEAARVLVDPVPS YKQVKQLKYVGMVLNEASR
eductase
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LWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVS I PQLHRDKT I WGDDVEE FRPER FENPSA I
PQHAFKPFGNGQRAC I GQQ
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TEQSAKKVRKKAENAHN T PS LVL .6.
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megateriuml
. YGSNMGTAEGTARDLAD IAMS KGFAPQVAT LD S HAGNL
PREGAVL IVTASYNGHPPDNAKQFVDWLDQASADEVKGVRYSV oe
amino acid FGCGDKNWAT TYQKVPAF I DET LAAKGAEN IADRGEADAS
DDFEGTYEEWREHMWS DVAAYFNLD I ENSEDNKS T L S LQFV oe
[P450 activity DSAADMPLAKMHGAFSTNVVASKELQQPGSARSTRHLE I EL PKEAS YQEGDHLGVI
PRNYEGIVNRVTARFGLDASQQ I RS
shown in
EAEEEKLAHLPLAKTVSVEELSQYVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVSAKRLTMLESLEKY
PA
italics, P450 CEMKF SEF IAL S PS I RPRYYS ISSS PRVDEKQAS I
TVSVVSGEAWSGYGEYKGIASNYLAESQEGDT I TCF I STPQSEFTS
reductase PKDPETPL
IMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRS PHEDYSYQEELENAQSEGI I TLHTAFSRMPNQP
activity shown KTYVQHVMEQDGKKL I ELLDQGAHFY I CGDGSQMAPAVEAT LMKS
YADVHQVSEADARLWSQQLEEKGRYAKDVWAG*
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Example 32: Amplification of Selected Beta Oxidation Activities
As noted previously, beta oxidation of fatty acids involves two acyl CoA
oxidase activities, encoded
by PDX4 and PDX5. Figures 15A-15C presented data regarding distribution of
fatty acids present
in yeast strains that were wild type with respect to acyl CoA oxidase activity
(e.g., PDX4:P0X5,
see Figure 15A), disrupted for PDX5 (e.g., PDX4:pox5), or disrupted for PDX4
(e.g., pox4:P0X5).
Wild-type C. tropicalis has two copies of PDX4 and two copies of PDX5. The
PDX4 enzyme has
broad substrate specificity (see Figure 15B), whereas the PDX5 enzyme (see
Figure 15C) has a
narrow substrate specificity with regard to fatty acid chain length. A
partially beta oxidation
disrupted strain was generated that contained no PDX4 activity (both alleles
are disrupted), and
two functional copies of PDX5, as described herein. The results of experiments
conducted with
the pox4:pox4/P0X5:P0X5 strain indicated that since the activity of the PDX5
enzyme drops off
dramatically with fatty acids shorter than C10, the beta-oxidation products of
the PDX5 strain are
the C8 diacid and C6 diacid (adipic). Additionally, if the strain is starved
for carbon (e.g., the only
possible source of energy is the C8 diacid), it will convert the C8 diacid to
the C6 diacid. Data
illustrating that amplification of the number of PDX 5 genes in the engineered
organism to Increase
beta oxidation activity, increases the C6/C8 ratio, is presented below and
shown in Figure 38.
Construction and shake flask evaluation of PDX5 amplified strains
Plasmid pAA166 (Pp0x4P0X5Tp0x4)
A PCR product containing the nucleotide sequence of PDX5 was amplified from C.
tropicalis
20336 genomic DNA using primers oAA540 and oAA541. The PCR product was gel
purified and
ligated into pCR-Blunt II-TOPO (Invitrogen), transformed into competent TOP10
E. coli cells
(Invitrogen) and clones containing PCR inserts were sequenced to confirm
correct DNA sequence.
One such plasmid was designated, pAA165. Plasmid pAA165 was digested with
BspQI and a 2-kb
fragment was isolated. Plasmid pAA073 which contained a PDX4 promoter and PDX4
terminator
was also digested with BspQI and gel purified. The isolated fragments were
ligated together to
generate plasmid pAA166. Plasmid pAA166 contains a Pp0x4P0X5Tp0x4 fragment.
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Plasmid pAA204 (thiolase deletion construct)
A PCR product containing the nucleotide sequence of a short-chain thiolase
(e.g., acetyl-coA
acetyltransferase) was amplified from C. tropicalis 20336 genomic DNA using
primers oAA640 and
oAA641. The PCR product was gel purified and ligated into pCR-Blunt II-TOPO
(Invitrogen),
transformed into competent TOP10 E. coli cells (Invitrogen) and clones
containing PCR inserts
were sequenced to confirm correct DNA sequence. One such plasmid was
designated, pAA184.
A URA3 PCR product was amplified from pAA061 using primers oAA660 and oAA661.
The PCR
product was gel purified and ligated into pCR-Blunt II-TOPO (Invitrogen),
transformed as described
and clones containing PCR inserts were sequenced to confirm the correct DNA
sequence. One
such plasmid was designated pAA192. Plasmid pAA184 was digested with
BgIII/Sall and gel
purified. Plasmid pAA192 was digested with BgIII/Sall and a 1.5 kb fragment
was gel purified. The
isolate fragments were ligated together to create pAA199. An alternative PuRA3
PCR product was
amplified from plasmid pAA061 using primers oAA684 and oAA685. The PCR product
was gel
purified and ligated into pCR-Blunt II-TOPO (Invitrogen), transformed as
described and clones
containing PCR inserts were sequenced. One such plasmid was designated,
pAA201. Plasmid
pAA199 was digested with Sall and gel purified. Plasmid pAA201 was digested
with Sall and a
0.43 kb PuRA3 was gel purified. The isolated fragments were ligated to create
plasmid pAA204 that
contains a direct repeat of P
URA3.
Plasmid pAA221 (Pp0x4P0X5Tp0x4 in thiolase deletion construct)
A PCR product containing the nucleotide sequence of Pp0x4P0X5Tp0x4 was
amplified from plasmid
pAA166 DNA using primers oAA728 and oAA729. The PCR product was gel purified
and ligated
into pCR-Blunt II-TOPO, transformed as described and clones containing PCR
inserts were
sequenced to confirm the sequence of the insert. One such plasmid was
designated, pAA220.
Plasmid pAA204 was digested with BgIII, treated with shrimp alkaline
phosphatase (SAP), and a
6.5 kb fragment was gel purified. Plasmid pAA220 was digested with BglIl and a
2.7 kb fragment
containing Pp0x4P0X5Tp0x4 was gel purified. The isolated fragments were
ligated to create
plasmid pAA221.
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Strain sAA617 (Pp0x4P0X5Tp0x4 in sAA451)
Strain sAA451 is a ura-, partially [3-oxidation blocked strain (ura3/ura3
pox4a::ura3/pox4b::ura3
PDX5/P0X5). Plasmid pAA221 was digested with EcoRI to release a DNA fragment
containing
PP0X4P0X5Tp0x4 in a thiolase deletion construct. The DNA was column purified
and transformed to
strain sAA451 to plate on SCD-ura plate. After two days, colonies were
streaked out on YPD
plates, single colonies selected and again streaked out on YPD plates. Single
colonies were
selected from the second YPD plates and characterized by colony PCR. The
insertion of
PP0X4P0X5TP0X4 in strain sAA451, disrupting the short-chain thiolase gene, was
confirmed by PCR
and one such strain was designated sAA617.
Strain sAA620
Strain sAA617 was grown overnight on YPD medium and plated on SCD+URA+5-F0A,
to select
for loop-out of URA3. Colonies were streaked out onto YPD plates twice as
described for strain
sAA617, and single colonies characterized by colony PCR. The loop-out of URA3
by direct
repeats of PURA3 was confirmed by PCR. One such strain was designated sAA620.
Strain
sAA620 has one additional copy of PDX5 under control of the PDX4 promoter.
Plasmid pAA156
A PCR product containing the nucleotide sequence of CYP52A19 was amplified
from C. tropicalis
strain 20336 genomic DNA, using primers oAA525 and oAA526. The PCR product was
gel
purified and ligated into pCR-Blunt II-TOPO, transformed as described, and
clones containing PCR
inserts were sequenced to confirm correct DNA sequence. One such plasmid was
designated,
pAA144. Plasmid pAA144 was digested with BspQI and a 1.7-kb fragment was
isolated. Plasmid
pAA073, which includes a PDX4 promoter and PDX4 terminator, also was digested
with BspQI
and gel purified. The isolated fragments were ligated together to generate
plasmid, pAA156.
Plasmid pAA156 included Pp0x4CYP52A19Tp0x4 fragment and URA3.
Strain sAA496
Plasmid pAA156 was digested with Clal and column purified. Strain sAA451 was
transformed with
this linearized DNA and plated on SCD-ura plate. Colonies were checked for
CYP52A19
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integration. Colonies positive for plasmid integration were further analyzed
by qPCR to determine
the number of copies of CYP52A19 integrated. One such strain, designated
sAA496 contained
about 13 copies of the monooxygenase activity encoded by CYP52A19.
Strains sAA632 and sAA635
Strain sAA620 was transformed with linearized pAA156 DNA and plated on SCD-ura
plates.
Several colonies were checked for CYP52A19 integration. Colonies positive for
plasmid
integration were further analyzed by qPCR to determine the number of copies of
CYP52A19
integrated. One such strain, designated sAA632 contained about 27 copies of
the
monooxygenase activity encoded by CYP52A19. Another strain, designated sAA635,
contained
about 12 copies of the monooxygenase activity encoded by CYP52A19.
Shake flask characterization of sAA496, sAA632 and sAA635
250mL glass bottom-baffled flasks containing 50mL of 5P92 media were
inoculated with a 5mL
overnight YPD culture (OD = 0.4). After 24h incubation at 30 C, with shaking
at 25Orpm (2" throw
incubator), sterile antifoam B was added to a final concentration of 0.1% to
dissipate foam. The
cells were centrifuged and the cell pellet resuspended in 20mL of TB-lowN
media (yeast nitrogen
base without amino acids and without ammonium sulfate, 1.7g/L; yeast extract,
3.0g/L; potassium
phosphate monobasic, 1.0g/L; potassium phosphate dibasic, 1.0g/L). 1.6mL
coconut oil containing
20uL sterile antifoam B was added to start adipic acid production in cultures
grown at 30 C, with
shaking. Samples were taken every 24 hour for gas chromatographic (GC)
analysis.
Shown in the table below and in Figure 38, are the diacid profiles for strains
described herein, at
various time points. Strains with an additional copy of the PDX5 gene show an
increased
proportion of C6 in the C6 + C8 diacid pool at early time points of the
analysis. An increased
proportion of C6 at early time points may indicate that the additional copy of
PDX5 in these strains
increases beta-oxidation activity, allowing chain shortening of the coconut
oil feedstock to the C6
diacid rather than to the C8 diacid. At the 144h time point, the strain
without an additional copy of
PDX5 has the same proportion of C6 in the C6 + C8 diacid pool.
Strain name time C6 (g/L) C6+C8 (g/L) C6/(C6+C8)
sAA496 24h 0.23 1.11 20%
sAA632 24h 0.39 1.14 34%
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Strain name time C6 (g/L) C6+C8 (g/L) C6/(C6+C8)
sAA635 24h 0.34 0.98 35%
sAA496 48h 1.4 2.69 52%
sAA632 48h 2.34 3.09 76%
sAA635 48h 1.86 2.75 68%
sAA496 72h 2.6 4.24 61%
sAA632 72h 3.6 5.1 71%
sAA635 72h 2.78 4.27 65%
sAA496 144h 7.70 7.86 98%
sAA632 144h 8.55 8.79 97%
sAA635 144h 7.92 8.23 96%
Strains described herein (e.g., partially beta-oxidation blocked, increased
monooxygenase activity
(e.g., SEQ ID NO: 20 amplified) and increased PDX5 activity) have produced
greater than 50g/L of
adipic acid under fermentation conditions using coconut oil as the feedstock.
In some
embodiments, strains described herein have 1 or more, 5 or more, 10 or more,
15 or more, 20 or
more, or 25 or more additional copies a nucleotide sequences encoding a
monooxygenase activity,
a monooxygenase reductase activity, and/or a PDX5 activity as compared to a
strain native for
monooxygenase activity, monooxygenase reductase activity, and/or PDX5
activity. In certain
embodiments, strains described herein have 2 times or more, 5 times or more,
10 times or more,
times or more, 20 times or more, or 25 times or more monooxygenase activity,
monooxygenase
reductase activity, and/or a PDX5 activity, as compared to a strain native for
monooxygenase
activity, monooxygenase reductase activity, and/or PDX5 activity.
15 The polynucleotide sequences of the PDX4 and PDX5 genes isolated as
described herein are
presented below as SEQ ID NOS: 37 and 38, respectively. The amino acid
sequences encoded
by the polynucleotides of SEQ ID NOS 37 and 38, are presented as SEQ ID NOS:
39 and 40,
respectively.
SEQ ID NO Description Sequence
SEQ ID NO:37 acyl CoA oxidase,
ATGACTTTTACAAAGAAAAACGTTAGTGTATCACAAGGTCCTGACCC
PDX4 (C. tropicalis TAGATCATCCATCCAAAAGGAAAGAGACAGCTCCAAATGGAACCCTC
strain ATCC20336) AACAAATGAACTACTTCTTGGAAGGCTCCGTCGAAAGAAGTGAGTTG
nucleotide ATGAAGGCTTTGGCCCAACAAATGGAAAGAGACCCAATCTTGTTCAC
AGACGGCTCCTACTACGACTTGACCAAGGACCAACAAAGAGAATTGA
CCGCCGTCAAGATCAACAGAATCGCCAGATACAGAGAACAAGAATCC
ATCGACACTTTCAACAAGAGATTGTCCTTGATTGGTATCTTTGACCC
ACAGGTCGGTACCAGAATTGGTGTCAACCTCGGTTTGTTCCTTTCTT
GTATCAGAGGTAACGGTACCACTTCCCAATTGAACTACTGGGCTAAC
GAAAAGGAAACCGCTGACGTTAAAGGTATCTACGGTTGTTTCGGTAT
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SEQ ID NO Description Sequence
GACCGAATTGGCCCACGGTTCCAACGTTGCTGGTTTGGAAACCACCG
CCACATTTGACAAGGAATCTGACGAGTTTGTCATCAACACCCCACAC
AT TGGTGCCACCAAGTGGTGGAT TGGTGGTGC TGC TCAC TCCGCCAC
CCACTGTTCTGTCTACGCCAGATTGATTGTTGACGGTCAAGATTACG
GTGTCAAGACTTTTGTTGTCCCATTGAGAGACTCCAACCACGACCTC
ATGCCAGGTGTCACTGTTGGTGACATTGGTGCCAAGATGGGTAGAGA
TGGTATCGATAACGGTTGGATCCAATTCTCCAACGTCAGAATCCCAA
GAT TC T T TATGT TGCAAAAGT TC TGTAAGGT T TC TGC TGAAGGTGAA
GTCACCTTGCCACCTTTGGAACAATTGTCTTACTCCGCCTTGTTGGG
TGGTAGAGTCATGATGGTTTTGGACTCCTACAGAATGTTGGCTAGAA
TGTCCACCATTGCCTTGAGATACGCCATTGGTAGAAGACAATTCAAG
GGTGACAATGTCGATCCAAAAGATCCAAACGCTTTGGAAACCCAATT
GATAGATTACCCATTGCACCAAAAGAGATTGTTCCCATACTTGGCTG
CTGCCTACGTCATCTCCGCTGGTGCCCTCAAGGTTGAAGACACCATC
CATAACACCTTGGCTGAATTGGACGCTGCCGTTGAAAAGAACGACAC
CAAGGC TATC T T TAAGTC TAT TGACGACATGAAGTCAT TGT T TGT TG
AC TC TGGT TCC T TGAAGTCCAC TGCCAC T TGGT TGGGTGC TGAAGCC
AT TGACCAATGTAGACAAGCC TGTGGTGGTCACGGT TAC TCGTCC TA
CAACGGCTTCGGTAAAGCCTACAACGATTGGGTTGTCCAATGTACTT
GGGAAGGTGACAACAATGTCTTGGCCATGAGTGTTGGTAAGCCAATT
GTCAAGCAAGTTATCAGCATTGAAGATGCCGGCAAGACCGTCAGAGG
TTCCACCGCTTTCTTGAACCAATTGAAGGACTACACTGGTTCCAACA
GC TCCAAGGT TGT T T TGAACAC TGT TGC TGAC T TGGACGACATCAAG
AC TGTCATCAAGGC TAT TGAAGT TGCCATCATCAGAT TGTCCCAAGA
AGC TGC T TC TAT TGTCAAGAAGGAATC T T TCGAC TATGTCGGCGC TG
AATTGGTTCAACTCTCCAAGTTGAAGGCTCACCACTACTTGTTGACT
GAATACATCAGAAGAATTGACACCTTTGACCAAAAGGACTTGGTTCC
ATAC T TGATCACCC TCGGTAAGT TGTACGC TGCCAC TAT TGTC T TGG
ACAGATTTGCCGGTGTCTTCTTGACTTTCAACGTTGCCTCCACCGAA
GCCATCACTGCTTTGGCCTCTGTGCAAATTCCAAAGTTGTGTGCTGA
AGTCAGACCAAACGTTGTTGCTTACACCGACTCCTTCCAACAATCCG
ACATGAT TGTCAAT TC TGC TAT TGGTAGATACGATGGTGACATC TAT
GAGAACTACTTTGACTTGGTCAAGTTGCAGAACCCACCATCCAAGAC
CAAGGCTCCTTACTCTGATGCTTTGGAAGCCATGTTGAACAGACCAA
CC T TGGACGAAAGAGAAAGAT T TGAAAAGTC TGAT GAAACCGC TGC T
ATCTTGTCCAAGTAA
SEQ ID NO:39 acyl CoA oxidase, MTFTKKNVSVSQGPDPRS S I QKERDS
SKWNPQQMNYFLEGSVERSEL
PDX4 (C. tropicalis MKALAQQMERDP I LFTDGS YYDLTKDQQRELTAVK INRIARYREQE S
strain ATCC20336) I DTFNKRL SL IGI FDPQVGTRI GVNLGLFL SC IRGNGTTSQLNYWAN
amino acid EKETADVKG I YGCFGMTELAHGSNVAGLET TATFDKE S
DEFVINT PH
I GATKWW I GGAAHSATHC SVYARL IVDGQDYGVKTFVVPLRDSNHDL
MPGVTVGD I GAKMGRDG I DNGW I QF SNVRI PRFFMLQKFCKVSAEGE
VT L PPLEQL S YSALLGGRVMMVLDS YRMLARMS T IALRYAIGRRQFK
GDNVDPKDPNALETQL I DYPLHQKRLFPYLAAAYVI SAGALKVEDT I
HNTLAELDAAVEKNDTKAIFKS I DDMKS LFVDSGS LKS TATWLGAEA
I DQCRQACGGHGYS S YNGFGKAYNDWVVQC TWEGDNNVLAMSVGKP I
VKQVI S I EDAGKTVRGS TAFLNQLKDYTGSNS SKVVLNTVADLDD I K
TVI KAI EVAI I RL SQEAAS IVKKESFDYVGAELVQLSKLKAHHYLLT
EY I RRI DTFDQKDLVPYL I TLGKLYAAT IVLDRFAGVFLTFNVASTE
AI TALASVQ I PKLCAEVRPNVVAYTDS FQQS DMIVNSAI GRYDGD I Y
ENYFDLVKLQNPPSKTKAPYSDALEAMLNRPTLDERERFEKSDETAA
I L SK*
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SEQ ID NO Description Sequence
SEQ ID NO:38 acyl CoA oxidase, AT G C C TACCGAAC T TCAAAAAGAAAGAGAAC T CAC
CAAG T TCAACCC
PDX5 (C. tropicalis AAAGGAGTTGAACTACTTCTTGGAAGGTTCCCAAGAAAGATCCGAGA
strain ATCC20336) TCATCAGCAACATGGTCGAACAAATGCAAAAAGACCCTATCTTGAAG
n
GTCGACGCTTCATACTACAACTTGACCAAAGACCAACAAAGAGAAGT
ucleotide
CACCGCCAAGAAGATTGCCAGACTCTCCAGATACTTTGAGCACGAGT
ACCCAGACCAACAGGCCCAGAGATTGTCGATCCTCGGTGTCTTTGAC
CCACAAGTCTTCACCAGAATCGGTGTCAACTTGGGTTTGTTTGTTTC
CTGTGTCCGTGGTAACGGTACCAACTCCCAGTTCTTCTACTGGACCA
TAAATAAGGGTATCGACAAGTTGAGAGGTATCTATGGTTGTTTTGGT
ATGACTGAGTTGGCCCACGGTTCCAACGTCCAAGGTATTGAAACCAC
CGCCACTTTTGACGAAGACACTGACGAGTTTGTCATCAACACCCCAC
ACATTGGTGCCACCAAGTGGTGGATCGGTGGTGCTGCGCACTCCGCC
ACCCAC TGC TCCGTC TACGCCAGAT TGAAGGTCAAAGGAAAGGAC TA
CGGTGTCAAGACCTTTGTTGTCCCATTGAGAGACTCCAACCACGACC
TCGAGCCAGGTGTGACTGTTGGTGACATTGGTGCCAAGATGGGTAGA
GACGGTATCGATAACGGTTGGATCCAGTTCTCCAACGTCAGAATCCC
AAGATTCTTTATGTTGCAAAAGTACTGTAAGGTTTCCCGTCTGGGTG
AAGTCACCATGCCACCATCTGAACAATTGTCTTACTCGGCTTTGATT
GGTGGTAGAGTCACCATGATGATGGACTCCTACAGAATGACCAGTAG
AT TCATCACCAT TGCC T TGAGATACGCCATCCACAGAAGACAAT TCA
AGAAGAAGGACACCGATACCATTGAAACCAAGTTGATTGACTACCCA
TTGCATCAAAAGAGATTGTTCCCATTCTTGGCTGCCGCTTACTTGTT
CTCCCAAGGTGCCTTGTACTTAGAACAAACCATGAACGCAACCAACG
ACAAGTTGGACGAAGCTGTCAGTGCTGGTGAAAAGGAAGCCATTGAC
GC TGCCAT TGTCGAATCCAAGAAAT TGT TCGTCGC T TCCGGT TGT T T
GAAGTCCACCTGTACCTGGTTGACTGCTGAAGCCATTGACGAAGCTC
GTCAAGCTTGTGGTGGTCACGGTTACTCGTCTTACAACGGTTTCGGT
AAAGCCTACTCCGACTGGGTTGTCCAATGTACCTGGGAAGGTGACAA
CAACATCTTGGCCATGAACGTTGCCAAGCCAATGGTTAGAGACTTGT
TGAAGGAGCCAGAACAAAAGGGATTGGTTCTCTCCAGCGTTGCCGAC
TTGGACGACCCAGCCAAGTTGGTTAAGGCTTTCGACCACGCCCTTTC
CGGCTTGGCCAGAGACATTGGTGCTGTTGCTGAAGACAAGGGTTTCG
ACATTACCGGTCCAAGTTTGGTTTTGGTTTCCAAGTTGAACGCTCAC
AGATTCTTGATTGACGGTTTCTTCAAGCGTATCACCCCAGAATGGTC
TGAAGTCTTGAGACCTTTGGGTTTCTTGTATGCCGACTGGATCTTGA
CCAACTTTGGTGCCACCTTCTTGCAGTACGGTATCATTACCCCAGAT
GTCAGCAGAAAGATTTCCTCCGAGCACTTCCCAGCCTTGTGTGCCAA
GGTTAGACCAAACGTTGTTGGTTTGACTGATGGTTTCAACTTGACTG
ACATGATGACCAATGC TGC TAT TGGTAGATATGATGGTAACGTC TAC
GAACAC TAC T TCGAAAC TGTCAAGGC T T T GAAC C CAC CAGAAAACAC
CAAGGCTCCATACTCCAAGGCTTTGGAAGACATGTTGAACCGTCCAG
ACC T TGAAGTCAGAGAAAGAGGTGAAAAGTCCGAAGAAGC TGC TGAA
ATCTTGTCCAGTTAA
SEQ ID NO:40 acyl CoA oxidase, MPTELQKERELTKFNPKELNYFLEGSQERSE I I
SNMVEQMQKDP I LK
P0X5 (C. tropicalis VDASYYNLTKDQQREVTAKKIARLSRYFEHEYPDQQAQRLS I LGVFD
strain ATCC20336) PQVFTRIGVNLGLFVSCVRGNGTNSQFFYWT INKG I DKLRG I YGCFG
amino acid MTELAHGSNVQG I ET TATFDEDTDEFVINT PH I GATKWWI
GGAAHSA
THC SVYARLKVKGKDYGVKTFVVPLRDSNHDLE PGVTVGD I GAKMGR
DG I DNGWI QF SNVRI PRFFMLQKYCKVSRSGEVTMPP SEQL S YSAL I
GGRVTMMMDS YRMT SRF I T IALRYAIHRRQFKKKDTDT I ETKL I DYP
LHQKRLFPFLAAAYLFSQGALYLEQTMNATNDKLDEAVSAGEKEAID
AAIVESKKLFVASGCLKSTCTWLTAEAIDEARQACGGHGYS SYNGFG
KAY S DWVVQC TWEGDNN I LAMNVAKPMVRDLLKE PEQKGLVL S SVAD
LDDPAKLVKAFDHAL SGLARD I GAVAEDKGFD I TGPSLVLVSKLNAH
RFL I DGFFKRI T PEWSEVLRPLGFLYADWI LTNFGATFLQYG I I TPD
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SEQ ID NO Description Sequence
VSRK I S SEHFPALCAKVRPNVVGLTDGFNLTDMMTNAAIGRYDGNVY
EHYFETVKALNPPENTKAPYSKALEDMLNRPDLEVRERGEKSEEAAE
ILSS*
Example 33: Amplification of Lipase Activity and Analysis of Strains with
Increased Lipase Activity
The C. tropicalis genome contains multiple genes which encode lipase
activities. A lipase activity,
carried out by a lipase enzyme, liberates fatty acids from a glycerol
backbone. This activity is
particularly beneficial when using oils (e.g., plant based oils, coconut oil)
as the culture feedstock.
Amplification of endogenous lipase activity coding sequences in an engineered
organism has been
shown to improve the level of adipic acid production with respect to a
corresponding strain with a
native level of lipase activity. Without being limited by any theory, it is
believed the increased
adipic acid titers seen are the result of improved substrate utilization.
Cloning of C. tropicalis strain lipase activity coding sequences
BLAST searches were conducted in the C. tropicalis strain ATCC20962 genomic
database using
the gene sequence of a lipase activity encoded by Yarrowia lipolytica (LIP2;
GenBank Accession #
CAB91111.1). The corresponding C. tropicalis sequence was cloned under the
control of PDX4 or
PGK promoters as follows.
Primers were generated to clone the identified C. tropicalis lipase homolog
into vectors pAA73
(P0X4 promoter) and pAA105 (PGK promoter), respectively. For cloning the
lipase activity into
pAA73 vector, the nucleotide sequence encoding the lipase activity was PCR
amplified from C.
tropicalis strain ATCC20336 using primers oAA734 and oAA735 in a reaction
containing 5uL 10X
buffer, 2.0uL of oAA734 and 2 uL of oAA735 (10uM), 1.0uL genomic DNA, 1.0uL of
dNTPs, 1.0uL
of Pfu and 38uL sterile H20. The therrmocycling parameters used were 95 C for
2 minutes, 30
cycles of 95 C 20 seconds, 55 C 30 seconds, 72 C 1 minute, followed by 72 C 5
minutes and a
4 C hold. PCR product of the correct size was gel purified, ligated into pCR-
Blunt II-TOPO and
transformed into competent TOP10 E. coli cells (Invitrogen). Clones containing
PCR inserts were
sequenced to confirm correct DNA sequence. The resulting plasmid was
designated, pAA234.
Plasmid pAA234 was restriction enzyme digested with Xmal and Xbal, according
to manufacturer's
recommendations. DNA fragments were separated on a 0.8% agarose gel. The DNA
fragment
containing the nucleotide sequence encoding lipase activity was gel purified
and ligated into
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Xmal/Xbal digested vector pAA073, and transformed into TOP10 E. coli cells
(Invitrogen). Correct
plasmid structure, including the presence of the fragment carrying lipase
activity, was confirmed by
restriction digestion and designated as pAA235. pAA235 includes the lipase
activity coding
sequence under control of the PDX4 promoter.
For cloning the lipase activity into the pAA105 (PGK promoter) vector, PCR
amplification of the
lipase activity was performed as above using primers oAA736 and oAA737. PCR
product was gel
purified, digested with Sapl, ligated into pAA105 vector and transformed into
TOP10 E. coli cells
(Invitrogen). Correct plasmid structure, including the presence of the
fragment carrying lipase
activity, was confirmed by restriction digestion and designated as pAA236.
pAA236 includes the
lipase activity coding sequence under control of the PGK promoter. The lipase
activity cloned into
plasmids pAA235 and pAA236 was analyzed to determine the presence or absence
of any
potential secretion leader sequences. The N-terminal protein secretion leader
sequence was
predicted by SignalP (SignalP 3.0 Server) and the predicted cleavage site of
the lipase activity
leader sequence is most likely between pos. 15 and 16: SSA-GK, as shown in the
amino acid
sequence listing presented in the table below. The nucleotide sequence of the
lipase activity
cloned into pAA235 and pAA236 also is presented in the table below.
SEQ ID NO Description Sequence
SEQ ID NO:27 C. Tropicalis
ATGATTGTTTTATTCATCCTTGTATTTATTTGTCTATCTTCAGCCGGG
lipase activity,
AAACCAAACAAACCAGAAGCTCCAGCAAAAGATTACATCAAACTCGTT
(identified using GAATTCTCCAATTTTGCCGCCGTTGGCTACTGCGTTAATAGAGGTCTA
GCAAAGGGCCGTCTAGGAGACGAGGACTCCAACTGTGCCTTGTTGGCA
Y. lipolytica
TGCAAGAACGACTTCCTTGCGGACGTCGAGATTATTAAGATATTTGAC
BLAST search) TTCAACCGTCTTAATGAAGTTGGAACAGGTTACTATGCCTTGGACAGG
Nucleotide Seci
, AAGAGAAAGGCAATAATATTGGTATTTAGAGGGTCTGTCTCCCGACGT
GACTGGGCGACAGACATGGATTTCATCCCCACTTCTTACAAGCCAATT
GTGTATGAGGAAAACTTTGGTTGTGACCCCTACATTCTGACCGAATGC
AAGAACTGTCGTGTGCACCGTGGTTTCTACAATTTCTTGAAGGATAAC
TCTGCAGCAATTATCACCGAGGGAATTGCGTTGAAAGAAGAGTACCCG
GACTACCAGTTCTTGATCATTGGTCATTCTTTGGGCGCTGCCTTGACA
ATGTTGAGTGGCATCGAGTTCCAGTTGTTGGGGTACGATCCTTTGGTG
GTGACTTATGGTGGTCCAAAGGTGGGCAACCAAGAGTTTGCTGACTTC
ACGGACAACTTGTTTGACACGGATGAGGTGGACAATGAAATCGCCACC
AACCGTGATTTTTCAAGAGGATTCATTAGAGTGGTACACAAGTATGAT
ATAATACCATTCTTGCCGCCGTTGTTTAGTCACGCAGGGTACGAATAC
TTTATTGACAAGAGAGAGTTGCCCCATGAAGAATGTGATTTGGACAGA
CGAGGCATGGAGTACTCGGGGATATTTAAGAGATCGCTGACCATAAAA
CCGTCCACTTTATGGCCAGATAGGTTGGGGAAGTATGAACATACACAT
TATTTTAGAAGAATCACTAGTTGTAGGGACGACGAT
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SEQ ID NO Description Sequence
SEQ ID NO:28 C. Tropicalis MIVLF I LVF I CLS SAGKPNKPEAPAKDY I
KLVEFSNFAAVGYCVNRGL
lipase activity, AKGRLGDEDSNCALLACKNDFLADVE I I KI
FDFNRLNEVGTGYYALDR
(identified using KRKAI I LVFRGSVSRRDWATDMDF I PT SYKP IVYEENFGCDPY I LTEC
Y l KNCRVHRGFYNFLKDNSAAI I TEGIALKEEYPDYQFL I
IGHSLGAALT
ipolytica .
MLSG I EFQLLGYDPLVVTYGGPKVGNQEFADFTDNLFDTDEVDNE IAT
BLAST search) NRDFSRGFIRVVHKYDI I PFLPPLFSHAGYEYF I DKRELPHEECDLDR
Amino Acid Seci
, RGMEYSGIFKRSLT
IKPSTLWPDRLGKYEHTHYFRRITSCRDDD
Plasmids pAA235 and pAA236 were linearized with Clal and transformed into
competent C.
tropicalis cells of strain sAA329 (URA3 auxotroph). The integration of the
linearized plasmids was
confirmed by colony PCR using primers oAA734 and oAA281 for the pAA235
integration and
oAA736 and oAA281 for the pAA236 integration, respectively. Two transformants
from each
transformation were selected. Strains from the pAA235 integration were
designated as sAA574
and sAA575. Strains from the pAA236 integration were designated as sAA580 and
sAA581. The
strains were used to evaluate the effects of increased lipase activity on
adipic acid production.
One colony of each strain was inoculated into 5 mL 5P92 and grown overnight at
30 C with
shaking at about 200 rpm. The overnight culture was used to inoculate 50 mL of
5P92 medium, in
shake flasks (e.g., about OD600nm 0.4), and incubated under the same condition
for 24 hours. Cells
were harvested and resuspended in 20 mL of TB-lowN medium supplemented with
1.6 mL of
coconut oil and 20uL sterile antifoam B. Samples were taken daily and analyzed
for diacid
production by gas chromatography. The results are shown graphically in Figure
39. As shown in
Figure 39, 3 of the 4 strains with increased copy number of the lipase
activity nucleotide sequence
tested showed increased levels of adipic acid production, when compared to the
parental strain.
The results shown in Figure 39 represent the data from six days of
fermentation in 5P92 medium
supplemented with coconut oil as the carbon source.
A second BLAST search utilizing the nucleotide sequence of a lipase activity
from C. dubliniensis
also was performed in the C. tropicalis strain ATCC20962 database. The second
BLAST search
identified another C. tropicalis lipase activity homolog (e.g., 0RF7657), with
an amino acid
sequence different from the lipase activity identified using the Y.lipolytica
lipase activity sequence.
The second lipase activity is being cloned and over-expressed in C. tropicalis
for evaluation in
fermentation of adipic acid. The amino acid sequence of the second lipase
activity identified in C.
tropicalis strain ATCC20962 using the C. dubliniensis lipase activity sequence
in a BLAST search,
is presented below as SEQ ID NO: 29.
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SEQ ID NO: 29
MLRTVRHYSKVIN I KDKGEKAARVI TSEFAKLKDHYDAPKYPIVLCHGFSGFDRLG
LF PL PNLLEDT TAT TKTKE I TERSL I ELDYWYG IKDALENLGS TVF IAKVPAFGD I
RSRAI S LDKF I EKQCKALRQTE SKS S I YNKPDS SNDDTTTFKDKHQPIKVNL I SHS
MGGVDSRYL I SRI HNDNENYRVAS LT T I LT PHHGSECADF IVDL I GDNGVLKKVC P
PS I YQLT T LHMKKFNEVVKDDP SVQYF S FGARFNPRWYNLFGLTWLVMKYQ I EKEQ
ADRFKHMIDNDGLVSVES SKWGQY I GT LDEVDHLDL INWTNRARSVFDKVMFAQNP
NFNPIALYLE IADQLSKKGL
Example 34: Amplification of acyl-CoA carboxylase activity
Acetyl-CoA carboxylase (e.g., ACC) catalyzes the reaction that produces
Malonyl-CoA for fatty
acid synthesis. The reaction catalyzed by ACC is the committed step in fatty
acid synthesis.
Strains engineered to convert glucose to adipic acid using a native, mutant,
or specialized (e.g.,
hexanoate synthase) fatty acid synthase would benefit from increased carbon
flux through the
pathway, as contributed by increased acetyl-CoA carboxylase activity. The
nucleotide sequence
encoding ACC activity has been cloned and is being over expressed and
evaluated for contribution
to adipic acid conversion in C. tropicalis. The nucleotide sequence encoding
the ACC activity from
C. tropicalis strain ATCC 20336 is presented below as SEQ ID NO: 30.
ATGAGATGCCAAGTATCTCAACCATCACGATTTACTAACTTGCTTGTACATAGACT
CCCACGAACAC TAC T TAAT TATCCAGT TGTAAATACCC TAT T TAT TCC TAGACGTC
AT TAT TCCC T TAAT T T T TCAT TCAAGAACC TAC TAAAGAAAATGACAGATC T T TCC
CCAAGTCCAACAGACTCCCTTAATTACACACAGTTGCACTCATCCTTGCCATCACA
TTTCTTAGGTGGGAACTCGGTGCTCACCGCTGAGCCTTCTGCCGTGACAGATTTCG
TCAAAACACACCAAGGTCACACTGTTATCACCAAAGTCTTGATTGCCAACAACGGT
AT TGGTGCCGTCAAAGAAATAAGATCCGTCAGAAAATGGGCC TACGAAAC T T T TGG
TGACGAAAGAGC TATACAGT T TGTCGCCATGGCCAC TCCCGAAGATATGGAGGC TA
ACGCCGAGTACATTCGAATGGCCGACCAGTTTGTCGAGGTCCCAGGTGGTACCAAT
AACAACAACTACGCGAATGTTGACTTGATTGTCGAAATCGCTGAAAGAACCGATGT
CCACGCCGTTTGGGCTGGTTGGGGTCATGCCTCCGAAAACCCTTTGTTGCCAGRAA
GGT TGGCAGC T TCCCC TAAGAAGATCGTGT T TAT TGGTCC TCCAGGGTC TGCCATG
AGATCTTTGGGTGACAAGATTTCTTCCACCATTGTTGCACAACACGCCAAAGTGCC
ATGTATCCCATGGTCTGGTACTGGTGTCGAAGAGGTCCACGTCGACCCAGAAACCA
AGTTGGTGTCTGTTGACGACCACGTCTACGCCAAAGGTTGCTGTACCTCGCCAGAA
GACGGTTTGGAAAAAGCCAAACGTATCGGATTCCCAGTTATGGTTAAGGCATCCGA
AGGTGGTGGTGGTAAAGGTATCAGAAAAGTCGACCACGAAAAGGACTTCATCAGTT
TGTACAACCAGGCGGCTAACGAAATACCAGGGTCACCAATTTTCATCATGAAGTTG
GCCGGTGACGCCAGACACTTGGAAGTGCAATTGTTTGCCGATCAGTACGGTACCAA
CAT T TCGC T T T TCGGTAGAGAT TGT TC TGTGCAAAGAAGACATCAAAAGATCAT TG
AAGAAGCTCCAGTCACAATTGCCAACAAAGACACTTTTGTTGAGATGGAGAAAGCT
GCCGTCAGATTGGGTAAGTTGGTTGGTTACGTGTCTGCCGGTACCGTTGAATACCT
TTACTCCTACGCCGAAGACAAGTTCTACTTTTTGGAATTGAACCCAAGATTGCAAG
TTGAACATCCAACTACCGAAATGGTTTCCGGTGTCAACTTACCAGCCGCTCAGTTG
CAAATTGCTATGGGTCTCCCAATGCACAGAATCAGAGACATCAGATTGTTGTACGG
TGTTGATCCACACTCTGCCACTGAGATTGATTTCGAGTTCAAGTCCCCAAACTCAT
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TGATCACGCAAAGAAAGCCAGCTCCAAAGGGTCACTGTACCGCTTGTCGTATCACT
TCTGAAGATCCAGGTGAAGGGTTCAAGCCAAGTGGTGGTACTCTTCACGAGTTGAA
CTTCCGTTCTTCGTCCAATGTCTGGGGTTACTTCTCGGTCGCCAACCAATCTTCTA
TCCACTCCTTTGCTGATTCCCAGTTTGGTCACATTTTCGCCTTTGGTGAAAATCGT
CAAGCCTCTAGAAAGCACATGATTGTTGCCTTGAAGGAATTGAGTATCAGAGGTGA
CTTTAGAACCACTGTTGAATACTTGATCAAGTTGTTGGAGACTCCAGATTTCGCCG
ACAACACCATCACTACCGGTTGGTTGGATGAGTTGATCACCAAGAAGTTGACTGCC
GAAAGACCAGATCCTATCGTTGCTGTTGTCTGTGGTGCCGTCACCAAAGCCCACAT
CCAAGCCGAAGAAGACAAGAAGGAGTACATTGAGTCTTTGGAAAAGGGTCAAGTTC
CAAACAAGTCCTTGTTGAAAACTATCTTCCCAGTTGAGTTTATCTACGAAGGTGAA
AGATACAAGTTTACTGCCACCAAGTCCTCCGAAGACAAGTACACTTTGTTCCTCAA
CGGTTCTAGATGTGTCATTGGTGCTCGCTCATTGTCTGATGGTGGCTTGTTGTGTG
CTTTGGACGGTAAGTCCCACTCTGTCTACTGGAAGGAAGAAGCAGCGGCCACTAGA
TTGTCTGTTGACGGTAAGACTTGCTTGTTGGAAGTTGAAAACGACCCAACCCAATT
GAGAACTCCGTCTCCAGGTAAGTTGGTCAAGTACTTGGTTGAGAGTGGTGAACACG
TTGATGCCGGCCAATCTTATGCCGAAGTTGAAGTCATGAAGATGTGTATGCCTTTG
ATTGCACAAGAAAACGGTACTGTTCAATTGCTCAAACAACCAGGTTCCACTCTTAA
CGCTGGTGACATCTTGGCAATCTTGGCATTGGACGATCCATCTAAAGTTAAACACG
CCAAGCCATATGAAGGCACTTTGCCAGAGATGGGTGATCCAACTGTTACCGGTTCC
AAACCAGCTCACTTGTTCCAACATTACGACACCATCTTGAAGAACATCTTGGCTGG
TTACGATAACCAAGTCATTTTGAACTCCACTTTGAAGAACATGATGGAGATCTTGA
AGAACAAGGAGTTGCCTTATTCTGAATGGAGATTGCAAATCTCCGCCTTGCATTCA
AGAATCCCACCAAAGTTGGATGAGGCTTTGACGTCCTTGATTGAAAGAACCGAAAG
CAGAGGCGCCGAATTCCCAGCTCGTCAGATTTTGAAGCTCGTCAACAAGACTCTTG
GTGAACCAGGCAACGAATTGTTGGGCGATGTTGTTGCTCCTCTTGTCTCCATTGCC
AACCGCTACCAGAACGGCTTGGTTGAACACGAGTACGACTACTTTGCTTCATTGGT
TAACGAGTACTGCAATGTTGAACACTTCTTTAGTGGTGAAAACGTGAGAGAAGAAG
ATGTTATCTTGAGATTGAGAGACGAGAACAAGTCTGATTTGAAGAAGGTTATCAGC
ATTTGCTTGTCCCACTCCCGTGTCAGTGCTAAGAACAACTTGATTTTGGCCATCTT
GGAAGCTTATGAACCATTGTTGCAATCCAACTCTTCAACTGCCGTTGCCATTAGAG
ATTCTTTGAAGAAGATAGTCCAGTTGGATTCTCGTGCTTGTGCCAAGGTTGGTTTG
AAAGCTAGAGAACTTTTGATTCAATGTTCTTTGCCATCCATCAAGGAAAGATCTGA
CCAATTGGAACACATTTTGAGAAGTGCAGTCGTTGAGACTTCTTATGGTGAAGTTT
TCGCCAAGCACAGAGAACCTAAATTGGAAATCATCCAAGAAGTTGTCGAATCCAAG
CACGTTGTTTTCGATGTCTTGTCGCAATTTTTGGTCCACCAAGACTGCTGGGTTGC
CATTGCTGCTGCCGAAGTCTATGTTAGACGTTCCTACAGAGCTTATGATTTGGGTA
AGATCGATTACCACATTCATGACAGATTGCCAATTGTTGAATGGAAGTTCAAGTTG
GCTCAAATCGCAGGTTCCAGATACAACGCCGTCCAATCTGCCAGTGTTGGTGACGA
CTCGACCACTATGAAGCATGCTGCATCTGTTTCTGACTTGTCGTTTGTTGTTGATT
CCAAGAGCGAATCCACTTCCAGAACTGGTGTTTTGGTTCCAGCTAGACATTTGGAC
GATGTTGATGAGATTCTTTCTGCTGCATTGGAGTACTTCCAACCATCTGATGCACT
CTCTTTCCAAGCTAAGGGAGAAAGACCAGAGTTGTTGAATGTTTTGAACATTGTCA
TCACCGACATTGACGGTTACTCTGACGAAGATGAATGCTTGAAGAGAATTCATGAA
ATCTTGAACGAGTACGAAGACGATTTGGTCTTTGCTGGTGTTCGTCGTGTTACTTT
TGTTTTCGCCCACCAAGTTGGTTCTTATCCAAAGTACTACACCTTCACTGGTCCAG
TGTATGAAGAAAACAAGGTTATCAGACACATCGAACCAGCTTTGGCTTTCCAATTG
GAATTGGGAAGATTAGCCAACTTTGACATCAAGCCAATTTTCACCAATAACAGAAA
CATTCATGTTTACGAAGCTATTGGTAAGAATGCTCCTTCGGATAAGAGATTCTTCA
CTAGAGGTATTATTAGAGGTGGTGTCCTCAAAGATGAAATCAGTCTTACTGAGTAC
TTGATTGCTGAATCGAACAGATTGATCAGTGNTATCTTGGATACCTTGGAAGTTAT
TGACACTTCCAACTCCGATTTGAACCACATTTTCATCAACTTCTCCAACGTTTTCA
ACGTCCAACCAGCTGATGTTGAAGCTGCTTTTGCTTCATTCTTGGAAAGATTTGGT
AGAAGATTGTGGAGATTGAGAGTTACTGGTGCTGAAATCAGAATTGTCTGTACCGA
CCCACAGGGCAACTCATTCCCATTGCGTGCCATTATCAATAACGTTTCAGGTTATG
TTGTCAAGTCGGAATTGTACTTGGAAGTGAAGAACCCTAAGGGTGATTGGGTCTTC
AAATCCATTGGCCACCCTGGCTCAATGCACTTGCAACCAATCTCGACTCCATACCC
AGTCAAGGAATCCTTGCAGCCAAAACGTTACAGAGCTCACAACATGGGAACCACTT
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TTGTTTACGATTTCCCAGAGTTGTTCCGTCAAGCCACCATTTCCCAATGGAAGAAG
CACGGCAAGAAGGCTCCTAAAGATGTCTTCACTTCTTTGGAGTTGATCACCGATGA
AAACGATGCTTTGGTTGCCGTTGAAAGAGATCCAGGTGCCAACAAGATTGGTATGG
TTGGTTTCAAGGTCACTGCCAAGACCCCTGAATACCCACGCGGACGTTCATTCATC
ATTGTTGCCAATGATATCACCCACAAGATTGGTTCCTTTGGTCCAGATGAAGATGA
ATACTTCAACAAGTGTACCGACTTGGCCAGAAAGTTGGGTGTTCCAAGAATTTACC
TTTCTGCCAACTCCGGTGCCAGAATTGGTGTTGCTGAAGAGTTGATTCCATTGTAC
CAAGTTGCTTGGAACGAAGAAGGTAACCCAGATAAAGGTTTCAGATACTTGTACTT
GAACCCAGACGCCAAAGAAGCTTTGGAAAAAGACGGCAAGGGTGACACTATTGTTA
CTGAACGTATTGTCGAAGATGGTCAAGAACGTCACGTTATCAAGGCCATTATTGGT
GCTGAGAACGGCTTGGGTGTTGAATGTTTGAAAGGTTCCGGTTTGATTGCTGGTGC
CACTTCAAGAGCCTACAGAGACATCTTCACCATTACCTTGGTCACTTGTAGATCTG
TTGGTATTGGTGCCTATTTGGTCAGATTGGGTCAAAGAGCTATCCAAATTGAAGGT
CAACCAATCATTTTGACTGGTGCACCAGCTATCAACAAGTTGTTGGGTAGAGAAGT
TTACTCGTCGAACTTGCAATTGGGTGGTACCCAGATCATGTACAACAATGGTGTTT
CCCACTTAACTGCCAGTGACGATTTGGCTGGTGTTGAGAAGATCATGGAATGGTTG
TCCTACGTTCCAGCTAAGCGTGGTATGCCAGTACCAATCTTGGAAAGTGAAGATAC
CTGGGACAGAGACATTGACTACTACCCACCAAAGCAAGAAGCTTTCGACATCAGAT
GGATGATCGAAGGTAAGCAAGTTGAAGGTGAAGAGTTTGAATCTGGTTTGTTTGAC
AAAGGTTCATTCCAGGAAACTTTATCAGGATGGGCTAAAGGTGTTGTCGTTGGTAG
AGCTCGTCTCGGTGGTATCCCAATTGGTGTCATTGGTGTTGAGACCAGAACTATTG
AAAACATGATCCCAGCTGACCCAGCCAACCCAAGTTCCACTGAAGCCTTGATCCAA
GAAGCCGGTCAAGTCTGGTATCCAAACTCTGCGTTCAAGACCGCACAAGCCATTAA
CGACTTCAACAACGGTGAACAATTGCCATTGATGATCTTGGCCAACTGGAGAGGTT
TCTCTGGTGGTCAGAGAGATATGTACAACGAGGTCTTGAAGTACGGTTCCTTCATT
GTTGACGCTTTAGTTGATTTCAAGCAGCCAATCTTCACTTACATCCCACCAAATGG
TGAATTGAGAGGTGGCTCTTGGGTCGTTGTTGATCCAACCATCAACTCCGACATGA
TGGAAATGTATGCCGACGTTGACTCCAGAGCTGGTGTTTTGGAACCAGAAGGTATG
GTTGGTATCAAATACAGACGGGACAAGTTGTTGGCTACCATGCAAAGATTGGATCC
AACTTATGCCCAATTGAAGGAGAAGTTGAACGACTCGAGCTTGTCGCCAGAAGAAC
ATGCCCAAGTCAGCACCAAGATTGTCAAGCGTGAAAAGGCATTGTTGCCAATCTAT
GCCCAAATTTCTGTCCAGTTTGCCGACTTGCACGACAGATCCGGACGTATGATGGC
TAAAGGTGTCATTAGAAAAGAAATCAAGTGGGTTGACGCCAGACGTTTCTTCTTCT
GGAGATTGAGAAGAAGATTGAACGAAGAGTACGTTTTGAAGTTGATTGGTGAACAG
GTCAAGAATGCCAACAAGTTGGAAAAGGTTGCCAGGTTGAAGAGTTGGATGCCAAC
TGTTGACTACGACGATGACCAAGCTGTCAGTACTTGGATTGAAGAGAACCACGCCA
AATTGCAAAAGAGAGTTGAAGAATTGAGACAGGAGAAGAACAAGTCCGACATTGTC
AAATTGTTGCAAGAAGACCCATCAAACGCTGCCTCTGTTATGAGGGATTTCGTTGA
TAGATTGTCCGATGAAGAAAAGGAAAAGTTCCTTAAATCATTGAACTAG
Example 35: Cloning, Amplification and Overexpression of C. tropicalis fatty
acid synthase activity
Type l fatty acid synthases contain all of the active domains for fatty acid
synthase on one (e.g.,
alpha) or two (e.g., alpha and beta) polypeptides. Hexanoate synthase, a
specialized fatty acid
synthase enzyme, is unique in that the fatty acid product is the six carbon
long hexanoic acid
rather than a fatty acid with a 16 or 18 carbon chain length product produced
by native fungal fatty
acid synthases. Hexanoate synthase is composed of an A (HexA) and B (HexB)
subunit with the
same active domain organization as the native fungal fatty acid synthase alpha
(Fas2) and beta
(Fas1) subunits.
231
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Hexanoate synthase activity was introduced into an engineered strain by
placing the nucleotide
sequences of HexA and HexB under the control of the C. tropicalis PGK promoter
and integrating
the heterologous sequence into the C. tropicalis genome, as described herein.
To further provide
increased carbon flux through engineered pathways and increase the production
of adipic acid,
additional fatty acid synthase activities were cloned and amplified in
engineered strains. Mutant
fatty acid synthase activities that produce intermediate chain length products
also would beneficial
for increasing the conversion of carbon feedstocks to adipic acid. To ensure
that the fatty acids
produced are not converted to products shorter than 6 carbons (e.g., hexanoic
acid), the strains for
increased fatty acid synthesis activity also would include a partially blocked
beta oxidation pathway
(e.g., disrupted for PDX4 activity) to reduce or eliminate the degradation of
fatty acids to chain
lengths below 6 carbons. In some embodiments, FAS mutants can be identified by
using serially
passaged or mutagenized C. tropicalis strains and determining fatty acid
profiles by gas
chromatography.
Cloning of C. tropicalis FAS2 and FAS1
The native C. tropicalis FAS2 and FAS1 genes were cloned between the C.
tropicalis strain
ATCC20336 PGK promoter and terminator, and correct nucleotide sequence was
verified by DNA
sequencing. The vectors were linearized inside the URA3 selectable marker by
restriction enzyme
digestion and subsequently co-transformed into a ura- derivative of C.
tropicalis strain ATCC20962
by targeted single-crossover integration at the URA3 locus. Correct
integration of both the FAS2
and FAS1 constructs was confirmed by PCR. The nucleotide sequences of the C.
tropicalis FAS2
and FAS1 activities are presented in the table below as SEQ ID NOS: 31 and 32.
232
113VV1993VV3V33VISVV31V31119991V3IV99133991VVV911V339199VV9911331311VV91999VV33
1
VV991VVV9991V9V33VV9V3393VV199991V33199119VV933911119933V319119119319331VV99113
V
9911917/399VV9911VV9V331391199VV9VV31V9V91V93V1931VVV91133VV333113V9311VVV91V3V
V3
397/9VV33VV311933V1199VV33V3V13VV3391V91191VV3V91991VV3191199VVVV3VV931V339331V
V9
117/3313191397/9VV9V119VV9139331VV991V9113V9131V9V9119VV33113V3111V99VV9113VVVV
911
VaLIVV39111991993VV9113V933991V119V33VV9VV9VV33919119VV31911V9V9V3313V911911199
9
11317/3VV31113991VVV99VVVV333131133VV9V31919911V9VVVV931V199VV933911V31V3VV3VV3
39
397/917/91119913V199V9V33V99119911V11933919919131933V9119VV3313999911V9VV93313V
199
17/97/3VV31191133VVV991133111V9VV331VV91313V19119991V931119931113V19937/33VV133
1319
E-=1
IVV3391131VISIVV3139V33V9V33V3V933V13V9VV9VV331VVV319139199911911V9V9111133VV33
V
911917/11VV9V3V3339111VV9331VVV1311V911V3VV3V931V1993VVISSIVVVV9V3331V139139311
V3
3317/11V3391V99113V9991399V111991999VV9VVV331V93V93V131V3VIVV31199111393V91191V
9V
V3VVV131199VV33VV311V3311911911V91113V1311993391391993V1V9V3393V191V3VVVV33VIDV
I
97/9137/1199VV9VV331311V9V13133V33V1191191199VV33919919919V31V911199VV391131VVV
933
9199117/331V9913919913919933V1199111193VISVV3V99VV31133V3131993VV319139139331VV
99
1119937/99113V11133391319119VV3933V93VIVV9991399VV93VV33V9VV9VVVV311V3V3911311V
33
Lc)
317/337/9V3333V3311913VV31131119V33VV333911VV3113V9VV3V9V33911V3113911VVV9IV93V
913
C
137/933933V33111VVV91V9VV91V913VV3333V3VV33139913VV33139VV391131V3V99VV33VVIIVV
39
7/313VVV911VVV93V9V311991391191199VV911911VV39V93V1319VVV9V3911VV99119VV39V3V9V
9V
331139VV39VV33131V339VVV3V1119V9V3399113V9VV9VV3IIVIDVV33VV333911V9VV33V9IVVV91
9
0
9199VV333991VVV93V13193V19V3VV91119VVV9V1191311V99VVVV9339VV9VV93V1VV911V3VV199
9
(
VV33939131933V33V9VVV3319913V31VV9V1311193V99VV3V1919V333V9VV9IVV91133VVV39VV19
1
Lc)
cs)
3VV37/911V9119V3VV3199911V9V9VV3399119VV3V113V9V91999VV3VIVV9V333913V33V911V3V3
3V
IVV391V3119VV31V91113VV333VV331V9V3VV9IVIIVVV331VISIVV33V931V33VVV9V9V3V9119331
3
Lc)
OD
C
37/9119VV19911191111V1V91113VISIV3319111191V9VV3V9V3399913VV9913V13311V91V133VV
9V
0
7/39VVV911139V3131113VV33VVV31V199V9V3393V1311VV91991V3VV9339VV99919113V9911VV9
VV
7/97/3911119139331139VV9VVVVV9VVV31V1111319VV139V99V313VV9113V9319VV39113VIV9V1
399
1131997/9VIIVV3VV3V9V139911VVV9VV3VVVV913913V9113393V991113913919V3V91191193393
99
1993393391997/99139199339199339131139V3193191133111V199139131V313391VIVVV9V3139
91
7/337/3V991131133V9VV139VV93399V9131199911V9V39VV39133VV93VV33V9111391199119113
191
3137/9VV3V9V199339199911199311V9V331VV99113VIVVVV9V33913933V3IVV313111991991339
1V
9VV13113191V911V9V19911V911V3113V13113V9VV199911VV319919V3113313V9VV33119V3VV93
3
9911VV9VV9911V3333V1V9VV9V33VVVVV913313V331199311VV9VVV1999113V919911311VVV93VV
9
7/3319137/3319VV1993VVII9VIIIV9VVV31V1399VV33191VV331191391V9911V33VVV9VVVIIVVV
9V3
oo
13911991131937/331V9119113311399VV3197/33VV91V9V3331V331VV9139133399139131V3333
9V3
oo
31391393393197/3333913913913313V1391391391391391391397/33V3V139339V331393393391
39V
V9VV99VV9VVV33VV9VV99VVV331391131V9V39V331V9V339VV3VIDVIDIV9V99VV3393V99VV1313V
1
3917/11319VV9V9VVV39113319119393393V93V1331VV33VIVVV3399VV31V33VV9V3VV33991V199
33 9CEOZOOlV
991133VV3333113339931VVV931V31VV9VVV933V3VV3V3VV39VV9113113191V9VV333VVV931V991
V slleo!daq :ON
97/3197/331311393119V31V1139V11911VV9V3VV1191133V3V3331VIIVV9VV3VV911V9V9V339VV
91V µZSVd al (DS
ON
amanbas uoRdposaa 01 02S
tZ
Z C/) Z(J)
0 M 0 õsrT1
KJ
0 0
> 0 71
0
H= > 0
C) ..:", = C/) ci)
O0 -s
o
0.) 77.
0
0)C/)
=
nnH>HHon>nnnHoHH>J>,000nOOHnHH>J>n> HHnH>>>0 (j)
Ho HoHHH HH >a- oH onH>a-o> HnHo >> onH>nnO>n>nHnH>-> 0
0
H H On>0>HOnnO>->JO>n>on>a-n>JoHHHnnHH>o>00>a- _0
>0 onHnnHH oon>Hn000n>>-HnnH000nHH>nnoHn>> 0 c
nH>J.- n>oonHHciooHnHoHnon>0>H>nnHnon>.-JnOnOO =
>On>> HO
>a-HHHonnnHoHoH >H>nnonHn>JH -
O =
., Hono>Ji-inno n>JoHnHoon>00HG--)HnOOPOH>JH >>>>0
>nHH>a-H>onn>,>-n>a-O>JO>O>HH>>0>nH oppHi-in, noHo
HnHo>a-H>a- H> >> >a- H>J-nHn>a-HHO>H>on>HO =J>- HnOnnO>Pnn
HnoHnnHHHHnoHop>no>nHnHopnO>OOnn>nno>>-Hn
H= nHHHOH >nnOn>>0-0 onooH HH>nnHoG)HnO>HHO HO>
HHo>nn> nnoH HH>a-n H>oonn>n>noHoHHOH>HHO>n
OH>n>>nonnonnnoo HH>Honno>->onHOOPO>OOHnH
HH>a-nnOO >1-1 >HHnn>>nOnOOn>n>00OnnH>0 >0>on>>nnn
HHH>nnonHHnHH>a-oH>a- HnHHnHo>a-Hnn>nnoH>J-000>>H
OOHnOnOH OnnO>, HO>, n>nH>HHO>H>JOH>JHOH>>>HnO
HHOHHOOOHHHHHHHHOH 0
nnHO H Don nn>JHOH>PH>JOHn>> H>nnn>H>a- HnoHnnnH
H= o> nHHoHnon O>0>>n>>HOnO 00>->H>>000>H000
H H H 6-) >n nnH>nonn>>JoH6--)>J00000OnnoHnHn> >Ho
H= nH> >0-000>J-Ho>a-o>->n> n>nHonnoH>n Hon>HHHoH
O>>0 >n>>oHHHHnnon nnoH>J> >a- Honn H>JoHooH>o
>nno nHonnoo>nnnHHn>H>o on>>>Hn>.-JnoHHno>.-J6--)Hn
OnnnHn>nOnnHnOP>H>HOH> 6-) p 6-) H H H D,- H H >n>H>a-oHn
Hn>n>>nHO>J>JHH>J>00>nOOOHn>a-oHHonn>HonnH 6--) p 6-) p
>J-
O
HOOOHHHHHHOHHOOHHHOH >
H= 1-1 Hop IN-1-]6-)H6-),,Hppo,,-HH =,1-] ,J6-)1-]=Ji-ii-]
non>JHHnnH>HoHon
H= o>no>>>0 HO oHno>nn >H>a->o>nnonHHono Hon
H= Ho>>n>nn H onoonH Hnn >0>>>>H0H>J-HOHOn>nHn
H= H>J>Hnnon> H>a-n HHOO>O>nOO>OHHnonooH> >no>
HonHnHH>a-HHHonO >>>H>On>>, 6-) n po 6-) 6-) >0- H >0- H OnH>Hn
n= O HOnnH HO Hn>>n>0 1-ii-ii-
iG-)i-ini-innni-innHoG-) 6-) n H 6-) 6-)
n 6--) H n 6--) n H n n n n H H H 0
0 >0 0 n
>HHH> HnoHHnno pa) H Hn>0>>anHpHOni-11-11-10>ann
HHnnHonno>nHnnpHn>nno>.- H6--)H6--)onp-oHn>nHoHHH
= nnon 0
HHnonHH>JHnHHnnHoon>nno>HHnno
HOO
O0 >001-1>n
OH>on >>Honno>a-nH> HH>J00HHonoo>n
HnHHHHo
n>H>on>>>0 HO >00 ooHnHHHn>a-oHnHHHHnnHn >a-nH
>no>a-nHHno o>n>a-nnH>nHoo0>->OHHOHHo>a-nHo non
nonn>Hno> on>>a-noHooHooH>H>a-o0OHOO>OHHO>
HOOHH
>>OnnOH HH 0 H n H 6-
) H 6-) HnOHnO>H>nHOPHHnHH 6-)
n G) > n 0 > > 0 H HHHOHOOHHHOHO
= n n
n n H
n >a- n n H
>noo> HHon o = J n H H H>nn>>, H n H H 6-) H
6-) n
HnH>OG-)HHH >JHHHH >a- nHH>a- H H>a- oonnoonoHHoonno
>nOHH>Hnn n 6-) Hr=)0>ni-1
H>H>J>H>JOH>Hnn>
n n o > n o n n
Z9S9S0/ZIOZS9lIDcl 86881'0/10Z OM
S3-0-17T03 S600S830 VD
37/31997/7/1997/33V9V37/7/3191113397/7/919197/7/37/7/199117/1393V3V3V3117/7/919
911V3V97/7/3131131
117/337/31VVV937/919991131VVV397/97/7/931917/931V3V917/7/37/937/113331933113919
93119919119V
/99137/13117/7/997/7/317/7/97/37/93VVV9V3VVVV9917/317/97/99113197/337/391119991
7/9137/7/3337/937/19
el
7/7/37/19113117/339111391197/33VVV39917/9139137/7/97/37/37/7/9117/9111319117/7/
3117/337/3VVV3397/7/
In
3199117/7/911VVV91191197/3311991197/919997/7/3199VVVV917/1139137/911VVV97/7/337
/13997/7/37/911
In
o
V331119197/331V9V33911933313991111V3V937/39117/3337/7/33931937/397/7/3313997/7/
3319117/7/97/3
el
,--i
7/7/337/331317/31113937/391199137/7/9117/7/9137/19933991191137/991199137/97/7/3
997/3397/3391197/7/
o
el
33119911VVV937/131933VVV997/7/7/97/337/13917/7/3197/7/311917/7/37/7/13917/7/119
97/7/37/9337/97/7/31119
ci)
919931137/197/91191197/3331V97/7/3317/3917/91991393VIVV917/997/7/317/91197/7/33
9117/17/339997/93
c.)
V3317/13137/991131VVV9399117/7/337/7/917/911997/7/97/7/337/3117/7/33313391197/3
319937/3911317/191
a,
137/7/97/7/7/311937/97/7/937/931911919VVV991137/97/7/91317/7/399111313117/9VVV9
7/7/3113113117/7/911
1V9VVV937/99113197/339193117/331199339VVVV39997/337/97/33913991191191111137/337
/91393V9V
V97/7/9331117/9117/7/37/7/997/7/3397/7/37/3331197/7/37/7/31131197/9339197/311V3
VVV9V3337/937/7/91197/3
317/3113VVVV3911911331119919399339139131137/311V9VVV9V7/91197/97/7/97/91111137/
919937/191
137/7/7/97/7/1133111937/9117/9917/9VVV313197/7/97/7/31937/1917/91197/9119911V9V
37/7/33931VVV997/93
/1337/91VVV97/7/391137/919111919913113917/7/97/7/1991119917/339VVVV331137/9397/
37/7/91197/7/97/7/
Lc)
317/317/37/13V9V9V97/7/97/7/97/7/317/3397/7/9911339VVVV339VVV3311337/7/311317/1
37/37/937/99117/7/99V
C \ I
V991311917/319199V9V337/339117/97/7/37/3317/7/3397/919991VVV91317/97/119137/317
/317/199199337/7/
1
ro
o
3397/7/97/7/37/133VVV397/999197/7/97/917/97/333191997/33391931339117/911VVV97/7
/1397/7/991193113V
1
.i.
37/333997/997/7/339337/917/319V9V33119911191131919917/937/17/33917/7/337/3337/1
37/7/311V9V97/7/33
H
0
1991131199137/9113V1V3337/133V3V97/7/937/913119911199933119913911991131911V3VVV
33391V
(N
97/7/97/317/97/7/33137/1917/97/3313317/7/337/7/337/331117/97/7/911113137/337/33
991991997/97/19919913V In
Lc)
m
cs)
9917/7/3911319117/7/333119VVV3317/313997/7/339117/337/317/3197/7/3397/317/33937
/9117/9311997/337/7/ el
o
o
V91139991137/3937/3131999119V97/7/911V3VIVV9137/3391197/7/99111317/331191991391
99117/337/9
Lc)
OD
C \ I
111317/7/3317/7/3317/11999VVVV9V9V91197/997/7/117/9117/33117/1999917/7/3911917/
3117/3337/7/31937/1
0
117/91137/7/317/19991119937/11997/3397/7/317/37/933911911VVV337/9117/339VVV337/
917/317/997/39933
4
o
931117/11991991993397/117/7/911V3V317/139913917/7/911131339339117/31V3V9V3337/7
/119137/337/7/
33V3V917/1997/33319917/911333139V9V3999119117/7/393131197/7/337/37/7/31937/131V
9VVV9997/3331
137/97/7/319911137/7/33VVV3117/7/97/7/991199117/9397/13999197/7/317/33197/7/37/
9339131137/97/331131
7/7/7/937/397/7/31119917/197/917/937/9117/7/3337/7/33137/911313919913911911VVIV
V9V139199137/199V
/997/7/37/7/V9V3V3337/9111191999111993313191991997/3339931117/9911117/37/3337/3
3999V3V337/7/
337/33933VVV999137/31197/3391197/9337/317/91197/7/319313V9V3V9117/117/7/337/7/9
97/7/397/97/97/7/331
137/7/33139937/937/1337/17/937/13197/39117/97/7/911VVV9V33111319111933V3VV9VVV3
1917/917/99111
1V3V197/9137/33917/913991191137/313137/33117/337/39111117/7/33911311V9V37/7/331
19197/7/91197/7/
oo
o
1937/7/9197/3117/331191937/317/7/337/99117/99917/7/331399VVVV33VVV9V91137/7/911
37/7/31119937/111
oo
oo
333197/99333331991311131199111VVV9V33919917/7/319911197/31933911V3V39VVVV99VVV3
39113
.7r
o
V313137/7/337/97/37/7/9117/11197/7/97/91197/7/3337/317/337/91137/97/97/1197/319
11917/7/331317/337/97/199
m
,--i
7/7/919937/37/7/991133137/97/7/3911917/337/7/337/33911337/137/7/97/7/3337/11399
17/9111917/97/33919911
o
el
V311113917/911331319339139117/3313VVVV3VVV31133137/999133137/9331197/331117/139
337/1391
0
37/1199111997/7/333137/3199337/33199197/7/9119313V9V9V1117/7/9199133337/9111991
13119VVV391
ON
amanbas uoRdposaa 01 02S
el
0
In
0
In
0
el
,--i
o
el
ci)
c.)
a,
SVIVV93VIVVVVV999137/7/37/9911317/13997/7/317/9
VVVV91317/7/7/137/91137/937/131917/997/331137/17/7/997/7/137/1137/7/91117/3397/
7/139137/91137/7/13311V
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Cultures of positive transformants were grown overnight in YPD medium at 30 C
and 200rpm
shaking until stationary phase. Cells from 4mL of the culture were harvested
by centrifugation and
the cell pellet was frozen at -20 C until used. Cell pellets were treated for
lysis by resuspension in
cold yeast lysis buffer (50mM Tris pH 8.0, 0.1% Triton X-100, 0.5mM EDTA, 1X
ProCEASE
protease inhibitors), working on ice during the entire lysis procedure. The
resuspended cell pellet
solutions were added to prechilled screw-cap tubes containing zirconia beads
and cycled on a
Bead Beater (BioSpec Products) once for 2 minutes. A volume of the whole cell
extract (200uL)
was removed for later analysis and stored at 4 C. The remainder of the whole
cell extract was
removed to a new tube and centrifuged for 15 minutes at 16,000 x g 4 C to
pellet insoluble debris.
The supernatant soluble cell extract was removed to a new tube and stored at 4
C. Protein
concentration was determined by Bradford assay (Pierce). For SDS PAGE
analysis, a volume of
whole cell extract and soluble cell extract from each sample equivalent to
5Oug total protein was
acetone precipitated at -20 C for 2 hours and centrifuged for 15 minutes at
16,000 x g 4 C.
The protein pellet was washed with cold 80% acetone and recentrifuged. The
protein pellet was
allowed to air dry for 10 minutes before adding 50uL of 1XLDS sample buffer
(Invitrogen)
containing 50mM DTT and incubated overnight at 4 C. The samples were heated at
70 C for 10
minutes before SDS PAGE on 4-12% NuPAGE gels (Invitrogen) and transfer to
nitrocellulose.
lmmunodetection was performed using overnight incubation in 1:5,000 mouse anti-
6xHIS (Abcam)
primary antibody and four hour incubation in 1:5,000 donkey anti-mouse HRP
(Abcam) secondary
antibody. Signal development was performed with Supersignal Pico West reagent
(Pierce). For
NativePAGE analysis, samples of soluble cell extract were prepared at a final
concentration of
0.35ug/uL in yeast lysis buffer and 1X NativePAGE sample buffer (Invitrogen)
containing 0.025%
G250. Samples (3.5ug) were resolved on 3-12% NativePAGE gels (Invitrogen) and
transferred to
PVDF according to manufacturer's instructions. lmmunodetection of 6xHIS tagged
oligomeric
complexes was performed as described herein.
lmmunodetection of the expressed proteins is shown in Figures 40 and 41.
Denaturing SDS
PAGE (see Figure 40) shows immunoreactive bands at sizes expected for the Fas2
(206 kDa) and
Fas1 (229 kDa) subunits indicating successful over-expression of both FAS
subunits. Native
electrophoresis allowed detection of oligomeric complexes formed by the 6xHIS-
tagged subunits.
lmmunodetection of the native PAGE (see Figure 41) shows no detectable signal
at the size for the
individual subunits indicating that all subunits are in oligomeric complexes.
Two sizes of oligomeric
complexes are detected, one at an estimated size of about 600 kDa and another
with an estimated
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size of about 1.1 MDa. The detected large complex is smaller than the
predicted size for the native
alpha6beta6 FAS (2.6 MDa), however migration of large oligomeric complexes in
native PAGE is
known to frequently be subject to large migration error, thereby hampering
accurate size
estimation. qPCR analysis of strains engineered for amplified FAS activity
indicated that in some
strains about 2 additional copies of FAS2 and FAS1 subunits were present (data
not shown).
In some embodiments, strains engineered for conversion of glucose to adipic
acid include two or
more additional copies of each of the nucleotide sequences identical to SEQ ID
NOS: 31 and 32.
In certain embodiments, strains engineered for conversion of glucose to adipic
acid produce two or
more times the fatty acid synthase activity encoded by nucleotide sequences
identical to SEQ ID
NOS: 31 and 32, as compared to a strain with native fatty acid synthase
activity.
In some embodiments, strains engineered for conversion of paraffins (e.g.,
oils, petroleum
distillates, plant based oils, coconut oil) to adipic acid include two or more
additional copies of each
of the nucleotide sequences identical to SEQ ID NOS: 31 and 32. In certain
embodiments, strains
engineered for conversion of paraffins to adipic acid produce two or more
times the fatty acid
synthase activity encoded by nucleotide sequences identical to SEQ ID NOS: 31
and 32, as
compared to a strain with native fatty acid synthase activity.
Example 36: Cloning of the C. tropicalis ATCC20336 ZWF gene
Plasmid pAA246
A PCR product containing the ZWF (glucose 6 phosphate dehydrogenase)
nucleotide sequence
was amplified from C. tropicalis 20336 genomic DNA using primers oAA831 and
oAA832. The
PCR product was gel purified and ligated into pCR-Blunt II-TOPO (Invitrogen),
transformed into
competent TOP10 E. coli cells (Invitrogen) and clones containing PCR inserts
were sequenced to
confirm correct DNA sequence. One such plasmid was designated pAA246. Plasmid
pAA246 was
digested with BspQI and a 1.5-kb fragment was isolated. Plasmid pAA073 which
contained a
PDX4 promoter and PDX4 terminator also was digested with BspQI and gel
purified. The isolated
fragments were ligated together to generate plasmid pAA253. Plasmid pAA253
contains
PPDX4ZWFTP0X4 fragment and URA3.
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Strains sAA650 and sAA651
Plasmids pAA253 and pAA156 were digested with Clal and column purified. Strain
sAA329 was
transformed with this linearized DNA and plated on SCD-ura plate. Several
colonies were checked
for ZWF and CYP52A19 integration. From those colonies, qPCR was performed to
check the copy
number of ZWF and CYP52A19 integration. Strain sAA650 contains 5 copies of ZWF
and 9 copies
of CYP52A19. Strain sAA651 contains 5 copies of ZWF and 12 copies of CYP52A19.
The nucleotide and amino acid sequence encoded by the C. tropicalis strain
ATC20336 ZWF gene
are presented below as SEQ ID NOS: 33 and 34
ZWF nucleotide sequence SEQ ID NO: 33
ATGTCTTATGATTCATTCGGTGACTACGTCACTATCGTCGTTTTCGGTGCTTCCGG
TGACTTGGCCAGCAAAAAAACCTTCCCTGCCTTGTTTGGCTTGTTTAGAGAAAAGC
AATTGCCCCCAACCGTCCAGATCATTGGCTATGCCAGATCCCATTTGTCCGACAAG
GACTTCAAAACCAAGATCTCCTCCCACTTCAAGGGCGGCGACGAAAAAACCAAGCA
AGACTTCTTGAACTTGTGTACTTATATCAGCGACCCATACGACACTGACGATGGTT
ACAAGAGATTGGAAGCCGCCGCTCAAGAATACGAATCCAAGCACAACGTCAAGGTC
CC TGAAAGAT TGT T T TAC T TGGCC T TGCC TCC T TC TGTC T TCCACACCGTC TGTGA
GCAAGTCAAGAAGATCGTCTACCCTAAGGACGGTAAGCTCAGAATCATCATTGAAA
AGCCGTTCGGACGTGATTTGGCCACCTACCGTGAATTGCAAAAGCAAATCTCCCCA
TTGTTCACCGAAGACGAACTCTACAGAATTGACCACTACTTGGGTAAAGAAATGGT
CAAGAACTTGTTGGTTTTGAGATTCGGTAACGAATTGTTCAGTGGGATCTGGAACA
ACAAGCACATCACCTCGGTGCAAATCTCCTTCAAGGAACCCTTCGGTACCGAAGGT
AGAGGTGGCTACTTTGACAACATTGGTATCATCAGAGATGTCATGCAAAACCACTT
GT TGCAAGTC T TGACC T TGT TGACCATGGAAAGACCAGTC TC T T T TGACCCAGAAG
CTGTCAGAGACGAAAAGGTCAAGGTTTTGAAAGCTTTTGACAAGATTGACGTCAAC
GACGTTCTTTTGGGACAATACGCCAAGTCTGAGGATGGCTCCAAGCCAGGTTACTT
GGATGACTCCACCGTCAAGCCAAACTCCAAGGCTGTCACCTACGCCGCTTTCAGAG
TCAACATCCACAACGAAAGATGGGACGGTGTTCCAATTGTTTTGAGAGCCGGTAAG
GC T T TAGACGAAGGTAAAGT TGAAAT TAGAATCCAAT TCAAGCCAGT TGCCAAAGG
TATGT T TAAGGAGATCCAAAGAAACGAAT TGGT TAT TAGAATCCAACCAGACGAAG
CCATCTACTTGAAGATCAACTCCAAGATCCCAGGTATCTCCACCGAAACTTCCTTG
ACCGACTTGGACTTGACTTACTCCAAGCGTTACTCCAAGGACTTCTGGATCCCAGA
AGCATACGAAGCCTTGATCAGAGACTGTTACTTGGGCAACCACTCCAACTTTGTCA
GAGACGATGAATTGGAAGTTGCTTGGAAGCTCTTCACCCCATTGTTGGAAGCCGTT
GAAAAAGAAGACGAAGTCAGCTTGGGAACCTACCCATACGGATCCAAGGGTCCTAA
AGAATTGAGAAAGTACTTGGTCGACCACGGTTACGTCTTCAACGACCCAGGTACTT
ACCAATGGCCATTGACCAACACCGATGTCAAAGGTAAGATCTAAGAATAG
ZWF amino acid sequence SEQ ID NO: 34
MSYDSFGDYVT IVVFGASGDLASKKTFPALFGLFREKQL PPTVQ I I GYARSHL S DK
DFKTKI S SHFKGGDEKTKQDFLNLC TY I SDPYDTDDGYKRLEAAAQEYESKHNVKV
PERLFYLAL PP SVFHTVCEQVKKIVYPKDGKLRI I I EKPFGRDLATYRELQKQ I S P
LFTEDELYRI DHYLGKEMVKNLLVLRFGNELF SG IWNNKH I T SVQ I SFKEPFGTEG
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RGGYFDN IGI I RDVMQNHLLQVLT LLTMERPVS FDPEAVRDEKVKVLKAFDK I DVN
DVLLGQYAKSEDGSKPGYLDDS TVKPNSKAVTYAAFRVN I HNERWDGVP IVLRAGK
ALDEGKVE I RI QFKPVAKGMFKE I QRNELVI RI QPDEAI YLK INSK I PG I STETSL
TDLDLTYSKRYSKDFW I PEAYEAL I RDCYLGNHSNFVRDDELEVAWKLFT PLLEAV
EKEDEVS LGTYPYGSKGPKELRKYLVDHGYVFNDPGTYQWPLTNTDVKGK I
Example 37: Cloning of C. tropicalis ACH genes
ACH PCR product was amplified from C. tropicalis 20336 genomic DNA using
primers oAA1095
and oAA1096, shown in the table below. The PCR product was gel purified and
ligated into pCR-
Blunt II-TOPO (Invitrogen), transformed into competent TOP10 E. coli cells
(Invitrogen) and clones
containing PCR inserts were sequenced to confirm correct DNA sequence.
Sequence analysis of multiple transformants revealed the presence of allelic
sequences for the
ACH gene, which were designated ACHA and ACHB. A vector containing the DNA
sequence for
the ACHA allele was generated and designated pAA310 (see FIG. 51). A vector
containing the
DNA sequence for the ACHB allele was generated and designated pAA311 (see FIG.
52).
Primer sequence
oAA1095 CACACACCCGGGATGATCAGAACCGTCCGTTATCAAT
oAA1096 CACACATC TAGAC TC TC T TC TAT TC T TAAT TGCCGC T TCCAC TAAACGGCAAAGTC
TCCACG
Example 38: Cloning of C. tropicalis FAT1 gene
FAT1 PCR product was amplified from C. tropicalis 20336 genomic DNA using
primers oAA1023
and oAA1024, shown in the table below. The PCR product was gel purified and
ligated into pCR-
Blunt II-TOPO (Invitrogen), transformed into competent TOP10 E. coli cells
(Invitrogen) and clones
containing PCR inserts were sequenced to confirm correct DNA sequence. A
vector containing the
DNA sequence for the FAT1 gene was designated pAA296 (see FIG. 53).
Primer sequence
oAA1023 GATAT TAT TCCACC T TCCC T TCAT T
OAA1024 CCGTTAAACAAAAATCAGTCTGTAAA
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Example 39: Cloning of C. tropicalis ARE1 and ARE2 genes
ARE1 and ARE2 PCR products were amplified from C. tropicalis 20336 genomic DNA
using
primers oAA2006/oAA2007 and oAA1012/oAA1018, respectively, shown in the table
below. The
PCR products were gel purified and ligated into pCR-Blunt II-TOPO
(Invitrogen), transformed into
competent TOP10 E. coli cells (Invitrogen) and clones containing PCR inserts
were sequenced to
confirm correct DNA sequence. A vector containing the DNA sequence for the
ARE1 gene was
designated pAA318 (see FIG. 54). A vector containing the DNA sequence for the
ARE2 gene was
designated pAA301 (see FIG. 55).
Primer sequence
oAA1012 ATGTCCGACGACGAGATAGCAGGAATAGTCAT
oAA1018 TCAGAAGAGTAAATACAACGCACTAACCAAGCT
oAA2006 ATGCTGAAGAGAAAGAGACAACTCGACAAG
oAA2007 GTGGTTATCGGACTCTACATAATGTCAACG
Example 40: Construction of an optimized TESA gene for expression in C.
tropicalis
The gene sequence for the E. coli TESA gene was optimized for expression in C.
tropicalis by
codon replacement. A new TESA gene sequence was constructed using codons from
C. tropicalis
with similar usage frequency for each of the codons in the native E. coli TESA
gene (avoiding the
use of the CTG codon due to the alternative yeast nuclear genetic code
utilized by C. tropicalis).
The optimized TESA gene was synthesized with flanking BspQI restriction sites
and provided in
vector pIDTSMART-Kan (Integrated DNA Technologies). The vector was designated
as pAA287
(see FIG. 43). Plasmid pAA287 was cut with BspQI and the 555bp DNA fragment
was gel purified.
Plasmid pAA073 (see FIG. 44) also was cut with BspQI and the linear DNA
fragment was gel
purified. The two DNA fragments were ligated together to place the optimized
TESA gene under
the control of the C. tropicalis PDX4 promoter. The resulting plasmid was
designated pAA294 (see
FIG. 45).
Example 41: Cloning of C. tropicalis DGA1 gene
DGA1 PCR product was amplified from C. tropicalis 20336 genomic DNA using
primers oAA996
and oAA997, shown in the table below. The PCR product was gel purified and
ligated into pCR-
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Blunt II-TOPO (Invitrogen), transformed into competent TOP10 E. coli cells
(Invitrogen) and clones
containing PCR inserts were sequenced to confirm correct DNA sequence. A
vector containing the
DNA sequence of the DGA1 gene was designated pAA299 (see FIG. 56).
Primer Sequence
oAA996 ATGACTCAGGACTATAAAGACGATAGTCCTACGTCCACTGAGTTG
OAA997 CTATTCTACAATGTTTAATTCAACATCACCGTAGCCAAACCT
Example 42: Cloning of C. tropicalis LRO1 gene
LRO1 PCR product was amplified from C. tropicalis 20336 genomic DNA using
primers oAA998
and oAA999, shown in the table below. The PCR product was gel purified and
ligated into pCR-
Blunt II-TOPO (Invitrogen), transformed into competent TOP10 E. coli cells
(Invitrogen) and clones
containing PCR inserts were sequenced to confirm correct DNA sequence. A
vector containing the
DNA sequence of the LRO1 gene was designated pAA300 (see FIG. 57).
Primer sequence
oAA998 ATGTCGTCTTTAAAGAACAGAAAATC
oAA999 TTATAAATTTATGGCCTCTACTATTTCT
Example 43: Cloning of C. tropicalis ACS1 gene and construction of deletion
cassette
ACS1 PCR product was amplified from C. tropicalis 20336 genomic DNA using
primers oAA951
and oAA952, shown in the table below. The PCR product was gel purified and
ligated into pCR-
Blunt II-TOPO (Invitrogen), transformed into competent TOP10 E. coli cells
(Invitrogen) and clones
containing PCR inserts were sequenced to confirm the DNA sequence. One such
plasmid was
designated pAA275 (see FIG. 47). Plasmid pAA280 (see FIG. 46) was digested
with BamHI to
release a 2.0 kb PuRA3URA3TuRA3PuRA3 cassette. Plasmid pAA275 was digested
with BglIl and gel
purified. The two pieces were ligated together to generate plasmid pAA276 (see
FIG. 48) and
pAA282 (see FIG. 49). Plasmid pAA276 and pAA282 have the PuRA3URA3TuRA3PuRA3
cassette
inserted into the ACS gene in opposite orientations.
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Primer sequence
oAA951 CCTACTTCCACAGCTTTAATCTACTATCAT
OAA952 TTTAAGAAAACAACTAAGAGAAGCCAC
Example 44: Construction of Strain sAA722 (pox4a::ura3/pox4b::ura3 PDX5/P0X5
ACS1/acs1::
PURA3URA3TURA3PURA3)
Plasmid pAA276 was digested with BamHI/Xhol and column purified. Strain sAA329
(ura3/ura3
pox4a::ura3/pox4b::ura3 PDX5/P0X5) was transformed with the linearized DNA and
plated on
SCD-ura plate. Several colonies were checked for ACS1 disruption. One such
strain was
designated sAA722.
Example 45: Construction of Strain sAA741 (pox4a::ura3/pox4b::ura3 PDX5/P0X5
ACS1/acs1::PuR,43)
Strain sAA722 was grown in YPD media overnight and plated on 5-FOA plate.
Colonies that grew
in the presence of 5-FOA were PCR screened for the looping out of the URA3
gene leaving behind
only the URA3 promoter (FuRA3) in the ACS1 site. Out of 30 colonies analyzed,
only one strain
showed the correct genetic modification. The strain was designated sAA741.
Example 46: Construction of Strain sAA776 (pox4a::ura3/pox4b::ura3 PDX5/P0X5
acs1 ::PURA3URA3TURA3PURA3/aCS1 :: PURA3)
Plasmid pAA282 was digested with BamHI/Xhol and column purified. Strain sAA741
(see
Example 45) was transformed with the linearized DNA and plated on SCD-ura
plate. Several
colonies were checked for double ACS1 knockout by insertional inactivation.
One such strain was
designated sAA776.
Example 47: Construction of Strain sAA779 (pox4a::ura3/pox4b::ura3 PDX5/P0X5
acs1::
PuR,43/acs1:: PURA3)
Strain sAA776 (see Example 46) was grown in YPD media overnight and plated on
5-FOA plates.
Colonies that grew in the presence of 5-FOA were PCR screened for the looping
out of the URA3
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gene leaving behind only the URA3 promoter (PURA3) in both ACS1 copies. One
such strain was
designated sAA779.
Example 48: Construction of Strain sAA811 (pox4a::ura3/pox4b::ura3 PDX5/P0X5
acs1::
PuRaiacst: PURA3 ura3::3xPp0x4P450A19)
Plasmid pAA156 (see FIG. 50) containing a P450A19 integration cassette was
digested with Clal
and column purified. Strain sAA779 (see Example 47) was transformed with the
linearized DNA
and plated on SCD-ura plate. Several colonies were checked for P450A19
integration. From those
colonies, qPCR was performed to check the copy number of P450A19 integration.
One strain,
designated sAA811, contained 3 copies of P450A19.
Example 49: Construction of Strain sAA810 (pox4a::ura3/pox4b::ura3 PDX5/P0X5
acs1::
PuRaiacs1:: PURA3 ura3::5xPpox4P450A19 ura3::8xPpox4TESA)
Plasmid pAA156 containing a P450-A19 integration cassette was digested with
Clal and column
purified. Plasmid pAA294 containing a TESA integration cassette also was
digested with Clal and
column purified. Strain sAA779 was cotransformed with both linearized DNAs and
plated on SCD-
ura plate. Several colonies were checked for both P450A19 integration and TESA
integration.
Colonies that were positive for both TESA and P450A19 were further analyzed by
qPCR. qPCR
was performed to check the copy number of the P450A19 and TESA integration
events. One
strain, designated sAA810, contained 5 copies of P450A19 and 8 copies of TESA.
Example 50: Shake flask characterization of sAA235, sAA776, sAA810 and sAA811
Starter cultures (5mL) in 5P92-glycerol media (6.7g/L Difco yeast nitrogen
base, 3.0g/L Difco yeast
extract, 3.0g/L ammonium sulfate, 1.0g/L potassium phosphate monobasic, 1.0g/L
potassium
phosphate dibasic, 75g/L glycerol) were incubated overnight at 30 C, with
shaking at
approximately 25Orpm. The overnight cultures were used to inoculate 25mL fresh
5P92-glycerol
media to an initial OD600nm of 0.4 and incubated approximately 18 hours at 30
C, and 300rpm
shaking. Cells were pelleted by centrifugation for 10 minutes at 4,000xg, at 4
C, then resuspended
in TB-lowN media (1.7g/L Difco yeast nitrogen base without amino acids and
ammonium sulfate,
3.0g/L Difco yeast extract, 1.0g/L potassium phosphate monobasic, 1.0g/L
potassium phosphate
dibasic). 1 mL coconut oil was added to start the adipic acid production at 30
C, and shaking at
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approximately 300rpm, in 250 mL shake flasks. Incubation of the cultures
continued for 96 hours
and samples were taken for analysis of fatty acids and diacids by GC. The
experiment was
performed with shake flasks in triplicate.
Yields of product per consumed substrate (Yp/s) were determined with the
equation [adipic acid
(g/L)* final volume of culture in flask (L)] / [coconut oil added to the flask
(g)]. The calculation
assumes all of the coconut oil added to the flask was consumed. GC analysis
revealed that there
were diacids of chain length C6 to C14 in all flasks with the majority
represented as C8 and C6. All
of the diacids except for the C6 (adipic) were ignored for yield calculations.
The maximum
theoretical yield (Ymax) for the conversion of coconut oil to adipic acid was
calculated to be 0.6g
adipic acid/ g coconut oil. The percent of Y. (%Y.) was calculated as Y
= p/s = = Y
max * 100.
The results of the shake flask experiments are shown in FIG. 58. Based on
calculations for Yp/s
shown in the previous paragraph, the results indicate that the yield of adipic
acid on coconut oil
was higher in the strain with disrupted ACS1 genes (11.6 %Ymax) than the
parental strain (5.4
%Ymax). The yield of adipic acid on coconut oil was further improved by the
gene amplification of
P450A19 (27.1 %Ymax) and by the coordinate gene amplification of P450A19 and
TESA (33.5
%Ymax), as shown graphically in FIG. 58.
245
Example 51: Nucleotide and Amino Acid Sequences Used for Manipulations
described in Examples 37-50
SEQ ID NO: Sequence description Sequence
SEQ ID NO:42 Acyl-CoA Hydrolase ATGATCAGAACCGTCCGT TATCAATCCC TCAAGAGGT
TCAGACC TC TGGC T T TGTC TCC TGT T T T TCGTCCACGC TA
(ACHA) Nucleotide
CAACTCCCAGAAGGCCAATTTCCACCGTCCAGACCACCCTGGGTCCGACGAGCCAGCTGAAGCCGCCGACGCCGCCG
Seq
CCACGATCCTCGCCGAGTTGCGAGACAAGCAGACGAACCCGAACAAGGCCACCTGGCTCGATGCGTTAACGGAGCGG
oo
oo
GAGAAGTTGCGTGCCGAGGGCAAGACGATTGACAGTTTCAGCTACGTTGACCCCAAGACGACCGTCGTGGGGGAGAA
oo
GACACGCAGTGACTCGTTCTCGTTCTTGTTGTTGCCGTTCAAGGACGACAAGTGGTTGTGTGACGCGTACATCAATG
CGTTTGGCCGGTTGCGTGTAGCGCAGTTGTTCCAGGACTTGGACGCCTTGGCGGGGCGCATCGCGTACAGGCACTGT
TCCCCAGCGGAGCCCGTGAATGTCACGGCGAGCGTGGATAGGGTGTACATGGTGAAGAAAGTGGACGAGATTAACAA
TTACAATTTCGTGTTGGCGGGGTCCGTGACGTGGACCGGGAGATCGTCGATGGAGATCACGGTGAAAGGGTATGCTT
TTGAAGACGCCGTGCCGGATATAACGAACGAGGAGTCCTTGCCGGCAGAGAATGTGTTTTTGGCTGCTAATTTCACC
T TCGTGGCACGGAACCCAC T TACACACAAGTCC T T TGC TAT TAACAGAT TGT TGCCCGTGAC
TGAGAAGGAC TGGGT
CGACTATCGCCGTGCTGAGTCCCACAACGCCAAGAAGAAGTTGATGGCAAAGAACAAGAAGATCTTGGAGCCTACCG
CGGAAGAGTCCAAGTTGATCTACGACATGTGGAGATCGTCCAAGTCCTTACAGAACATCGAGAGGGCCAACGATGGG
ATCGCGTTCATGAAGGACACGACCATGAAGTCCACCTTGTTCATGCAGCCCCAGTACCGTAACAGACACTCATACAT
GAT T T TCGGAGGGTAC T TGT TAAGACAAAC T T TCGAAT TGGCC TAC TGTACCGCGGCAACGT T T
TCCC TGGCCGGGC co
CCCGTTTCGTCAGCTTGGACTCCACCACGTTCAAGAACCCCGTGCCCGTGGGGTCGGTGCTCACCATGGACTCGTCG
o
ATCTCGTACACGGAGCACGTGCACGAGGGAGTGGAGGAGATTGACGCGGACTCACCGTTCAACTTCAGCTTGCCTGC
CACGAACAAGATCTCGAAGAACCCCGAGGCGTTCTTGTCGGAACCCGGCACGTTGATTCAAGTCAAGGTCGACACAT
ACATCCAGGAGTTAGAGCAGAGTGTGAAGAAGCCCGCGGGTACGTTCATCTACTCGTTCTATGTTGATAAAGAAAGC
0
GT TAC TGT TGATGGAAAGGCGTCGT T T TGT TCAGT TATCCCGCAGACGTAC TCCGAGATGATGAC T
TATGTGGGCGG
GAGAAGAAGAGCCCAGGATACTGCTAACTACGTGGAGACTTTGCCGTTTAGTGGAAGCGGCAATTAA
0
UJ
SEQ ID NO:43 Acyl-CoA Hydrolase MI RTVRYQ S LKRFRP SAL S PVFRPRYNS
QKANFHRPDHPGS DE PAEAADAAAT I LAELRDKQTNPNKATWLDALTER
(ACHA) Amino Acid EKLRAEGKT I DS F S YVDPKT TVVGEKTRS DS F S FLLL
PFKDDKWLCDAY INAFGRLRVAQLFQDLDALAGRIAYRHC
Seq S PAEPVNVTASVDRVYMVKKVDE I NNYNFVLAGSVTWTGRS SME
I TVKGYAFE DAVPD I TNEESLPAENVFLAANFT
FVARNPLTHKS FAINRLL PVTEKDWVDYRRAE SHNAKKKLMAKNKK I LE PTAEE SKL I YDMWRS SKS
LQN I ERANDG
IAFMKDTTMKS TLFMQPQYRNRHSYMI FGGYLLRQTFELAYCTAATFS SAGPRFVS LDS
TTFKNPVPVGSVLTMDS S
I SYTEHVHEGVEE I DADS PFNF S L PATNK I SKNPEAFL SE PGT L I QVKVDTY I QELEQ
SVKKPAGT F I YS FYVDKE S
VTVDGKASFCSVI PQTYSEMMTYVGGRRRAQDTANYVETLPFSGSGN
SEQ ID NO:44 Acyl-CoA Hydrolase ATGATCAGAACCGTCCGT TATCAATCC T TCAAGAGGT
TCAAACC TC TGAC T T TATCCCCCGT T T TCCGTCCACGC TA
(ACHB) Nucleotide
CAACTCCCAGAAGGCCAATTTCCACCGTCCAGACCACGCTGGGTCCGACGAGCCAGCCGAAGCCGCCGACGCCGCTG
1-3
Seq
CCACGATCCTCGCCGAGTTGCGAGACAAGCAGACGAACCCGAACAAGGCCACCTGGCTCGATGCGTTAACGGAGCGG
ci)
GAGAAGTTGCGTGCCGAGGGCAAGACAATCGACAGCTTCAGCTACGTTGACCCCAAGACAACCGTCGTGGGGGAGAA
GACACGCAGCGACTCGTTCTCGTTCTTGTTGTTGCCGTTCAAGGACGACAAGTGGTTGTGTGACGCGTACATCAATG
CGTTTGGCCGGTTGCGTGTAGCGCAGTTGTTCCAGGACTTGGACGCCTTGGCGGGCCGCATCGCGTACAGGCACTGT
TCCCCCGCTGAGCCCGTGAATGTCACGGCGAGCGTGGATAGAGTGTATATGGTGAAGAAAGTGGACGAGATTAATAA
TTACAATTTCGTGTTGGCGGGGTCCGTGACGTGGACCGGGAGATCGTCGATGGAGATCACGGTCAAAGGGTATGCTT
TTGAAGACGCCGTGCCGGAGATAACTAACGAGGAGTCCTTGCCGGCAGAGAATGTGTTCTTGGCTGTTAATTTCACC
T TCGTGGCACGTAACCCAC TCACACACAAGTCC T TCGC TAT TAACAGAT TGT TGCCCGTGAC
TGAGAAGGAC TGGGT
n c
SEQ ID NO: Sequence description Sequence
CGATTATCGCCGTGCTGAGTCCCACAACGCCAAGAAGAAGTTGATGGCAAAGAACAAGAAGATCTTGGAGCCTACCC
CGGAAGAGTCCAAGTTGATCTACGACATGTGGAGATCGTCCAAGTCCTTACAGAACATCGAGAAGGCCAACGACGGG
ATCGCGTTCATGAAGGACACGATAATGAAGTCCACCTTGTTCATGCAGCCCCAGTACCGTAACAGACACTCATACAT
GAT T T TCGGTGGGTAT T TGT TAAGACAAAC T T TCGAAT TGGCC TAT TGTACCGCAGCAACGT T T
TCCC TGGCGGGAC
CCCGTTTCGTCAGCTTGGACTCCACCACGTTCAAGAACCCCGTGCCCGTGGGGTCGGTGCTCACCATGGACTCGTCG
ATCTCGTACACGGAGCACGTCCACGATGGCGTTGAGGAGATTGACGCCGACTCCCCGTTCAACTTCAGCTTGCCTGC
oe
CACGAACAAGATCTCGAAGAACCCCGAGGCGTTCTTGTCGGAGCCCGGCACGTTGATCCAAGTCAAGGTCGACACGT
oe
ACATCCAGGAGTTAGAGCAAAGTGTGAAGAAGCCTGCGGGAACGTTCATCTACTCGTTCTATGTTGATAAAGAGAGC
oe
GT TAC TGTGGATGGAAAGGCGTCGT T T TGT TCAGT TATCCCGCAGACGTAC TCCGAGATGATGAC T
TATGTGGGCGG
GAGAAGAAGAGCCCAGGATACTGCTAATTACGTGGAGACTTTGCCGTTTAGTGGAAGCGGCAATTAA
SEQ ID NO:45 Acyl-CoA Hydrolase MI RTVRYQ S FKRFKPLT L S PVFRPRYNS
QKANFHRPDHAGS DE PAEAADAAAT I LAELRDKQTNPNKATWLDALTER
(ACHB) EKLRAEGKT I DS F S YVDPKT TVVGEKTRS DS F S FLLL
PFKDDKWLCDAY INAFGRLRVAQLFQDLDALAGRIAYRHC
S PAEPVNVTASVDRVYMVKKVDE I NNYNFVLAGSVTWTGRS SME I TVKGYAFEDAVPE I
TNEESLPAENVFLAVNFT
FVARNPLTHKS FAINRLL PVTEKDWVDYRRAE SHNAKKKLMAKNKK I LE PT PEE SKL I YDMWRS SKS
LQN I EKANDG
IAFMKDT IMKS TLFMQPQYRNRHSYMI FGGYLLRQT FELAYC TAAT F S LAGPRFVS LDS
TTFKNPVPVGSVLTMDS S
I SYTEHVHDGVEE I DADS PFNF S L PATNK I SKNPEAFL SE PGT L I QVKVDTY I QELEQ
SVKKPAGT F IYS FYVDKE S
VTVDGKASFCSVI PQTYSEMMTYVGGRRRAQDTANYVETLPFSGSGN
n.)
co
SEQ ID NO:46 E. co/iAcyl-COA
ATGGCCGATACATTGCTCATCTTGGGTGACTCTTTGTCTGCAGGGTATCGGATGTCCGCATCTGCCGCATGGCCTGC
Thioesterase (TESA) AC TCC TCAATGACAAATGGCAAAGCAAGACATCGGTCGTGAATGCATC TATC TC
TGGCGATACC TCGCAGCAGGGGT
gene without signal
TGGCCCGTCTCCCAGCCTTGTTGAAGCAACATCAACCACGTTGGGTCTTGGTCGAATTGGGCGGCAATGATGGTCTC
n.)
peptide sequence
AGAGGTTTTCAACCTCAACAGACCGAGCAGACATTGCGTCAAATCCTCCAAGACGTGAAGGCAGCAAACGCCGAACC
0
optimized for C.
TCTCTTGATGCAGATAAGATTGCCTGCCAACTATGGTCGTAGATACAATGAAGCCTTTTCTGCAATCTACCCGAAGC
oI
tropicalis
TTGCAAAGGAGTTTGACGTCCCATTGTTGCCGTTTTTGATGGAAGAGGTGTACCTTAAGCCTCAGTGGATGCAAGAC
Nucleotide Seq
GATGGTATCCATCCGAACCGTGATGCACAACCATTCATCGCAGATTGGATGGCCAAACAACTCCAACCTTTGGTCAA
n.)
TCATGATAGCTAA
SEQ ID NO:47 E. colt Acyl-CoA MADTLL I LGDSLSAGYRMSASAAWPALLNDKWQSKT SVVNAS
I SGDT SQQGLARLPALLKQHQPRWVLVELGGNDGL
Thioesterase (TESA) RGFQ PQQTEQT LRQ I LQDVKAANAE PLLMQ I RL PANYGRRYNEAF SAI
Y PKLAKEFDVPLL PFLMEEVYLKPQWMQD
without signal peptide DGIHPNRDAQPF IADWMAKQLQPLVNHDS
Amino Acid Seq
SEQ ID NO:48 Acyl-CoA Synthetase
ATGGGTGCCCCTTTAACAGTCGCCGTTGGCGAAGCAAAACCAGGCGAAACCGCTCCAAGAAGAAAAGCCGCTCAAAA
(ACS1) Nuc. Seq
AATGGCCTCTGTCGAACGCCCAACAGACTCAAAGGCAACCACTTTGCCAGACTTCATTGAAGAGTGTTTTGCCAGAA
ACGGCACCAGAGATGCCATGGCCTGGAGAGACTTGGTCGAAATCCACGTCGAAACCAAACAGGTTACCAAAATCATT
GACGGCGAACAGAAAAAGGTCGATAAGGACTGGATCTACTACGAAATGGGTCCTTACAACTACATATCCTACCCCAA
GT TGT TGACGT TGGTCAAGAAC TAC TCCAAGGGT T TGT TGGAGT TGGGC T
TGGCCCCAGATCAAGAATCCAAGT TGA
TGATCTTTGCCAGTACCTCCCACAAGTGGATGCAGACCTTCTTAGCCTCCAGTTTCCAAGGTATCCCCGTTGTCACC
GCCTACGACACCTTGGGTGAGTCGGGCTTGACCCACTCCTTGGTGCAAACCGAATCCGATGCCGTGTTCACCGACAA
CCAATTGTTGTCCTCCTTGATTCGTCCTTTGGAGAAGGCCACCTCCGTCAAGTATGTCATCCACGGGGAAAAGATTG
ACCCTAACGACAAGAGACAGGGCGGCAAAATCTACCAGGATGCGGAAAAGGCCAAGGAGAAGATTTTACAAATTAGA
CCAGATAT TAAAT T TAT T TC T T TCGACGAGGT TGT TGCAT TGGGTGAACAATCGTCCAAAGAAT
TGCAT T TCCCAAA
ACCAGAAGACCCAATCTGTATCATGTACACCTCGGGTTCCACCGGTGCTCCAAAGGGTGTGGTTATCACCAATGCCA
ACATTGTTGCCGCCGTGGGTGGTATCTCCACCAATGCTACTAGAGACTTGGTTAGAACTGTCGACAGAGTGATTGCA
SEQ ID NO: Sequence description Sequence
TTTTTGCCATTGGCCCACATTTTCGAGTTGGCCTTTGAGTTGGTTACCTTCTGGTGGGGGGCTCCATTGGGTTACGC
CAATGTCAAGACTTTGACCGAAGCCTCCTGCAGAAACTGTCAGCCAGACTTGATTGAATTCAAACCAACCATCATGG
TTGGTGTTGCTGCCGTTTGGGAATCGGTCAGAAAGGGTGTCTTGTCTAAATTGAAACAGGCTTCTCCAATCCAACAA
AAGATCTTCTGGGCTGCATTCAATGCCAAGTCTACTTTGAACCGTTATGGCTTGCCAGGCGGTGGGTTGTTTGACGC
TGTCTTCAAGAAGGTTAAAGCCGCCACTGGTGGCCAATTGCGTTATGTGTTGAATGGTGGGTCCCCAATCTCTGTTG
CB;
ATGCCCAAGTGTTTATCTCCACCTTGCTTGCGCCAATGTTGTTGGGTTACGGTTTGACTGAAACCTGTGCCAATACC
oe
ACCATTGTCGAACACACGCGCTTCCAGATTGGTACTTTGGGTACCTTGGTTGGATCTGTCACTGCCAAGTTGGTTGA
oe
TGTTGCTGATGCTGGATACTACGCCAAGAACAACCAGGGTGAAATCTGGTTGAAAGGCGGTCCAGTTGTCAAGGAAT
oe
AC TACAAGAACGAAGAAGAAACCAAGGC TGCAT TCACCGAAGATGGC TGGT TCAAGAC TGGTGATAT
TGGTGAATGG
ACCGCCGACGGTGGTTTGAACATCATTGACCGTAAGAAGAACTTGGTCAAGACTTTGAATGGTGAATACATTGCTTT
GGAGAAATTGGAAAGTATTTACAGATCCAACCACTTGATTTTGAACTTGTGTGTTTACGCTGACCAAACCAAGGTCA
AGCCAAT TGC TAT TGTC T TGCCAAT TGAAGCCAAC T TGAAGTC TATGT TGAAGGACGAAAAGAT
TATCCCAGATGC T
GAT TCACAAGAAT TGAGCAGC T TGGT TCACAACAAGAAGGT TGCCCAAGC TGTC T TGAGACAC T TGC
TCCAAACCGG
TAAACAACAAGGTTTGAAAGGTATTGAATTGTTGCAGAATGTTGTCTTGTTGGATGACGAGTGGACCCCACAGAATG
GT T T TGT TAC T TC TGCCCAAAAGT TGCAGAGAAAGAAGAT T T TAGAAAGT TGTAAAAAAGAAGT
TGAAGAGGCATAC
AAGTCGTC T TAG
0
SEQ ID NO:49 Acyl-CoA Synthetase MGAPLTVAVGEAKPGE TAPRRKAAQKMASVERPT DSKAT T L
PDF I EECFARNGTRDAMAWRDLVE I HVE TKQVTK I I n.)
co
(ACS1)
DGEQKKVDKDW I YYEMGPYNY I S Y PKLLT LVKNY
SKGLLELGLAPDQE SKLMI FAS T SHKWMQT FLAS SFQGI PVVT
A. A. Seq
AYDT LGE SGLTHS LVQTE S DAVFT DNQLL S SLI RPLEKAT
SVKYVI HGEK I DPNDKRQGGK I YQDAEKAKEK I LQ I R
PD I KF I SFDEVVALGEQS SKELHF PKPEDP I C IMYTSGSTGAPKGVVI TNANIVAAVGGI
STNATRDLVRTVDRVIA
n.)
FL PLAH I FELAFELVT FWWGAPLGYANVKT LTEASCRNCQ PDL I EFKPT
IMVGVAAVWESVRKGVLSKLKQAS P I QQ 0
K I FWAAFNAKS T LNRYGL PGGGLFDAVFKKVKAATGGQLRYVLNGGS PI SVDAQVF I
STLLAPMLLGYGLTETCANT
oI
T IVEHTRFQ I GT LGT LVGSVTAKLVDVADAGYYAKNNQGE IWLKGGPVVKEYYKNEEE
TKAAFTEDGWFKTGD I GEW
TADGGLN I I DRKKNLVKT LNGEY IALEKLE S I YRSNHL I LNLCVYADQTKVKP IAIVL P I
EANLKSMLKDEK I I PDA
n.)
DSQELS S LVHNKKVAQAVLRHLLQTGKQQGLKGI ELLQNVVLLDDEWT PQNGFVT SAQKLQRKK I LE
SCKKEVEEAY
KS S
SEQ ID NO:50 Long-chain Acyl-CoA ATGTCAGGAT TAGAAATAGCCGC TGC TGCCATCC T
TGGTAGTCAGT TAT TGGAAGCCAAATAT T TAAT TGCCGACGA
Synthetase (FAT1)
CGTGCTGTTAGCCAAGACAGTCGCTGTCAATGCCCTCCCATACTTGTGGAAAGCCAGCAGAGGTAAGGCATCATACT
Nuc. Seq
GGTACTTTTTCGAGCAGTCCGTGTTCAAGAACCCAAACAACAAAGCGTTGGCGTTCCCAAGACCAAGAAAGAATGCC
CCCACCCCCAAGACCGACGCCGAGGGATTCCAGATCTACGACGATCAGTTTGACCTAGAAGAATACACCTACAAGGA
AT TGTACGACATGGT T T TGAAGTAC TCATACATC T TGAAGAACGAGTACGGCGTCAC
TGCCAACGACACCATCGGTG
TTTCTTGTATGAACAAGCCGCTTTTCATTGTCTTGTGGTTGGCATTGTGGAACATTGGTGCCTTGCCTGCGTTCTTG
AACTTCAACACCAAGGACAAGCCATTGATCCACTGTCTTAAGATTGTCAACGCTTCGCAAGTTTTCGTTGACCCGGA
CTGTGATTCCCCAATCAGAGATACCGAGGCTCAGATCAGAGAGGAATTGCCACATGTGCAAATAAACTACATTGACG
AGTTTGCCTTGTTTGACAGATTGAGACTCAAGTCGACTCCAAAACACAGAGCCGAGGACAAGACCAGAAGACCAACC
GATACTGACTCCTCCGCTTGTGCATTGATTTACACCTCGGGTACCACCGGTTTGCCAAAAGCCGGTATCATGTCCTG
CB;
GAGAAAAGCCTTCATGGCCTCGGTTTTCTTTGGCCACATCATGAAGATTGACTCGAAATCGAACGTCTTGACCGCCA
TGCCCTTGTACCACTCCACCGCGGCCATGTTGGGGTTGTGTCCTACTTTGATTGTCGGTGGCTGTGTCTCCGTGTCC
CAGAAATTCTCCGCTACTTCGTTCTGGACCCAGGCCAGATTATGTGGTGCCACCCACGTGCAATACGTCGGTGAGGT
CTGTCGTTACTTGTTGAACTCCAAGCCTCATCCAGACCAAGACAGACACAATGTCAGAATTGCCTACGGTAACGGGT
TGCGTCCAGATATATGGTCTGAGTTCAAGCGCAGATTCCACATTGAAGGTATCGGTGAGTTCTACGCCGCCACCGAG
SEQ ID NO: Sequence description Sequence
TCCCCTATCGCCACCACCAACTTGCAGTACGGTGAGTACGGTGTCGGCGCCTGTCGTAAGTACGGGTCCCTCATCAG
C T TGT TAT TGTC TACCCAGCAGAAAT TGGCCAAGATGGACCCAGAAGACGAGAGTGAAATC
TACAAGGACCCCAAGA
CCGGGTTCTGTACCGAGGCCGCTTACAACGAGCCAGGTGAGTTGTTGATGAGAATCTTGAACCCTAACGACGTGCAG
AAATCC T TCCAGGGT TAT TATGGTAACAAGTCCGCCACCAACAGCAAAATCC TCACCAATGT T T
TCAAAAAAGGTGA
CGCGTGGTACAGATCCGGTGACTTGTTGAAGATGGACGAGGACAAATTGTTGTACTTTGTCGACAGATTAGGTGACA
CB;
CTTTCCGTTGGAAGTCCGAAAACGTCTCCGCCACCGAGGTCGAGAACGAATTGATGGGCTCCAAGGCCTTGAAGCAG
oe
TCCGTCGTTGTCGGTGTCAAGGTGCCAAACCACGAAGGTAGAGCCTGTTTTGCCGTCTGTGAAGCCAAGGACGAGTT
oe
GAGCCATGAAGAAATCTTGAAATTGATTCACTCTCACGTGACCAAGTCTTTGCCTGTGTATGCTCAACCTGCGTTCA
oe
TCAAGATTGGCACCATTGAGGCTTCGCACAACCACAAGGTTCCTAAGAACCAATTCAAGAACCAAAAGTTGCCAAAG
GGTGAAGACGGCAAGGATTTGATCTACTGGTTGAATGGCGACAAGTACCAGGAGTTGACTGAAGACGATTGGTCTTT
GAT T TGTACCGGTAAAGCCAAAT TG
SEQ ID NO:51 Long-chain Acyl-CoA MSGLE IAAAAI LGSQLLEAKYL
IADDVSLAKTVAVNALPYLWKASRGKASYWYFFEQSVFKNPNNKALAFPRPRKNA
Synthetase (FAT1) PT PKT DAEGFQ I YDDQFDLEEYTYKELYDMVLKY S Y I
LKNEYGVTANDT I GVSCMNKPLF IVLWLALWN I GAL PAFL
A.A. Seq NFNTKDKPL I HCLK IVNAS QVFVDPDCDS P I RDTEAQ I
REEL PHVQ INY I DEFALFDRLRLKS TPKHRAEDKTRRPT
DT DS SACAL I YT SGT TGL PKAGIMSWRKAFMASVFFGH IMK I DSKSNVLTAMPLYHS TAAMLGLC
PT L IVGGCVSVS
QKF SAT S FWTQARLCGATHVQYVGEVCRYLLNSKPHPDQDRHNVRIAYGNGLRPD IWSEFKRRFH I EGI
GEFYAATE
S PIATTNLQYGEYGVGACRKYGSL I SLLLS TQQKLAKMDPEDE SE I YKDPKTGFC TEAAYNE
PGELLMRI LNPNDVQ n.)
co
KS FQGYYGNKSATNSK I LTNVFKKGDAWYRSGDLLKMDEDKLLYFVDRLGDT
FRWKSENVSATEVENELMGSKALKQ
SVVVGVKVPNHEGRACFAVCEAKDELSHEE I LKL I HSHVTKS L PVYAQ PAF I K I GT I
EASHNHKVPKNQFKNQKL PK
GEDGKDL I YWLNGDKYQELTEDDWS L I C TGKAKL
o
n.)
o
SEQ ID NO: Sequence description Sequence
SEQ ID NO:52 Acyl-CoA Sterol acyl
ATGTCCGACGACGAGATAGCAGGAATAGTCATTGAAATCGACGATGACGTGAAATCCACGTCTTCGTTCCAGGAAGA
transferase (ARE1) AC TAGTCGAGGT TGAAATGTCCAAC TCGTCCAT
TAACGAATCCCAGACCGATGAGTCGTACCGTCC TGAAGAAACC T
Nuc. Seq CAT TGCAT
TACAGGAGGAAGTCCCACAGGACCCCGTCAGAGGAGTCGT TCC TAGAGATCACCAAGAACGTGAATGAT
CCGGATCTAGTTTCCAAGATTGAGAACCTAAGGGGCAAAGTAAGCCAACGGGAAGACAGGTTGAGGAAGCACTACCT
TCACACC TCCCAGGACGTCAAGT TC T TGTCCCGGT TCAACGACATCAAGT TCAAGC TGAAC
TCCGCGACGAT TC TAG
AT TCGGATGCGT T T TACAAGAGTGAATAC T T TGGAGTC T TGACCATC T TC TGGGTGGT
TATCGCAC TC TACATAT TG
oe
TCAACGTTGTCAGATGTTTACTTTGGCATGGCCAAGCCCTTACTGGACTGGATCATCATAGGAATGTTCAAGCAGGA
oe
C T TGGTGAAAGT TGCAC TCGT TGATC T TGCCATGTACC TATCC TCGTAT T T TCC T TAT T TC
T TGCAGGT TGCATGCA oe
AACGGGGTGATGTATCTTGGCATGGTCTTGGATGGGCAATACAGGGGGTTTACAGCTTGGTGTTTTTGACGTTCTGG
ACGGTAGTTCCGCAGGAGTTGGCCATGGATCTTCCTTGGATTGCACGAATTTTCTTGATCTTGCATTGCTTGGTGTT
TAT TATGAAGATGCAGTCGTATGGGCAT TACAATGGATACC T T TGGGATGTGTATCAGGAAGGAT TGGCC
TC TGAGG
CTGATCTCAGGGACCTTTCTGAGTATGATGAAGATTTCCCCCTGGATCACGTGGAGGTTCTAGAACAGAGCTTGTGG
TTTGCCAAACACGAGTTGGAGTTTCAATCGAATGGAACTGCTGAGAGGAAGGACCACCATCACCATGTATTCGACGA
AAAGGATGTCAACAAACCAATACGTGTCTTGCAAGAAGAGGGAATTATCAAGTTTCCGGCAAACATCAACTTCAAGG
AT TAT T TCGAGTACAGTATGT TCCCAACGC TAGTC TACACGT TGAGC T TCCCCCGAAC TCGACAGAT
TAGATGGACG
TATGTGTTGCAGAAGGTTTTGGGAACATTTGCCTTAGTGTTTGCCATGATTATCGTCGCCGAAGAGAGTTTCTGCCC
CTTGATGCAAGAAGTTGATCAGTACACAAAATTGCCAACCAACCAAAGGTTCCCAAAATACTTCGTCGTTCTTTCCC
n.)
AC T TGATAT TACCGC TCGGCAAGCAGTAC T TGC TC TCAT TCATCC TCATC TGGAATGAAAT TC
TCAACGGCATAGCG
GAGT TAAGCAGGT T TGGCGACCGGCAT T TC TACGGCGC T TGGTGGTCGAGCGTCGAT TACATGGAC
TAT TCAAGAAA
ATGGAACACCATCGTGCACCGATTCCTCCGTCGGCACGTTTACAATTCGAGCATTCACATCCTCGGTATTTCCAGGA
CGCAAGCCGCGATAGT TACAC T T T TGC T T TC TGCCACAATCCACGAAC TCGT TATGTACGTCC TAT
T TGGCAAAT TA n.)
0
CGAGGGTACC TAT TCC T TACGATGC T TGTCCAGATCCCCATGACCGTCACC TCCAAGT
TCAACAACCGTGT T TGGGG
oI
CAACATCATGT TC TGGT TGACGTAT T TATC TGGCCCCAGC T TGGT TAGTGCGT TGTAT T TAC TC
T TC TAG
SEQ ID NO:53 Acyl-CoA Sterol acyl MS DDE IAGIVIE I DDDVKS T S SFQEELVEVEMSNS S
INE SQT DE S YRPEET S LHYRRKSHRT P SEE S FLE I TKNVND
n.)
transferase (ARE1) PDLVSK I ENLRGKVSQREDRLRKHYLHT SQDVKFL SRFND I
KFKLNSAT I LDS DAFYKSEYFGVLT I FWVVIALY I L
Seq S TLS DVYFGMAKPLLDW I I I GMFKQDLVKVALVDLAMYL S
SYFPYFLQVACKRGDVSWHGLGWAIQGVYSLVFLTFW
TVVPQELAMDL PW IARI FL I LHCLVF IMKMQS YGHYNGYLWDVYQEGLASEADLRDL SEYDEDF
PLDHVEVLEQS LW
FAKHELEFQSNGTAERKDHHHHVFDEKDVNKP I RVLQEEGI I KF PAN INFKDYFEYSMF PT LVYT L S
F PRTRQ I RWT
YVLQKVLGTFALVFAMI IVAEE S FC PLMQEVDQYTKL PTNQRF PKYFVVL SHL I L PLGKQYLL SFIL
IWNE I LNGIA
EL SRFGDRHFYGAWWS SVDYMDYSRKWNT IVHRFLRRHVYNS SIHILGI SRTQAAIVT LLL SAT I
HELVMYVLFGKL
RGYLFLTMLVQ I PMTVTSKFNNRVWGNIMFWLTYLSGPSLVSALYLLF
SEQ ID NO:54 Acyl-CoA Sterol acyl
ATGTCCGACGACGAGATAGCAGGAATAGTCATTGAAATCGACGATGACGTGAAATCTACGTCTTCGTTCCAGGAAGA
transferase (ARE2) CC TAGTCGAGGT TGAGATGTCCAAC TCGTCCAT
TAACGAATCCCAGACGGATGAGT TGTCGTACCGTCC TGAAGAAA
Nuc. Seq
TCTCATTGCATTCGAGAAGGAAGTCCCACAAGACCCCGTCAGATGAGTCGTTCCTAGAGATCACCAAGAACGTGAAT
GATCCGGATC TAGTC TCCAAGAT TGAGAAC T TAAGGGGCAAAGTAAGCCAACGGGAAGACAGGT
TGAGGAAACAC TA
CC TCCACACATCCCAGGACGTCAAGT TC T TGTC TCGGT TCAACGACATCAAGT TCAAGC TGAAC
TCCGCGACGAT TC
TAGATTCGGATGCGTTTTACAAGAGCGAGCACTTTGGAGTCTTGACTATCTTCTGGGTGGTTATCGGACTCTACATA
ATGTCAACGTTGTCAGACATGTATTTTGGCATGGCCAAGCCCTTACTGGACTGGATAATCATAGGAATGTTCAAGAA
GGAT T TGATGCAAGT TGCAC TCGT TGATC T TGTCATGTAC T TATCC TCGTAT T T TCC T TAT T
TCC TACAGGT TGCAT
GCAAGACCGGAGCTATATCTTGGCATGGTCTTGGATGGGCCATACAGGGGGTTTACAGCTTGGTGTTTTTAACTTTC
TGGGCGGTACTTCCGCTGGAGCTGGCCATGGATCTTCCTTGGATTGCACGAGTTTTCTTGATCTTGCATTGCTTGGT
SEQ ID NO: Sequence description Sequence
GT T TAT TATGAAGATGCAATCATATGGACAT TACAATGGATACC T T TGGGATGTATATCAGGAAGGAT
TGGTC TCGG
AAGCTGATCTCACGGCTGTTTCTGAGTATGATGATGATTTCCCCCTGGATCACGGGGAGGTTCTAGAACAGAGCTTG
TGGTTCGCCAAACACGAGTTGGAGTTTCAATCTAATGGAACTACGGAGAGGAAGGATCACCATCATCATGTATTCGA
CGAAAAGGATGTCAACAAACCAATGCGTGTCTTGCAAGAAGAGGGAATTATCAAATTTCCGGCAAACATCAATTTCA
AGGAT TAT T TCGAGTACAGTATGT TCCCCACGC TAGTC TACACAT TGAAC T TCCCCAGAAT
TCGACATAT TAGATGG
GCGTATGTGTTGCAGAAAGTTTTGGGAACATTTGCCTTAGTGTTTGCCATGATTATCGTCGCCGAAGAGAGTTTCTG
oe
TCCCTTGATGCAAGAAGTTGAACAGTACACAAGATTGCCAACCAACCAAAGGTTCTCAAAGTACTTCGTCGTTCTTT
oe
CCCACTTGATATTGCCCCTCGGCAAACAGTACTTGCTCTCGTTTATCCTCATTTGGAACGAAATTCTCAACGGGATA
oe
GCGGAGT TAAGCAGGT T TGGGGATCGCCAT T TC TACGGCGCC TGGTGGTCAAGCGTCGAC TACATGGAC
TAT TCAAG
AAAATGGAACACGATCGTGCACCGATTCCTCCGCCGGCACGTTTACAATTCGACCATTCGCATCCTCGGTATTTCCA
GGACCCAAGCCGCGATAAT TACAC T T T TGC T T TCAGCCACAATCCACGAAC TCGT TATGTACATCC
TAT T TGGAAAA
T TACGAGGGTACC TAT TCC T TACGATGC T TGTCCAGATCCCCATGACAGTCACCGCCAAGT
TCAACAACCGT T TGTG
GGGCAACATCATGTTCTGGTTGACGTATTTATCTGGCCCCAGCTTGGTTAGTGCGTTGTATTTACTCTTCTGA
SEQ ID NO:55 Acyl-CoA Sterol acyl MS DDE IAGIVIE I DDDVKST S SFQEDLVEVEMSNS S
INE SQT DEL S YRPEE I S LHSRRKSHKT P S DE S FLE I TKNVN
transferase (ARE2) DPDLVSK I ENLRGKVSQREDRLRKHYLHT SQDVKFL SRFND I
KFKSNSAT I LDS DAFYKSEHFGVLT I FWVVI GLY I
Seq MS T L S DMYFGMAKPL S DW I I I
GMFKKDLMQVALVDLVMYL S SYFPYFLQVACKTGAI SWHGLGWAIQGVYSLVFLTF
WAVL P SE SAMDL PW IARVFL I LHCLVF IMKMQ S YGHYNGYLWDVYQEGLVSEADLTAVSEYDDDF P
S DHGEVLEQ S L n.)
co
WFAKHELEFQSNGTTERKDHHHHVFDEKDVNKPMRVLQEEGI I KF PAN INFKDYFEY SMF PT LVYT LNF
PRI RH I RW
AYVLQKVLGTFALVFAMI IVAEESFCPLMQEVEQYTRLPTNQRFSKYFVVLSHL I LPLGKQYLLSFIL IWNE I
LNGI
AELSRFGDRHFYGAWWS SVDYMDYSRKWNT IVHRFLRRHVYNST I RI LGI SRTQAAI I T LLL SAT I
HELVMY I LFGK
n.)
LRGYLFLTMLVQ I PMTVTAKFNNRLWGNIMFWLTYLSGPSLVSALYLLF
0
SEQ ID NO:56 Diacylglycerol
ATGACTCAGGACTATAAAGACGATAGTCCTACGTCCACTGAGTTGGACACTAACATAGAAGAGGTGGAAAGCACTGC
oI
acyltrangerase
AACCCTAGAGTCGGAACTCAGACAGAGAAAACAGACCACGGAAACTCCAGCATCAACCCCACCACCACCTCCACAAC
(DGA1)
AACAGCAGGCGCATAAGAAAGCCCTGAAGAATGGCAAGAGGAAGAGACCATTTATAAACGTGGCGCCGCTCAACACC
n.)
Nuc. Seq
CCGTTGGCTCACAGGCTCGAGACTTTGGCTGTTGTTTGGCACTGTGTCAGTATCCCGTTCTTTATGTTTTTGTTCTT
GC T TACGGTC TCCATGGGGT TGC T TGGGTGGT TC T T TATCAT T T TGCCATAT T TCAT T
TGGTGGTACGGT T TCGAC T
TGCACACTCCATCGAATGGTAAAGTTGTCTATCGTGTGCGCAACTCGTTCAAGAATTTCATCATTTGGGACTGGTTT
GTCAAGTATTTCCCGATTGAAGTGCACAAGACGGTCGAGTTGGATCCTACTTTTAGCGAATTGCCTGTGGAAGAGAG
CGGCGACAGTTCGGACGACGACGAACAAGACTTGGTGTCTGAGCACAGCAGAACTTTGGTTGATCAAATCTTCAAGT
TTTTCGGGTTGAAGAAACGCTTGAATGACACCTCCCTGGGCAAACCAGAGACATTCAAGAATGTGCCTACGGGTCCA
AGGTATATTTTTGGGTACCACCCACACGGAGTGATTTCTATGGGGGCAGTGGGGTTGTTTGCCAACAACGCCTTGAG
GAACGAACCATATACGCCAATTTCCAAATGGTTAAAACCATTCTTCCACGACAGCTCCAAGGGCGAGAGATTGTTCC
CTGGTATTGGCAATATCTTCCCATTGACGCTTACCACACAGTTTGCGCTCCCATTTTACCGTGACTACTTGATGGCT
TTGGGGATCACTAGTGCATCGGCTAAAAACATTAGAAGCTTGATCAACAATGGAGACAACTCTGTGTGTCTCGTCGT
TGGCGGTGCACAAGAATCGTTGTTGAACAATATGATTGCCAAGCACGCCAGAGTCGGGTACGGTTACAAAGAGAGCC
TAGATATTCATGGCGACCAGTCCGAAGAAGAAGAAGAAGAAGAGGATGATACCAAGCAGCTAGAGAACCCAAGTCCT
AAACGTGAAGTGCAATTGGTCTTGAACAAACGTAAAGGTTTTGTGAAGTTGGCTATCGAACTAGGAAATGTTTCCTT
GGTGCC TAT T T T TGCAT TCGGAGAAGC TGATGT T TACAGAT TGGCCCAGCCAGCACCAGGC TCGT
TC T TGTACAAGT
TCCAGCAATGGATGAAGGCAACTTTTCAATTCACCATCCCATTGTTTAGTGCTCGAGGCGTGTTCATCTATGATTTC
GGATTGTTGCCATTCAGAAACCCAATAAACATTTGCGTCGGTAGACCCGTCTACATTCCGCACAACGTCTTGCAAGA
ATACAAGCAAAAGCACCCAGAGGAGTTTGCCGAAGAGGAACCTGCCAGTACCCCGATGAAGAAGTCTGGATCTTTCA
SEQ ID NO: Sequence description Sequence
CCGATATGTTCAAAGCTGGTGAAAAGAAGCCCAAGACTTCAAGTATCAAGACTAAAATCCCACCTGCATTACTAGAC
AAGTACCACAAGCTATACGTCGACGAGTTGAAGAAGGTCTATGAAGAGAACAAGGAAAGGTTTGGCTACGGTGATGT
TGAATTAAACATTGTAGAATAG
SEQ ID NO:57 Diacylglycerol MTQDYKDDS PT S TELDTN I EEVE S TAT LE SELRQRKQT
TE T PAS TPPPPPQQQQAHKKASKNGKRKRPF INVAPLNT
acyltrangerase PLAHRLETLAVVWHCVS I PFFMFLFLLTVSMGLLGWFF I I
LPYF I WWYGFDLHT PSNGKVVYRVRNSFKNF I I WDWF
(DGA1) VKYFP I EVHKTVELDPT F SEL PVEE S GDS
SDDDEQDLVSEHSRTLVDQ I FKFFGLKKRLNDT S SGKPETFKNVPTGP
oe
Seq RY I FGYHPHGVI SMGAVGLFANNALRNEPYT P I
SKWLKPFFHDS SKGERLF PGI GN I FPLTLTTQFALPFYRDYLMA oe
oe
LGI T SASAKN I RS L INNGDNSVC LVVGGAQE S LLNNMIAKHARVGYGYKE S LD I HGDQ
SEEEEEEEDDTKQLENP S P
KREVQLVLNKRKGFVKLAIELGNVSLVP I FAFGEADVYRLAQPAPGSFLYKFQQWMKATFQFT I PLFSARGVF
I YDF
GLLPFRNP IN I CVGRPVY I PHNVLQEYKQKHPEEFAEEE PAS T PMKKS GS FT DMFKAGEKKPKT S
S I KTK I PPALLD
KYHKLYVDELKKVYEENKERFGYGDVELNIVE
SEQ ID NO:58 Diacylglycerol
ATGTCGTCTTTAAAGAACAGAAAATCCGCAAGCGTCGCCACAAGCGATACAGAAGACTCAGAAACAGAGGCAGTATC
acyltrangerase
CTCCTCAATTGATCCCAACGGCACCATATTGCGACCAGTCCTACATGACGAACCCCACCACAGCCATCACCACCACA
(LR01)
ACATAACTAGACCAGTATTGGAGGACGATGGCAGCATCCTGGTGTCCAGAAGATCGTCGATCTCCAAATCCGACGAC
Nuc. Seq
CTGCAGGCAAAGCAAAAGAAGAAGAAACCCAAGAAGAAGATCTTGGAGTCTCGTCGGGTCATGTTTATCTTTGGTAC
CC TCAT TGGGT TAATC T T TGCGTGGGCGT T TACCACAGACACGCATCC T T TCAATGGCGAC T
TGGAGAAGT T TATCA
o
AC T T TGACCAGC TCAACGGGATC T T TGACGAC TGGAAGAAC TGGAAGGATATC T
TGCCCAACAGCATCCAGACGTAC
co
TTGCAGGAATCGGGCAAGGGCGAAGATAACGACGGGTTGCATGGTCTGGCCGATTCCTTCTCCGTCGGGCTCCGCTT
GAAAGCCCAGAAGAACTTCACTGACAACCACAATGTCGTGTTGGTTCCTGGTGTGGTGAGCACGGGGTTGGAATCGT
GGGGAACAACCACCACCGGTGAT TGTCCATC TATCGGATAC T TCAGGAAGAGAT TGTGGGGATCAT T T
TATATGT TA
n.)
AGGACAATGATTTTGGAGAAAACGTGCTGGTTGAAGCATATCCAGTTGGACGAGAAGACGGGGTTGGATCCTCCCAA
0
TAT TAAGGTCCGTGCGGCGCAGGGT T TCGAAGCGGCAGAT T TC T T TATGGC TGGGTAC TGGATC
TGGAACAAGATC T
oI
TGCAGAAC T TGGCGGT TAT TGGGTACGGACCAAATAACATGGTGAGTGC TAGT TATGAC TGGAGAT TGGC
T TACAT T
GACTTGGAGAGAAGAGATGGATATTTTTCGAAACTTAAAGCGCAGATTGAGTTGAATAACAAGTTGAACAACAAGAA
n.)
GAC TGTGT TGAT TGGCCAC TCGATGGGGACCCAGAT TAT T T TC TAC T T T T
TGAAATGGGTCGAAGCCACCGGGAAAC
CATAC TATGGCAATGGCGGACCAAAC TGGGTGAATGATCATAT TGAGTCGAT TAT TGACATCAGTGGGTCGAC
T T TG
GGTACCCCCAAGAGTAT TCC TGTGT TGATC TC TGGGGAAATGAAAGACACCGT TCAAT TGAACGCGT
TGGCGGT T TA
CGGGTTGGAGCAATTTTTCAGCAGGCGTGAAAGAGTCGATATGTTGCGTACATTTGGTGGCGTTGCCAGTATGTTAC
CCAAGGGGGGAGACAAGATATGGGGCAACTTGACGCATGCGCCAGATGATCCAATTTCCACATTCAGTGATGACGAA
GT TACGGACAGCCACGAACC TAAAGATCGT TC T T T TGGTACGT T TATCCAAT TCAAGAACCAAAC
TAGCGACGC TAA
GCCATACAGGGAGATCACCATGGCTGAAGGTATCGATGAATTGTTGGACAAATCACCAGACTGGTATTCCAAGAGAG
TCCGTGAGAACTACTCTTACGGCATTACAGACAGCAAGGCGCAATTAGAGAAGAACAACAATGACCACCTGAAGTGG
TCGAACCCAT TAGAAGC TGCC T TGCC TAAAGCACCCGACATGAAGATC TAT TGT T TC TACGGAGT
TGGAAATCC TAC
CGAAAGGGCATACAAGTATGTGACTGCCGATAAAAAAGCCACGAAATTGGACTACATAATAGACGCCGACGATGCCA
ATGGAGTCATAT TAGGAGACGGAGACGGCAC TGT T TCGT TAT TAACCCAC
TCGATGTGCCATGAGTGGGCCAAGGGA
GACAAGTCGAGATACAACCCAGCCAACTCGAAGGTTACCATTGTTGAAATCAAGCACGAGCCAGACAGATTTGATTT
ACGAGGCGGCGCCAAGACTGCGGAACATGTTGATATTTTGGGGAGTGCCGAGTTGAACGAGTTGATTTTGACTGTGG
TTAGCGGGAACGGGGACGAGATTGAGAATAGATATGTCAGCAACTTAAAAGAAATAGTAGAGGCCATAAATTTATAA
SEQ ID NO: Sequence description Sequence
SEQ ID NO:59 Diacylglycerol MS SLKNRKSASVATSDTEDSETEAVS S S I DPNGT I
LRPVLHDE PHHSHHHHN I TRPVLEDDGS I SVSRRS S I SKS DD
acyltransferase SQAKQKKKKPKKKI LE SRRVMF I FGTL I GL I
FAWAFT TDTHPFNGDLEKF INFDQLNGI FDDWKNWKD I L PNS I QTY
(LR01)
LQESGKGEDNDGLHGSADSFSVGLRLKAQKNFTDNHNVVLVPGVVSTGLESWGTTTTGDCPS I GYFRKRLWGS
FYML 0
r..)
A.A. Seq RTMI LEKTCWLKH I QLDEKTGLDPPN I
KVRAAQGFEAADFFMAGYWIWNKI LQNLAVI GYGPNNMVSASYDWRLAY I
1¨,
DLERRDGYF SKLKAQ I ELNNKLNNKKTVL I GHSMGTQ I I FYFLKWVEATGKPYYGNGGPNWVNDH IES
I IDI SGSTL ,..,
-a-,
GT PKS I PVL I SGEMKDTVQLNALAVYGLEQFF SRRERVDMLRTFGGVASML PKGGDKIWGNLTHAPDDP I
STFSDDE .6.
oe
VTDSHE PKDRS FGTF I QFKNQT S DAKPYRE I TMAEGIDELLDKS PDWYSKRVRENYSYGI
TDSKAQLEKNNNDHSKW oe
SNPLEAAL PKAPDMKI YCFYGVGNPTERAYKYVTADKKATKLDY I I DADDANGVI LGDGDGTVS
LLTHSMCHEWAKG oe
DKSRYNPANSKVT IVE I KHE PDRFDLRGGAKTAEHVD I LGSAELNEL I LTVVSGNGDE I
ENRYVSNLKE IVEAINL
n
o
n.)
m
in
o
o
N
lo
un
in
t...)
n.)
o
H
11.
O
(A
I
N)
in
IV
n
,-i
cp
w
w
-a-,
u,
c7,
u,
c7,
w
CA 02850095 2014-03-25
WO 2013/048898
PCT/US2012/056562
Example 52: Production of genetically modified Candida tropicalis strains
Strain sAA779 (pox4a::ura3/pox4b::ura3 PDX5/P0X5 acs1:: PURA3/acs1:: PURA3)
Strain sAA776 was grown in YPD media overnight and plated on 5-FOA plate.
Colonies
that grew in the presence of 5-FOA were PCR screened for the looping out of
the URA3 gene
leaving behind only the URA3 promoter (PURA3) in both ACS1 copies. One such
strain was
named sAA779. As such both alleles of the ACS1 gene were disrupted.
Strain sAA865 (pox4a::ura3/pox4b::ura3 PDX5/P0X5 acs1:: PURA3/acs1:: PURA3
fat1-
A1::URA3/FAT1)
The full-length coding sequence ofthe Fat1 gene was amplified from C.
tropicalis
(ATCC20336) genomic DNA using primers oAA1023 and oAA1024. The 2,086 bp PCR
product
was gel purified and ligated into pCR-Blunt II-TOPO (Invitrogen), transformed
into competent
TOP10 E. coli cells (Invitrogen) and clones containing PCR inserts were
sequenced to confirm
correct DNA sequence. One such plasmid was named pAA296.
Deletion of each FAT1 allele was achieved by transforming cells with linear
DNA cassettes
constructed by overlap extension PCR (OE-PCR). The deletion cassette for the
first FAT1 allele in
sAA779 was created from three DNA fragments. The first DNA fragment (FAT1 5'
homology) was
amplified from plasmid pAA296 using primers oAA2055 and oAA2056. The second
DNA fragment
(PURA3URA3TURA3PURA3) was amplified from plasmid pAA298 using primers oAA2057
and
oAA2068. The third DNA fragment (FAT1 3' homology) was amplified from plasmid
pAA296 using
primers oAA2069 and oAA2060. The location of primer annealing sites in pAA296
that amplify
FAT1 DNA fragments are shown in Figure 59. All three DNA fragments were
combined in the
same reaction to generate the full-length deletion cassette (Figure 60) by OE-
PCR using primers
oAA2055 and oAA2060.
Strain sAA779 was transformed with the full-length deletion cassette and
plated on SCD-
Ura plate. Several colonies were screened by PCR for integration of the
deletion cassette at the
first FAT1 allele. One such strain was named sAA865.
Strain sAA869 (pox4a::ura3/pox4b::ura3 PDX5/P0X5 acs1:: PURA3/acs1:: PURA3
fat1-
A1::PURA3/FAT1)
Strain sAA865 was grown in YPD media overnight and plated on 5-FOA plate.
Colonies
that grew in the presence of 5-FOA were PCR screened for the looping out of
the URA3 gene
254
CA 02850095 2014-03-25
WO 2013/048898
PCT/US2012/056562
leaving behind only the URA3 promoter (PURA3) in the first FAT1 allele. One
such strain was
named sAA869.
Strain sAA875 (pox4a::ura3/pox4b::ura3 PDX5/P0X5 acs1:: PURA3/acs1:: PURA3
fat1-
.8,1::PURA3/fat1-.82::URA3)
The deletion of the second FAT1 allele in sAA869 was performed by
transformation with a
deletioin cassette created by OE-PCR. The deletion cassette for the second
FAT1 allele was
constructed from three DNA fragments. The first DNA fragment (FAT1 5'
homology) was amplified
from plasmid pAA296 using primers oAA2070 and oAA2071. The second DNA fragment
(PURA3URA3TURA3PURA3) was amplified from plasmid pAA298 using primers oAA2072
and
oAA2073. The third DNA fragment (FAT1 3' homology) was amplified from plasmid
pAA296 using
primers oAA2074 and oAA2075. The location of primer annealing sites in pAA296
that amplify
FAT1 DNA fragments are shown in Figure 59. All three DNA fragments were
combined in the
same reaction to create the full-length deletion (Figure 60) cassette by OE-
PCR using primers
oAA2070 and oAA2071.
Strain sAA869 was transformed with the full-length deletion cassette and
plated on SCD-
Ura plate. Several colonies were screened by PCR for integration of the
deletion cassette at the
second FAT1 allele. One such strain was named sAA875.
255
SEQ ID NO: Description Sequence
SEQ ID NO: 60 Cytochrome P450
ATGGCCACACAAGAAATCATCGATTCTGTACTTCCGTACTTGACCAAATGGTACACTGTGATTACTG
0
monooxygenase
CAGCAGTATTAGTCTTCCTTATCTCCACAAACATCAAGAACTACGTCAAGGCAAAGAAATTGAAATG
n.)
o
1-,
CYP52Al2
TGTCGATCCACCATACTTGAAGGATGCCGGTCTCACTGGTATTCTGTCTTTGATCGCCGCCATCAAG
c,.)
Nucleotide Seq.
GCCAAGAACGACGGTAGATTGGCTAACTTTGCCGATGAAGTTTTCGACGAGTACCCAAACCACACCT
-a-,
.6.
TCTACTTGTCTGTTGCCGGTGCTTTGAAGATTGTCATGACTGTTGACCCAGAAAACATCAAGGCTGT
oe
oe
CTTGGCCACCCAATTCACTGACTTCTCCTTGGGTACCAGACACGCCCACTTTGCTCCTTTGTTGGGT
oe
GACGGTATCTTCACCTTGGACGGAGAAGGTTGGAAGCACTCCAGAGCTATGTTGAGACCACAGTTTG
CTAGAGACCAGATTGGACACGTTAAAGCCTTGGAACCACACATCCAAATCATGGCTAAGCAGATCAA
GT TGAACCAGGGAAAGAC T T TCGATATCCAAGAAT TGT TC T T TAGAT T TACCGTCGACACCGC TAC
T
GAGTTCTTGTTTGGTGAATCCGTTCACTCCTTGTACGATGAAAAATTGGGCATCCCAACTCCAAACG
AAATCCCAGGAAGAGAAAACTTTGCCGCTGCTTTCAACGTTTCCCAACACTACTTGGCCACCAGAAG
TTACTCCCAGACTTTTTACTTTTTGACCAACCCTAAGGAATTCAGAGACTGTAACGCCAAGGTCCAC
CAC T TGGCCAAGTAC T T TGTCAACAAGGCC T TGAAC T T TAC TCC TGAAGAAC
TCGAAGAGAAATCCA
n
AGTCCGGTTACGTTTTCTTGTACGAATTGGTTAAGCAAACCAGAGATCCAAAGGTCTTGCAAGATCA
AT TGT TGAACAT TATGGT TGCCGGAAGAGACACCAC TGCCGGT T TGT TGTCC T T TGC T T TGT T
TGAA o
n.)
TTGGCTAGACACCCAGAGATGTGGTCCAAGTTGAGAGAAGAAATCGAAGTTAACTTTGGTGTTGGTG
co
in
AAGACTCCCGCGTTGAAGAAATTACCTTCGAAGCCTTGAAGAGATGTGAATACTTGAAGGCTATCCT
o
o
un
TAACGAAACCTTGCGTATGTACCCATCTGTTCCTGTCAACTTTAGAACCGCCACCAGAGACACCACT
in
cA
TTGCCAAGAGGTGGTGGTGCTAACGGTACCGACCCAATCTACATTCCTAAAGGCTCCACTGTTGCTT
n.)
o
ACGTTGTCTACAAGACCCACCGTTTGGAAGAATACTACGGTAAGGACGCTAACGACTTCAGACCAGA
H
11.
oI
AAGATGGTTTGAACCATCTACTAAGAAGTTGGGCTGGGCTTATGTTCCATTCAACGGTGGTCCAAGA
GTCTGCTTGGGTCAACAATTCGCCTTGACTGAAGCTTCTTATGTGATCACTAGATTGGCCCAGATGT
co
1
TTGAAACTGTCTCATCTGATCCAGGTCTCGAATACCCTCCACCAAAGTGTATTCACTTGACCATGAG
n.)
in
TCACAACGATGGTGTCTTTGTCAAGATGTAA
SEQ ID NO: 61 Cytochrome P450 MATQE I I DSVLPYLTKWYTVI TAAVLVFL I S
TN I KNYVKAKKLKCVDP PYLKDAGLTGI S SL IAAIK
monooxygenase AKNDGRLANFADEVFDEY PNHT FYL SVAGALK
IVMTVDPEN I KAVLATQFT DF S LGTRHAHFAPLLG
CYP52Al2 DGI FT LDGEGWKHSRAMLRPQFARDQ I
GHVKALE PH I Q IMAKQ I KLNQGKT FD I QELFFRFTVDTAT
A.A. Seq. EFLFGESVHSLYDEKLGI PT PNE I
PGRENFAAAFNVSQHYLATRSYSQTFYFLTNPKEFRDCNAKVH
HLAKYFVNKALNFT PEELEEKSKSGYVFLYELVKQTRDPKVLQDQLLN IMVAGRDT TAGLL S FALFE
IV
LARHPEMWSKLREE I EVNFGVGEDSRVEE I TFEALKRCEYLKAI LNETLRMYPSVPVNFRTATRDTT
n
L PRGGGANGT DP I Y I PKGS TVAYVVYKTHRLEEYYGKDANDFRPERWFEPS TKKLGWAYVPFNGGPR
1-3
VCLGQQFALTEASYVI TRLAQMFE TVS SDPGLEYPPPKC I HLTMSHNDGVFVKM
ci)
SEQ ID NO: 62 Cytochrome P450
ATGACTGTACACGATATTATCGCCACATACTTCACCAAATGGTACGTGATAGTACCACTCGCTTTGA
n.)
o
1-,
monooxygenase
TTGCTTATAGAGTCCTCGACTACTTCTATGGCAGATACTTGATGTACAAGCTTGGTGCTAAACCATT
r..)
CYP52A13
TTTCCAGAAACAGACAGACGGCTGTTTCGGATTCAAAGCTCCGCTTGAATTGTTGAAGAAGAAGAGC
-a-,
u,
cA
Nucleotide Seq.
GACGGTACCCTCATAGACTTCACACTCCAGCGTATCCACGATCTCGATCGTCCCGATATCCCAACTT
un
cA
TCACATTCCCGGTCTTTTCCATCAACCTTGTCAATACCCTTGAGCCGGAGAACATCAAGGCCATCTT
n.)
GGCCACTCAGTTCAACGATTTCTCCTTGGGTACCAGACACTCGCACTTTGCTCCTTTGTTGGGTGAT
GGTATCTTTACGTTGGATGGCGCCGGCTGGAAGCACAGCAGATCTATGTTGAGACCACAGTTTGCCA
SEQ ID NO: Description Sequence
GAGAACAGATTTCCCACGTCAAGTTGTTGGAGCCACACGTTCAGGTGTTCTTCAAACACGTCAGAAA
GGCACAGGGCAAGACTTTTGACATCCAGGAATTGTTTTTCAGATTGACCGTCGACTCCGCCACCGAG
TTTTTGTTTGGTGAATCCGTTGAGTCCTTGAGAGATGAATCTATCGGCATGTCCATCAATGCGCTTG
0
n.)
AC T T TGACGGCAAGGC TGGC T T TGC TGATGC T T T TAAC TAT TCGCAGAAT TAT T TGGC T
TCGAGAGC =
1¨,
GGTTATGCAACAATTGTACTGGGTGTTGAACGGGAAAAAGTTTAAGGAGTGCAACGCTAAAGTGCAC
,..,
-a-,
AAGTTTGCTGACTACTACGTCAACAAGGCTTTGGACTTGACGCCTGAACAATTGGAAAAGCAGGATG
.6.
oe
GT TATGTGT T T T TGTACGAAT TGGTCAAGCAAACCAGAGACAAGCAAGTGT TGAGAGACCAAT TGT T
oe
GAACATCATGGTTGCTGGTAGAGACACCACCGCCGGTTTGTTGTCGTTTGTTTTCTTTGAATTGGCC
oe
AGAAACCCAGAAGT TACCAACAAGT TGAGAGAAGAAAT TGAGGACAAGT T TGGAC TCGGTGAGAATG
CTAGTGTTGAAGACATTTCCTTTGAGTCGTTGAAGTCCTGTGAATACTTGAAGGCTGTTCTCAACGA
AACCTTGAGATTGTACCCATCCGTGCCACAGAATTTCAGAGTTGCCACCAAGAACACTACCCTCCCA
AGAGGTGGTGGTAAGGACGGGT TGTC TCC TGT T T TGGTGAGAAAGGGTCAGACCGT TAT T TACGGTG
TCTACGCAGCCCACAGAAACCCAGCTGTTTACGGTAAGGACGCTCTTGAGTTTAGACCAGAGAGATG
GT T TGAGCCAGAGACAAAGAAGC T TGGC TGGGCC T TCC TCCCAT TCAACGGTGGTCCAAGAATC TGT
TTGGGACAGCAGTTTGCCTTGACAGAAGCTTCGTATGTCACTGTCAGGTTGCTCCAGGAGTTTGCAC
n
AC T TGTC TATGGACCCAGACACCGAATATCCACC TAAGAAAATGTCGCAT T TGACCATGTCGC T T T T
o
CGACGGTGCCAATAT TGAGATGTAT TAG
n.)
co
SEQ ID NO: 63 Cytochrome P450 MTVHD I IATYFTKWYVIVPLAL
IAYRVLDYFYGRYLMYKLGAKPFFQKQTDGCFGFKAPLELLKKKS in
o
r..) monooxygenase DGTL I DFT LQRI HDLDRPD I PT FT F PVF
S INLVNT LE PEN I KAI LATQFNDFSLGTRHSHFAPLLGD o
ko
un
in
--.1 CYP52A13 GI FT LDGAGWKHSRSMLRPQFAREQ I SHVKLLE
PHVQVFFKHVRKAQGKT FD I QELFFRLTVDSATE
n.)
A.A. Seq. FLFGESVESLRDES I GMS
INALDFDGKAGFADAFNY S QNYLASRAVMQQLYWVLNGKKFKECNAKVH 0
H
KFADYYVNKALDLT PEQLEKQDGYVFLYELVKQTRDKQVLRDQLLN IMVAGRDT TAGLL S FVFFELA
11.
oI
RNPEVTNKLREE I EDKFGLGENASVED I S FE S LKSCEYLKAVLNE T LRLY P SVPQNFRVATKNT T
L P
co
I
RGGGKDGLS PVLVRKGQTVI YGVYAAHRNPAVYGKDALEFRPERWFE PE TKKLGWAFL PFNGGPRI C
n.)
LGQQFALTEAS YVTVRLLQEFAHL SMDPDTEY P PKKMSHLTMS LFDGAN I EMY
in
SEQ ID NO: 64 Cytochrome P450
ATGACTGCACAGGATATTATCGCCACATACATCACCAAATGGTACGTGATAGTACCACTCGCTTTGA
monooxygenase
TTGCTTATAGGGTCCTCGACTACTTTTACGGCAGATACTTGATGTACAAGCTTGGTGCTAAACCGTT
CYP52A14 T T TCCAGAAACAAACAGACGGT TAT T TCGGAT
TCAAAGC TCCAC T TGAAT TGT TAAAAAAGAAGAGT
Nucleotide Seq.
GACGGTACCCTCATAGACTTCACTCTCGAGCGTATCCAAGCGCTCAATCGTCCAGATATCCCAACTT
TTACATTCCCAATCTTTTCCATCAACCTTATCAGCACCCTTGAGCCGGAGAACATCAAGGCTATCTT
GGCCACCCAGTTCAACGATTTCTCCTTGGGCACCAGACACTCGCACTTTGCTCCTTTGTTGGGCGAT
IV
GGTATCTTTACCTTGGACGGTGCCGGCTGGAAGCACAGCAGATCTATGTTGAGACCACAGTTTGCCA
n
,-i
GAGAACAGATTTCCCACGTCAAGTTGTTGGAGCCACACATGCAGGTGTTCTTCAAGCACGTCAGAAA
GGCACAGGGCAAGACTTTTGACATCCAAGAATTGTTTTTCAGATTGACCGTCGACTCCGCCACTGAG
ci)
n.)
T T T T TGT T TGGTGAATCCGT TGAGTCC T TGAGAGATGAATC TAT TGGGATGTCCATCAATGCAC T
TG o
1¨,
n.)
AC T T TGACGGCAAGGC TGGC T T TGC TGATGC T T T TAAC TAC TCGCAGAAC TAT T TGGC T
TCGAGAGC -a-,
GGTTATGCAACAATTGTACTGGGTGTTGAACGGGAAAAAGTTTAAGGAGTGCAACGCTAAAGTGCAC
un
cA
un
AAGT T TGC TGAC TAT TACGTCAGCAAGGC T T TGGAC T TGACACC TGAACAAT
TGGAAAAGCAGGATG cA
n.)
GT TATGTGT TC T TGTACGAGT TGGTCAAGCAAACCAGAGACAGGCAAGTGT TGAGAGACCAGT TGT T
GAACATCATGGTTGCCGGTAGAGACACCACCGCCGGTTTGTTGTCGTTTGTTTTCTTTGAATTGGCC
AGAAACCCAGAGGTGACCAACAAGTTGAGAGAAGAAATCGAGGACAAGTTTGGTCTTGGTGAGAATG
SEQ ID NO: Description Sequence
CTCGTGTTGAAGACATTTCCTTTGAGTCGTTGAAGTCATGTGAATACTTGAAGGCTGTTCTCAACGA
AACTTTGAGATTGTACCCATCCGTGCCACAGAATTTCAGAGTTGCCACCAAAAACACTACCCTTCCA
AGGGGAGGTGGTAAGGACGGGTTATCTCCTGTTTTGGTCAGAAAGGGTCAAACCGTTATGTACGGTG
0
n.)
TCTACGCTGCCCACAGAAACCCAGCTGTCTACGGTAAGGACGCCCTTGAGTTTAGACCAGAGAGGTG
=
1¨,
GT T TGAGCCAGAGACAAAGAAGC T TGGC TGGGCC T TCC T TCCAT TCAACGGTGGTCCAAGAAT T
TGC ,..,
-a-,
TTGGGACAGCAGTTTGCCTTGACAGAAGCTTCGTATGTCACTGTCAGATTGCTCCAAGAGTTTGGAC
.6.
oe
AC T TGTC TATGGACCCCAACACCGAATATCCACC TAGGAAAATGTCGCAT T TGACCATGTCCC T T T T
oe
CGACGGTGCCAACAT TGAGATGTAT TAG
oe
SEQ ID NO: 65 Cytochrome P450 MTAQD I IATY I TKWYVIVPLAL
IAYRVLDYFYGRYLMYKLGAKPFFQKQTDGYFGFKAPLELLKKKS
monooxygenase
DGTLIDFTLERIQALNRPDIPTFTFPIFSINLISTLEPENIKAILATQFNDFSLGTRHSHFAPLLGD
CYP52A14 GI FT LDGAGWKHSRSMLRPQFAREQ I SHVKLLE
PHMQVFFKHVRKAQGKT FD I QELFFRLTVDSATE
A.A. Seq. FLFGESVESLRDES I GMS
INALDFDGKAGFADAFNY SQNYLASRAVMQQLYWVLNGKKFKECNAKVH
KFADYYVSKALDLT PEQLEKQDGYVFLYELVKQTRDRQVLRDQLLN IMVAGRDT TAGLL S FVFFELA
RNPEVTNKLREE I EDKFGLGENARVED I S FE S LKSCEYLKAVLNE T LRLY P SVPQNFRVATKNT T
L P
RGGGKDGLS PVLVRKGQTVMYGVYAAHRN PAVYGKDALE FRPERWFE PE TKKLGWAFL PFNGGPRI C
n
LGQQFALTEAS YVTVRLLQEFGHL SMDPNTEY P PRKMSHLTMS LFDGAN I EMY
o
SEQ ID NO: 66 Cytochrome P450
ATGTCGTCTTCTCCATCGTTTGCCCAAGAGGTTCTCGCTACCACTAGTCCTTACATCGAGTACTTTC
N)
co
monooxygenase
TTGACAACTACACCAGATGGTACTACTTCATACCTTTGGTGCTTCTTTCGTTGAACTTTATAAGTTT
in
o
r..) CYP52A15 GC TCCACACAAGGTAC T TGGAACGCAGGT
TCCACGCCAAGCCAC TCGGTAAC T T TGTCAGGGACCC T o
ko
un
in
oe Nucleotide Seq.
ACGTTTGGTATCGCTACTCCGTTGCTTTTGATCTACTTGAAGTCGAAAGGTACGGTCATGAAGTTTG
n.)
CTTGGGGCCTCTGGAACAACAAGTACATCGTCAGAGACCCAAAGTACAAGACAACTGGGCTCAGGAT
0
H
TGTTGGCCTCCCATTGATTGAAACCATGGACCCAGAGAACATCAAGGCTGTTTTGGCTACTCAGTTC
11.
oI
AATGATTTCTCTTTGGGAACCAGACACGATTTCTTGTACTCCTTGTTGGGTGACGGTATTTTCACCT
co
1
TGGACGGTGCTGGCTGGAAACATAGTAGAACTATGTTGAGACCACAGTTTGCTAGAGAACAGGTTTC
n.)
TCACGTCAAGTTGTTGGAGCCACACGTTCAGGTGTTCTTCAAGCACGTTAGAAAGCACCGCGGTCAA
in
ACGTTCGACATCCAAGAATTGTTCTTCAGGTTGACCGTCGACTCCGCCACCGAGTTCTTGTTTGGTG
AGTC TGC TGAATCC T TGAGGGACGAATC TAT TGGAT TGACCCCAACCACCAAGGAT T TCGATGGCAG
AAGAGAT T TCGC TGACGC T T TCAAC TAT TCGCAGAC T TACCAGGCC TACAGAT T T T TGT
TGCAACAA
ATGTACTGGATCTTGAATGGCTCGGAATTCAGAAAGTCGATTGCTGTCGTGCACAAGTTTGCTGACC
AC TATGTGCAAAAGGC T T TGGAGT TGACCGACGATGAC T TGCAGAAACAAGACGGC TATGTGT TC T T
GTACGAGT TGGC TAAGCAAACCAGAGACCCAAAGGTC T TGAGAGACCAGT TAT TGAACAT T T TGGT T
IV
GCCGGTAGAGACACGACCGCCGGTTTGTTGTCATTTGTTTTCTACGAGTTGTCAAGAAACCCTGAGG
n
,-i
TGTTTGCTAAGTTGAGAGAGGAGGTGGAAAACAGATTTGGACTCGGTGAAGAAGCTCGTGTTGAAGA
GATCTCGTTTGAGTCCTTGAAGTCTTGTGAGTACTTGAAGGCTGTCATCAATGAAACCTTGAGATTG
ci)
n.)
TACCCATCGGTTCCACACAACTTTAGAGTTGCTACCAGAAACACTACCCTCCCAAGAGGTGGTGGTG
o
1¨,
n.)
AAGATGGATAC TCGCCAAT TGTCGTCAAGAAGGGTCAAGT TGTCATGTACAC TGT TAT TGC TACCCA
-a-,
CAGAGACCCAAGTATCTACGGTGCCGACGCTGACGTCTTCAGACCAGAAAGATGGTTTGAACCAGAA
un
cA
un
AC TAGAAAGT TGGGC TGGGCATACGT TCCAT TCAATGGTGGTCCAAGAATC TGT T TGGGTCAACAGT
cA
n.)
TTGCCTTGACCGAAGCTTCATACGTCACTGTCAGATTGCTCCAGGAGTTTGCACACTTGTCTATGGA
CCCAGACACCGAATATCCACCAAAATTGCAGAACACCTTGACCTTGTCGCTCTTTGATGGTGCTGAT
GT TAGAATGTAC TAA
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
CONTENANT LES PAGES 1 A 258
NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des
brevets
JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
THIS IS VOLUME 1 OF 2
CONTAINING PAGES 1 TO 258
NOTE: For additional volumes, please contact the Canadian Patent Office
NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:
