Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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MICROORGANISMS AND METHODS FOR PRODUCING ACRYLATE
AND OTHER PRODUCTS FROM HOMOSERINE
FIELD OF THE INVENTION
This invention relates to microorganisms that convert a carbon source to
acrylate or other
desirable products using homoserine and 2-keto-4-hydroxybutyrate as
intermediates. The
invention provides genetically engineered microorganisms that carry out the
conversion, as well
as methods for producing acrylate by culturing the microorganisms. Also
provided are
microorganisms and methods for converting homoserine to 3-hydroxypropionyl-
CoA, 3-
hydroxypropionate (3HP), poly-3-hydroxypropionate and 1,3-propanediol.
BACKGROUND OF THE INVENTION
One organic chemical used to make super absorbent polymers (used in diapers),
plastics,
coatings, paints, adhesives, and binders (used in leather, paper and textile
products) is acrylic
acid. Acrylic acid (IUPAC: prop-2-enoic acid) is the simplest unsaturated
carboxylic acid.
Traditionally, acrylic acid is made from propene. Propene itself is a
byproduct of oil
refining from petroleum (i.e., crude oil) and of natural gas production.
Disadvantages associated
with traditional acrylic acid production are that petroleum is a nonrenewable
starting material and
that the oil refining process pollutes the environment. Synthesis methods for
acrylic acid
utilizing other starting materials have not been adopted for widespread use
due to expense or
environmental concerns. These starting materials included, for example,
acetylene, ethenone and
ethylene cyanohydrins.
To avoid petroleum-based production, researchers have proposed other methods
for
producing acrylic acid involving the fermentation of sugars by engineered
microorganisms.
Straathof et al., Appl Microbiol Biotechnol, 67: 727-734 (2005) discusses a
conceptual
fermentation process for acrylic acid production from sugars. The process
proposed in the article
proceeds via a [3-a1anine, methylcitrate, malonyl-CoA or methylmalonate-CoA
intermediate in
the microorganism. Another process described in Lynch, U.S. Patent Publication
No.
2011/0125118 relates to using synthesis gas components as a carbon source in a
microbial system
to produce 3-hydroxypropionic acid, with subsequent conversion of the 3-
hydroxyproprionic acid
to acrylic acid.
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Methods to manufacture other organic chemicals in genetically engineered
microorganisms have been proposed. See, for example, U.S. Patent Publication
No.
2011/0014669 published January 20, 2011 relating to microorganisms for
converting L-
glutamate to 1,4-butanediol.
Since at least four million metric tons of acrylic acid are produced annually,
there remains
a need in the art for cost-effective, environmentally-friendly methods for its
production from
renewable carbon sources.
SUMMARY OF THE INVENTION
Homoserine is an intermediate in the biosynthesis of the amino acids threonine
and
methionine. Homoserine is naturally made from glucose in the bacterium E. coli
and many other
organisms. Figure 1 set out an illustration of the steps converting glucose to
homoserine.
The present invention utilizes homoserine and 2-keto-4-hydroxybutyrate as
intermediates
to make acrylate (the chemical form of acrylic acid at neutral pH) and other
products of interest.
Figures 2 and 3 set out examples of contemplated pathways for making acrylate,
3-
hydroxypropionate, poly-3-hydroxypropionate, 1,3-propanediol and 3-
hydroxypropionyl-CoA
from homoserine. Microorganisms do not naturally make acrylate and the other
products, but
microorganisms (such as bacteria, yeast, fungi and algae) are genetically
modified according to
the invention to carry out the conversions in the pathways. Microorganisms
include, but are not
limited to, an E. coli bacterium.
Producing acrylate
In a first aspect, the invention provides a first type of microorganism, one
that converts
homoserine to acrylate, wherein the microorganism expresses recombinant genes
encoding a
deaminase or transaminase; a dehydrogenase or decarboxylase; a dehydratase;
and a thioesterase,
a phosphate transferase/kinase combination, or an acyl-CoA transferase.
The deaminase or transaminase catalyzes a reaction to convert homoserine to 2-
keto-4-
hydroxybutyrate. In some embodiments, the deaminase or transaminase is an
aminotransferase,
an L-amino acid oxidase or an L-amino acid dehydrogenase. Aminotransferases
include, but are
not limited to, a glutamate-oxaloacetate aminotransferase, a glutamate-
pyruvate
aminotransferase, an L-aspartate:2-oxoglutarate aminotransferase, and an L-
alanine:2-
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oxoglutarate aminotransferase. Amino acid sequences of some aminotransferases
known in the
art are set out in SEQ ID NOs: 2, 4, 6, 8, 10 and 12. Exemplary DNA sequences
encoding those
aminotransferases are respectively set out in SEQ ID NOs: 1, 3, 5, 7, 9 and
11. Amino acid
sequences of some L-amino acid oxidases known in the art are set out in SEQ ID
NOs: 14 and 16.
The dehydrogenase catalyzes a reaction to convert 2-keto-4-hydroxybutyrate to
3-
The dehydratase catalyzes a reaction to convert 3-hydroxypropionyl-CoA to
acryloyl-
CoA. In some embodiments, the dehydratase is a 3-hydroxypropionyl-CoA-
dehydratase. The
The thioesterase, the phosphate transferase/kinase combination or the acyl-CoA
transferase catalyzes a reaction to convert acryloyl-CoA to acrylate. In some
embodiments, the
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acrylate kinase known in the art is set out in SEQ ID NO: 52. An exemplary DNA
sequence
encoding the acrylate kinase is set out in SEQ ID NO: 51. The amino acid
sequence of an acyl-
CoA transferase known in the art is set out in SEQ ID NO: 46. An exemplary DNA
sequence
encoding the acyl-CoA transferase is set out in SEQ ID NO: 45.
In a second aspect, the invention provides a first type of method, one for
producing
acrylate in which the first type of microorganism is cultured to produce
acrylate. The first type
of method for producing acrylate converts homoserine to 2-keto-4-
hydroxybutyrate, 2-keto-4-
hydroxybutyrate to 3-hydroxypropionyl-CoA, 3-hydroxypropionyl-CoA to acryloyl-
CoA and
then acryloyl-CoA to acrylate.
In a third aspect, the invention provides a second type of microorganism, one
that
converts homoserine to acrylate, wherein the microorganism expresses
recombinant genes
encoding: a deaminase or transaminase, a decarboxylase, a dehydrogenase, a
dehydratase and a
thioesterase.
The deaminase or transaminase catalyzes a reaction to convert homoserine to 2-
keto-4-
hydroxybutyrate. In some embodiments, the deaminase or transaminase is an
aminotransferase,
an L-amino acid oxidase or an L-amino acid dehydrogenase. Aminotransferases
include, but are
not limited to, a glutamate-oxaloacetate aminotransferase, a glutamate-
pyruvate
aminotransferase, an L-aspartate:2-oxoglutarate aminotransferase, an L-
alanine:2-oxoglutarate
aminotransferase. Amino acid sequences of some aminotransferases known in the
art are set out
in SEQ ID NOs: 2, 4, 6, 8, 10 and 12. Exemplary DNA sequences encoding those
aminotransferases are respectively set out in SEQ ID NOs: 1, 3, 5, 7, 9 and
11. Amino acid
sequences of some L-amino acid oxidases known in the art are set out in SEQ ID
NOs: 14 and 16.
Exemplary DNA sequences encoding those L-amino acid oxidases are respectively
set out in
SEQ ID NOs: 13 and 15. Amino acid sequences of some L-amino acid
dehydrogenases known in
the art are set out in SEQ ID NOs: 18 and 20. Exemplary DNA sequences encoding
those L-
amino acid dehydrogenases are set out in SEQ ID NOs: 17 and 19.
The decarboxylase catalyzes a reaction to convert 2-keto-4-hydroxybutyrate to
3-
hydroxy-propionaldehyde. In some embodiments, the decarboxylase is a 2-keto
acid
decarboxylase. The 2-keto acid decarboxylases include, but are not limited to,
the 2-keto acid
decarboxylase KdcA set out in SEQ ID NO: 54 and its derivatives. An exemplary
DNA
sequence encoding KdcA is set out in SEQ ID NO: 53.
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The dehydrogenase catalyzes a reaction to convert 3-hydroxy-propionaldehyde to
3-
hydroxypropionyl-CoA. In some embodiments, the dehydrogenase is a
propionaldehyde
dehydrogenase. Propionaldehyde dehydrogenases include, but are not limited to,
a PduP. Amino
acid sequences of some PduP propionaldehyde dehydrogenases known in the art
are set out in
5 SEQ ID NOs: 60 and 62. Exemplary DNA sequences encoding the PduP
propionaldehyde
dehydrogenases are respectively set out in SEQ ID NOs: 59 and 61.
The dehydratase catalyzes a reaction to convert 3-hydroxypropionyl-CoA to
acryloyl-
CoA. In some embodiments, the dehydratase is a 3-hydroxypropionyl-CoA
dehydratase. The
amino acid sequence of 3-hydroxypropionyl-CoA dehydratase known in the art is
set out in SEQ
ID NO: 48. An exemplary DNA sequence encoding the 3-hydroxypropionyl-CoA
dehydratase is
set out in SEQ ID NO: 47.
The thioesterase catalyzes a reaction to convert acryloyl-CoA to acrylate. In
some
embodiments, the thioesterase is an acryloyl-CoA thioesterase. Acryloyl-CoA
thioesterases
include, but are not limited to E. coli TesB set out in SEQ ID NO: 90, the
Clostridium
propionicum-derived thioesterase including an E324D substitution set out in
SEQ ID NO: 92 and
the Megasphaera elsdenii-derived thioesterase including an E325D substitution
set out in SEQ
ID NO: 94. Exemplary DNA sequences encoding these acryloyl-CoA thioesterases
are
respectively set out in SEQ ID NOs: 89, 91 (codon-optimized for E. coli) and
93 (codon-
optimized for E. coli).
In a fourth aspect, the invention provides a second type of method, one for
producing
acrylate in which the second type of microorganism is cultured to produce
acrylate. The second
type of method for producing acrylate converts homoserine to 2-keto-4-
hydroxybutyrate, 2-keto-
4-hydroxybutyrate to 3-hydroxy-propionaldehyde, 3-hydroxy-propionaldehyde to 3-
hydroxypropionyl-CoA, 3-hydroxy-propionyl-CoA to acryloyl-CoA and then
acryloyl-CoA to
acrylate.
Producing 3-hydroxypropionate
In a fifth aspect, the invention provides a third type of microorganism, one
that converts
homoserine to 3-hydroxypropionate, wherein the microorganism expresses
recombinant genes
encoding: a deaminase or transaminase, a dehydrogenase or decarboxylase, and
acyl-CoA
transferase or athioesterase.
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The deaminase or transaminase catalyzes a reaction to convert homoserine to 2-
keto-4-
hydroxybutyrate. In some embodiments, the deaminase or transaminase is an
aminotransferase,
an L-amino acid oxidase or an L-amino acid dehydrogenase. Aminotransferases
include, but are
not limited to, a glutamate-oxaloacetate aminotransferase, a glutamate-
pyruvate
aminotransferase, an L-aspartate:2-oxoglutarate aminotransferase, an L-
alanine:2-oxoglutarate
aminotransferase. Amino acid sequences of some aminotransferases known in the
art are set out
in SEQ ID NOs: 2, 4, 6, 8, 10 and 12. Exemplary DNA sequences encoding those
aminotransferases are respectively set out in SEQ ID NOs: 1, 3, 5, 7, 9 and
11. Amino acid
sequences of some L-amino acid oxidases known in the art are set out in SEQ ID
NOs: 14 and 16.
Exemplary DNA sequences encoding those L-amino acid oxidases are respectively
set out in
SEQ ID NOs: 13 and 15. Amino acid sequences of some L-amino acid
dehydrogenases known in
the art are set out in SEQ ID NOs: 18 and 20. Exemplary DNA sequences encoding
those L-
amino acid dehydrogenases are set out in SEQ ID NOs: 17 and 19.
The dehydrogenase or decarboxylase catalyzes a reaction to convert 2-keto-4-
hydroxybutyrate to 3-hydroxypropionyl-CoA. In some embodiments, the
dehydrogenase is a 2-
keto acid dehydrogenase (or an alpha keto acid dehydrogenase). Dehydrogenases
include, but
are not limited to, a pyruvate dehydrogenase, a 2-keto-glutarate dehydrogenase
or a branched
chain keto acid dehydrogenase. A pyruvate dehydrogenase known in the art is
the pyruvate
dehydrogenase PDH, the amino acid sequences of the subunits of which are set
out in SEQ ID
NOs: 30, 32 and 34. Exemplary DNA sequences encoding those subunits are
respectively set out
in SEQ ID NOs: 29, 31 and 33. A 2-keto-glutarate dehydrogenase known in the
art similarly
comprises three subunits, the amino acid sequences of which are set out in SEQ
ID NOs: 36, 38
and 40. Exemplary DNA sequences encoding those subunits are respectively set
out in SEQ ID
NOs: 35, 37 and 39. A branched chain keto acid dehydrogenase known in the art
is the branched
chain keto acid dehydrogenase BI(D, the amino acid sequences of the subunits
of which are set
out in SEQ ID NOs: 22, 24, 26 and 28. Exemplary DNA sequences encoding those
subunits are
respectively set out in SEQ ID NOs: 21, 23, 25 and 27.
The acyl-CoA transferase or the acyl-CoA thioesterase catalyzes a reaction to
convert 3-
hydroxypropionyl-CoA to 3-hydroxypropionate. Contemplated thioesterases
include, but are not
limited to E. coli TesB set out in SEQ ID NO: 90, the C. propionicum-derived
thioesterase
including an E324D substitution set out in SEQ ID NO: 92 and the M. elsdenii-
derived
thioesterase including an E325D substitution set out in SEQ ID NO: 94.
Exemplary (codon-
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optimized for E. coli) DNA sequences encoding these thioesterases are
respectively set out in
SEQ ID NOs: 89, 91(codon-optimized for E. coli) and 93 (codon-optimized for E.
coli).
In a sixth aspect, the invention provides a third type of method, one for
producing 3-
hydroxypropionate in which the third type of microorganism is cultured to
produce 3-
hydroxypropionate . The third type of method converts homoserine to 2-keto-4-
hydroxybutyrate,
2-keto-4-hydroxybutyrate to 3-hydroxy-propionaldehyde,3-hydroxy-
propionaldehyde to 3-
hydroxypropionyl-CoA and then 3-hydroxypropionyl-CoA to 3-hydroxypropionate.
In a seventh aspect, the invention provides a fourth type of microorganism,
one that
converts homoserine to 3-hydroxypropionate, wherein the microorganism
expresses recombinant
genes encoding: a deaminase or transaminase, a decarboxylase and a
dehydrogenase.
The deaminase or transaminase catalyzes a reaction to convert homoserine to 2-
keto-4-
hydroxybutyrate. In some embodiments, the deaminase or transaminase is an
aminotransferase,
an L-amino acid oxidase or an L-amino acid dehydrogenase. Aminotransferases
include, but are
not limited to, a glutamate-oxaloacetate aminotransferase, a glutamate-
pyruvate
aminotransferase, an L-aspartate:2-oxoglutarate aminotransferase, an L-
alanine:2-oxoglutarate
aminotransferase. Amino acid sequences of some aminotransferases known in the
art are set out
in SEQ ID NOs: 2, 4, 6, 8, 10 and 12. Exemplary DNA sequences encoding those
aminotransferases are respectively set out in SEQ ID NOs: 1, 3, 5, 7, 9 and
11. Amino acid
sequences of some L-amino acid oxidases known in the art are set out in SEQ ID
NOs: 14 and 16.
Exemplary DNA sequences encoding those L-amino acid oxidases are respectively
set out in
SEQ ID NOs: 13 and 15. Amino acid sequences of some L-amino acid
dehydrogenases known in
the art are set out in SEQ ID NOs: 18 and 20. Exemplary DNA sequences encoding
those L-
amino acid dehydrogenases are set out in SEQ ID NOs: 17 and 19.
The decarboxylase catalyzes a reaction to convert 2-keto-4-hydroxybutyrate to
3-
hydroxy-propionaldehyde. In some embodiments the decarboxylase is a 2-keto
acid
decarboxylase. The 2-keto acid decarboxylases include, but are not limited to,
the 2-keto acid
decarboxylase KdcA set out in SEQ ID NO: 54 and its derivatives. An exemplary
DNA
sequence encoding KdcA is set out in SEQ ID NO: 53.
The dehydrogenase catalyzes a reaction to convert 3-hydroxy-propionaldehyde to
3-
hydroxypropionate. In some embodiments, the dehydrogenase is an aldehyde
dehydrogenase.
Amino acid sequences of aldehyde dehydrogenases known in the art are set out
in SEQ ID NOs:
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56 and 58. Exemplary DNA sequences encoding the aldehyde dehydrogenases are
respectively
set out in SEQ ID NOs: 55 and 57.
In an eighth aspect, the invention provides a fourth type of method, one for
producing 3-
hydroxypropionate in which the fourth type of microorganism is cultured to
produce 3-
hydroxypropionate . The fourth type of method converts homoserine to 2-keto-4-
hydroxybutyrate, 2-keto-4-hydroxybutyrate to 3-hydroxy-propionaldehyde, 3-
hydroxy-
propionaldehyde to 3-hydroxypropionate.
In a ninth aspect, the invention provides a fifth type of microorganism, one
that converts
homoserine to 3-hydroxypropionate, wherein the microorganism expresses
recombinant genes
encoding: a deaminase or transaminase, a decarboxylase, a dehydrogenase, and a
acyl-CoA
transferase or a thioesterase.
The deaminase or transaminase catalyzes a reaction to convert homoserine to 2-
keto-4-
hydroxybutyrate. In some embodiments, the deaminase or transaminase is an
aminotransferase,
an L-amino acid oxidase or an L-amino acid dehydrogenase. Aminotransferases
include, but are
not limited to, a glutamate-oxaloacetate aminotransferase, a glutamate-
pyruvate
aminotransferase, an L-aspartate:2-oxoglutarate aminotransferase, an L-
alanine:2-oxoglutarate
aminotransferase. Amino acid sequences of some aminotransferases known in the
art are set out
in SEQ ID NOs: 2, 4, 6, 8, 10 and 12. Exemplary DNA sequences encoding those
aminotransferases are respectively set out in SEQ ID NOs: 1, 3, 5, 7, 9 and
11. Amino acid
sequences of some L-amino acid oxidases known in the art are set out in SEQ ID
NOs: 14 and 16.
Exemplary DNA sequences encoding those L-amino acid oxidases are respectively
set out in
SEQ ID NOs: 13 and 15. Amino acid sequences of some L-amino acid
dehydrogenases known in
the art are set out in SEQ ID NOs: 18 and 20. Exemplary DNA sequences encoding
those L-
amino acid dehydrogenases are set out in SEQ ID NOs: 17 and 19.
The decarboxylase catalyzes a reaction to convert 2-keto-4-hydroxybutyrate to
3-
hydroxy-propionaldehyde. In some embodiments, the decarboxylase is a 2-keto
acid
decarboxylase. The 2-keto acid decarboxylases include, but are not limited to,
the 2-keto acid
decarboxylase KdcA set out in SEQ ID NO: 54 and its derivatives. An exemplary
DNA
sequence encoding KdcA is set out in SEQ ID NO: 53.
The dehydrogenase catalyzes a reaction to convert 3-hydroxy-propionaldehyde to
3-
hydroxypropionyl-CoA. In some embodiments, the dehydrogenase is a
propionaldehyde
dehydrogenase. Propionaldehyde dehydrogenases include, but are not limited to,
a PduP. Amino
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acid sequences of some PduP propionaldehyde dehydrogenases known in the art
are set out in
SEQ ID NOs: 60 and 62. Exemplary DNA sequences encoding the PduP
propionaldehyde
dehydrogenases are respectively set out in SEQ ID NOs: 59 and 61.
The 3-hydroxypropionyl-CoA transferase or thioesterase catalyzes a reaction to
convert
3-hydroxypropionyl-CoA to 3-hydroxypropionate. Contemplated thioesterases
include, but are
not limited to E. coli TesB set out in SEQ ID NO: 90, the C. propionicum-
derived thioesterase
including an E324D substitution set out in SEQ ID NO: 92 and the M. elsdenii-
derived
thioesterase including an E325D substitution set out in SEQ ID NO: 94.
Exemplary DNA
sequences encoding these acryloyl-CoA thioesterases are respectively set out
in SEQ ID NOs:
89, 91 (codon-optimized for E. coli) and 93 (codon-optimized for E. coli).
In a tenth aspect, the invention provides a fifth type of method, one for
producing 3-
hydroxypropionate in which the fifth type of microorganism is cultured to
produce 3-
hydroxypropionate. The fifth type of method for producing acrylate converts
homoserine to 2-
keto-4-hydroxybutyrate, 2-keto-4-hydroxybutyrate to 3-hydroxy-propionaldehyde,
3-hydroxy-
propionaldehyde to 3-hydroxypropionyl-CoA, and 3-hydroxy-propionyl-CoA to 3-
hydroxypropionate.
Producing poly-3-hydroxypropionate
In a eleventh aspect, the invention provides a sixth type of microorganism,
one that
converts homoserine to poly-3-hydroxypropionate, wherein the microorganism
expresses
recombinant genes encoding: a deaminase or transaminase, a dehydrogenase or
decarboxylase,
and a PHA synthase.
The deaminase or transaminase catalyzes a reaction to convert homoserine to 2-
keto-4-
hydroxybutyrate. In some embodiments, the deaminase or transaminase is an
aminotransferase,
an L-amino acid oxidase or an L-amino acid dehydrogenase. Aminotransferases
include, but are
not limited to, a glutamate-oxaloacetate aminotransferase, a glutamate-
pyruvate
aminotransferase, an L-aspartate:2-oxoglutarate aminotransferase, an L-
alanine:2-oxoglutarate
aminotransferase. Amino acid sequences of some aminotransferases known in the
art are set out
in SEQ ID NOs: 2, 4, 6, 8, 10 and 12. Exemplary DNA sequences encoding those
aminotransferases are respectively set out in SEQ ID NOs: 1, 3, 5, 7, 9 and
11. Amino acid
sequences of some L-amino acid oxidases known in the art are set out in SEQ ID
NOs: 14 and 16.
Exemplary DNA sequences encoding those L-amino acid oxidases are respectively
set out in
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SEQ ID NOs: 13 and 15. Amino acid sequences of some L-amino acid
dehydrogenases known in
the art are set out in SEQ ID NOs: 18 and 20. Exemplary DNA sequences encoding
those L-
amino acid dehydrogenases are set out in SEQ ID NOs: 17 and 19.
The dehydrogenase or decarboxylase catalyzes a reaction to convert 2-keto-4-
5 hydroxybutyrate to 3-hydroxypropionyl-CoA. In some embodiments, the
dehydrogenase is a 2-
keto acid dehydrogenase (or an alpha keto acid dehydrogenase). Dehydrogenases
include, but
are not limited to, a pyruvate dehydrogenase, a 2-keto-glutarate dehydrogenase
or a branched
chain keto acid dehydrogenase. A pyruvate dehydrogenase known in the art is
the pyruvate
dehydrogenase PDH, the amino acid sequences of the subunits of which are set
out in SEQ ID
10 NOs: 30, 32 and 34. Exemplary DNA sequences encoding those subunits are
respectively set out
in SEQ ID NOs: 29, 31 and 33. A 2-keto-glutarate dehydrogenase known in the
art similarly
comprises three subunits, the amino acid sequences of which are set out in SEQ
ID NOs: 36, 38
and 40. Exemplary DNA sequences encoding those subunits are respectively set
out in SEQ ID
NOs: 35, 37 and 39. A branched chain keto acid dehydrogenase known in the art
is the branched
chain keto acid dehydrogenase BI(D, the amino acid sequences of the subunits
of which are set
out in SEQ ID NOs: 22, 24, 26 and 28. Exemplary DNA sequences encoding those
subunits are
respectively set out in SEQ ID NOs: 21, 23, 25 and 27.
The PHA synthase catalyzes a reaction to convert 3-hydroxypropionyl-CoA to
poly-3-
hydroxyalkanoate containing 3-hydroxypropionate monomers. The polymer may have
a
molecule of Coenzyme A (CoA) at the carboxy end. The amino acid sequence of a
PHA
synthase known in the art is set out in SEQ ID NO: 42. An exemplary DNA
sequence encoding
the PHA synthase is set out in SEQ ID NO: 41.
In a twelfth aspect, the invention provides a sixth type of method, one for
producing poly-
3-hydroxypropionate in which the sixth type of microorganism is cultured to
produce poly-3-
hydroxypropionate . The sixth type of method converts homoserine to 2-keto-4-
hydroxybutyrate,
2-keto-4-hydroxybutyrate to 3-hydroxypropionyl-CoA and 3-hydroxypropionyl-CoA
to poly-3-
hydroxypropionate.
In thirteenth aspect, the invention provides a seventh type of microorganism,
one that
converts homoserine to poly-3-hydroxypropionate, wherein the microorganism
expresses
recombinant genes encoding: a deaminase or transaminase, a decarboxylase, a
dehydrogenase
and a PHA synthase.
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The deaminase or transaminase catalyzes a reaction to convert homoserine to 2-
keto-4-
hydroxybutyrate. In some embodiments, the deaminase or transaminase is an
aminotransferase,
an L-amino acid oxidase or an L-amino acid dehydrogenase. Aminotransferases
include, but are
not limited to, a glutamate-oxaloacetate aminotransferase, a glutamate-
pyruvate
.. aminotransferase, an L-aspartate:2-oxoglutarate aminotransferase, an L-
alanine:2-oxoglutarate
aminotransferase. Amino acid sequences of some aminotransferases known in the
art are set out
in SEQ ID NOs: 2, 4, 6, 8, 10 and 12. Exemplary DNA sequences encoding those
aminotransferases are respectively set out in SEQ ID NOs: 1, 3, 5, 7, 9 and
11. Amino acid
sequences of some L-amino acid oxidases known in the art are set out in SEQ ID
NOs: 14 and 16.
.. Exemplary DNA sequences encoding those L-amino acid oxidases are
respectively set out in
SEQ ID NOs: 13 and 15. Amino acid sequences of some L-amino acid
dehydrogenases known in
the art are set out in SEQ ID NOs: 18 and 20. Exemplary DNA sequences encoding
those L-
amino acid dehydrogenases are set out in SEQ ID NOs: 17 and 19.
The decarboxylase catalyzes a reaction to convert 2-keto-4-hydroxybutyrate to
3-
.. hydroxy-propionaldehyde. In some embodiments, the decarboxylase is a 2-keto
acid
decarboxylase. The 2-keto acid decarboxylases include, but are not limited to,
the 2-keto acid
decarboxylase KdcA set out in SEQ ID NO: 54 and its derivatives. An exemplary
DNA
sequence encoding KdcA is set out in SEQ ID NO: 53..
The dehydrogenase catalyzes a reaction to convert 3-hydroxy-propionaldehyde to
3-
.. hydroxypropionyl-CoA. In some embodiments, the dehydrogenase is a
propionaldehyde
dehydrogenase. Propionaldehyde dehydrogenases include, but are not limited to,
a PduP. Amino
acid sequences encoding of PduP propionaldehyde dehydrogenases known in the
art are set out in
SEQ ID NOs: 60 and 62. Exemplary DNA sequences encoding the PduP
propionaldehyde
dehydrogenases are respectively set out in SEQ ID NOs: 59 and 61.
The PHA synthase catalyzes a reaction to convert 3-hydroxypropionyl-CoA to
poly-3-
hydroxyalkanoate containing 3-hydroxypropionate monomers. The polymer may have
a
molecule of Coenzyme A (CoA) at the carboxy end. The amino acid sequence of a
PHA
synthase known in the art is set out in SEQ ID NO: 42. An exemplary DNA
sequence encoding
the PHA synthase is set out in SEQ ID NO: 41.
In a fourteenth aspect, the invention provides a seventh type of method, one
for producing
poly-3-hydroxypropionate in which the seventh type of microorganism is
cultured to produce
poly-3-hydroxypropionate . The seventh type of method converts homoserine to 2-
keto-4-
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hydroxybutyrate, 2-keto-4-hydroxybutyrate to 3-hydroxy-propionaldehyde, 3-
hydroxy-
propionaldehyde to 3-hydroxypropionyl-CoA and then 3-hydroxy-propionyl-CoA to
poly-3-
hydroxypropionate.
Producing 3-hydroxypropionyl-CoA
In a fifteenth aspect, the invention provides a eighth type of microorganism
that converts
homoserine to 3-hydroxypropionyl-CoA, wherein the microorganism expresses
recombinant
genes encoding: a deaminase or transaminase, and a dehydrogenase or
decarboxylase.
The deaminase or transaminase catalyzes a reaction to convert homoserine to 2-
keto-4-
hydroxybutyrate. In some embodiments, the deaminase or transaminase is an
aminotransferase,
an L-amino acid oxidase or an L-amino acid dehydrogenase. Aminotransferases
include, but are
not limited to, a glutamate-oxaloacetate aminotransferase, a glutamate-
pyruvate
aminotransferase, an L-aspartate:2-oxoglutarate aminotransferase, an L-
alanine:2-oxoglutarate
aminotransferase. Amino acid sequences of some aminotransferases known in the
art are set out
in SEQ ID NOs: 2, 4, 6, 8, 10 and 12. Exemplary DNA sequences encoding those
aminotransferases are respectively set out in SEQ ID NOs: 1, 3, 5, 7, 9 and
11. Amino acid
sequences of some L-amino acid oxidases known in the art are set out in SEQ ID
NOs: 14 and 16.
Exemplary DNA sequences encoding those L-amino acid oxidases are respectively
set out in
SEQ ID NOs: 13 and 15. Amino acid sequences of some L-amino acid
dehydrogenases known in
the art are set out in SEQ ID NOs: 18 and 20. Exemplary DNA sequences encoding
those L-
amino acid dehydrogenases are set out in SEQ ID NOs: 17 and 19.
The dehydrogenase or decarboxylase catalyzes a reaction to convert 2-keto-4-
hydroxybutyrate to 3-hydroxypropionyl-CoA. In some embodiments, the
dehydrogenase is a 2-
keto acid dehydrogenase (or an alpha keto acid dehydrogenase). Dehydrogenases
include, but
are not limited to, a pyruvate dehydrogenase, a 2-keto-glutarate dehydrogenase
or a branched
chain keto acid dehydrogenase. A pyruvate dehydrogenase known in the art is
the pyruvate
dehydrogenase PDH, the amino acid sequences of the subunits of which are set
out in SEQ ID
NOs: 30, 32 and 34. Exemplary DNA sequences encoding those subunits are
respectively set out
in SEQ ID NOs: 29, 31 and 33. A 2-keto-glutarate dehydrogenase known in the
art similarly
comprises three subunits, the amino acid sequences of which are set out in SEQ
ID NOs: 36, 38
and 40. Exemplary DNA sequences encoding those subunits are respectively set
out in SEQ ID
NOs: 35, 37 and 39. A branched chain keto acid dehydrogenase known in the art
is the branched
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chain keto acid dehydrogenase BKD, the amino acid sequences of the subunits of
which are set
out in SEQ ID NOs: 22, 24, 26 and 28. Exemplary DNA sequences encoding those
subunits are
respectively set out in SEQ ID NOs: 21, 23, 25 and 27.
In a sixteenth aspect, the invention provides an eighth type of method, one
for producing
3-hydroxypropionyl-CoA in which the eighth type of microorganism is cultured
to produce 3-
hydroxypropionyl-CoA. The eighth type of method converts homoserine to 2-keto-
4-
hydroxybutyrate and then converts 2-keto-4-hydroxybutyrate to 3-
hydroxypropionyl-CoA.
In a seventeenth aspect, the invention provides a ninth type of microorganism,
one that
converts homoserine to 3-hydroxypropionyl-CoA, wherein the microorganism
expresses
recombinant genes encoding: a deaminase or transaminase, a decarboxylase, and
a
dehydrogenase.
The deaminase or transaminase catalyzes a reaction to convert homoserine to 2-
keto-4-
hydroxybutyrate. In some embodiments, the deaminase or transaminase is an
aminotransferase,
an L-amino acid oxidase or an L-amino acid dehydrogenase. Aminotransferases
include, but are
not limited to, a glutamate-oxaloacetate aminotransferase, a glutamate-
pyruvate
aminotransferase, an L-aspartate:2-oxoglutarate aminotransferase, an L-
alanine:2-oxoglutarate
aminotransferase. Amino acid sequences of some aminotransferases known in the
art are set out
in SEQ ID NOs: 2, 4, 6, 8, 10 and 12. Exemplary DNA sequences encoding those
aminotransferases are respectively set out in SEQ ID NOs: 1, 3, 5, 7, 9 and
11. Amino acid
sequences of some L-amino acid oxidases known in the art are set out in SEQ ID
NOs: 14 and 16.
Exemplary DNA sequences encoding those L-amino acid oxidases are respectively
set out in
SEQ ID NOs: 13 and 15. Amino acid sequences of some L-amino acid
dehydrogenases known in
the art are set out in SEQ ID NOs: 18 and 20. Exemplary DNA sequences encoding
those L-
amino acid dehydrogenases are set out in SEQ ID NOs: 17 and 19.
The decarboxylase catalyzes a reaction to convert 2-keto-4-hydroxybutyrate to
3-
hydroxy-propionaldehyde. In some embodiments, the decarboxylase is a 2-keto
acid
decarboxylase. The 2-keto acid decarboxylases include, but are not limited to,
the 2-keto acid
decarboxylase KdcA set out in SEQ ID NO: 54 and its derivatives. An exemplary
DNA
sequence encoding KdcA is set out in SEQ ID NO: 53.
The dehydrogenase catalyzes a reaction to convert 3-hydroxy-propionaldehyde to
3-
hydroxypropionyl-CoA. In some embodiments, the dehydrogenase is a
propionaldehyde
dehydrogenase. Propionaldehyde dehydrogenases include, but are not limited to,
a PduP. Amino
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acid sequences encoding of PduP propionaldehyde dehydrogenases known in the
art are set out in
SEQ ID NOs: 60 and 62. Exemplary DNA sequences encoding the PduP
propionaldehyde
dehydrogenases are respectively set out in SEQ ID NOs: 59 and 61.
In an eighteenth aspect, the invention provides a ninth type of method, one
for producing
3-hydroxypropionyl-CoA in which the ninth type of microorganism is cultured to
produce 3-
hydroxypropionyl-CoA. The ninth type of method converts homoserine to 2-keto-4-
hydroxybutyrate, 2-keto-4-hydroxybutyrate to 3-hydroxy-propionaldehyde, and 3-
hydroxy-
propionaldehyde to 3-hydroxypropionyl-CoA.
Producing 1,3-propanediol
In a ninteenth aspect, the invention provides an tenth type of microorganism,
one that
converts homoserine to 1,3-propanediol, wherein the microorganism expresses
recombinant
genes encoding: a deaminase or transaminase, a decarboxylase and a 1,3-
propanediol
dehydrogenase or aldehyde reductase.
The deaminase or transaminase catalyzes a reaction to convert homoserine to 2-
keto-4-
hydroxybutyrate. In some embodiments, the deaminase or transaminase is an
aminotransferase,
an L-amino acid oxidase or an L-amino acid dehydrogenase. Aminotransferases
include, but are
not limited to, a glutamate-oxaloacetate aminotransferase, a glutamate-
pyruvate
aminotransferase, an L-aspartate:2-oxoglutarate aminotransferase, an L-
alanine:2-oxoglutarate
aminotransferase. Amino acid sequences of some aminotransferases known in the
art are set out
in SEQ ID NOs: 2, 4, 6, 8, 10 and 12. Exemplary DNA sequences encoding those
aminotransferases are respectively set out in SEQ ID NOs: 1, 3, 5, 7, 9 and
11. Amino acid
sequences of some L-amino acid oxidases known in the art are set out in SEQ ID
NOs: 14 and 16.
Exemplary DNA sequences encoding those L-amino acid oxidases are respectively
set out in
SEQ ID NOs: 13 and 15. Amino acid sequences of some L-amino acid
dehydrogenases known in
the art are set out in SEQ ID NOs: 18 and 20. Exemplary DNA sequences encoding
those L-
amino acid dehydrogenases are set out in SEQ ID NOs: 17 and 19.
The decarboxylase catalyzes a reaction to convert 2-keto-4-hydroxybutyrate to
3-
hydroxy-propionaldehyde. In some embodiments, the decarboxylase is a 2-keto
acid
decarboxylase. The 2-keto acid decarboxylases include, but are not limited to,
the 2-keto acid
decarboxylase KdcA set out in SEQ ID NO: 54 and its derivatives. An exemplary
DNA
sequence encoding KdcA is set out in SEQ ID NO: 53.
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The 1,3-propanediol dehydrogenase or aldehyde reductase catalyzes a reaction
to convert
3-hydroxypropionaldehyde to 1,3-propanediol. Amino acid sequences of some 1,3-
propanediol
dehydrogenases know in the art are set out in SEQ ID NOs: 64, 66, 68, 70 and
72. Exemplary
DNA sequence encoding the 1,3-propanediol dehydrogenases are respectively set
out in SEQ ID
5 NOs: 63, 65, 67, 69 and 71.
In a twentieth aspect, the invention provides an tenth type of method, one for
producing
1,3-propanediol in which the tenth type of microorganism is cultured to
produce 1,3-propanediol.
The tenth type of method converts homoserine to 2-keto-4-hydroxybutyrate, 2-
keto-4-
hydroxybutyrate to 3-hydroxy-propionaldehyde and then 3-hydroxy-
propionaldehyde to 1,3-
10 propanediol.
Increasing the carbon flow to homoserine
In a twenty-first aspect, the invention provides microorganisms that include
further
genetic modifications in order to increase the carbon flow to homoserine
which, in turn, increases
15 the production of acrylate or other products of the invention. The
microorganisms exhibit one or
more of the following characteristics.
In some embodiments, the microorganism exhibits increased carbon flow to
oxaloacetate
in comparison to a corresponding wild-type microorganism. The microorganism
expresses a
recombinant gene encoding, for example, phosphoenolpyruvate carboxylase or
pyruvate
carboxylase (or both). The phosphoenolpyruvate caroxylases include, but are
not limited to, the
phosphoenolpyruvate carboxylase set out in SEQ ID NO: 84. An exemplary DNA
sequence
encoding the phosphoenolpyruvate carboxylase is set out in SEQ ID NO: 83. The
pyruvate
carboxylases include, but are not limited to, the pyruvate carboxylases set
out in SEQ ID NOs: 86
and 88. Exemplary DNA sequences encoding the pyruvate carboxylases are set out
in SEQ ID
NO: 85 and 87.
In some embodiments, the microorganism exhibits reduced aspartate kinase
feedback
inhibition in comparison to a corresponding wild-type microorganism. The
microorganism
expresses one or more of the genes encoding the polypeptides including, but
not limited to,
5345F ThrA (SEQ ID NO: 76), T352I LysC (SEQ ID NO: 78) and MetL (SEQ ID NO:
74).
Exemplary coding sequences encoding the polypeptides are respectively set out
in SEQ ID NO:
75, SEQ ID NO: 77 and SEQ ID NO: 73.
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In some embodiments, the microorganism exhibits reduced lysA gene expression
or
diaminopimelate decarboxylase activity in comparison to a corresponding wild-
type
microorganism. In some embodiments, the microorganism exhibits reduced dapA
expression or
dihydropicolinate synthase activity in comparison to a corresponding wild type
organism. An
exemplary DNA sequence of a lysA coding sequence known in the art is set out
in SEQ ID NO:
113. It encodes the amino acid sequence set out in SEQ ID NO: 114. An
exemplary DNA
sequence of a dapA coding sequence known in the art is set out in SEQ ID NO:
115. It encodes
the amino acid sequence set out in SEQ ID NO: 116.
In some embodiments, the microorganism exhibits reduced metA gene expression
or
homoserine succinyltransferase activity in comparison to a corresponding wild-
type
microorganism. An exemplary DNA sequence of a metA coding sequence known in
the art is set
out in SEQ ID NO: 79. It encodes the amino acid sequence set out in SEQ ID NO:
80.
In some embodiments, the microorganism exhibits reduced thrB gene expression
or
homoserine kinase activity in comparison to a corresponding wild-type
microorganism. An
exemplary DNA sequence of a thrB coding sequence known in the art is set out
in SEQ ID NO:
81. It encodes the amino acid sequence set out in SEQ ID NO: 82.
In some embodiments, the microorganism does not express an eda gene. An
exemplary
DNA sequence of an eda coding sequence known in the art is set out in SEQ ID
NO: 43. It
encodes the amino acid sequence set out in SEQ ID NO: 44.
In an twenty-second aspect, the invention provides an methods of culturing the
further
modified microorganisms to produce products of the invention.
Thioesterases
In a twenty-third aspect, the invention provides a thioesterase that
hydrolyzes an
intermediate of a metabolic pathway described herein to produce a desired end
product. In this
regard, a microorganism of the invention expresses a recombinant gene
comprising a nucleic acid
sequence encoding a thioesterase with activity against Coenzyme A (CoA)
attached to a two-,
three- or four-carbon chain, such as a three- or four-carbon chain comprising
a double bond (e.g.,
a three- or four-carbon chain comprising a double bond between C2 and C3). In
some
embodiments, the thioesterase hydrolyzes acryloyl-CoA to form acrylic acid.
Alternatively (or in
addition), in some embodiments the thioesterase hydrolyzes crotonoyl-CoA to
form crotonic
acid.
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This aspect of the invention is predicated, at least in part, on the use of
thioesterases with
activity against substrates with short carbon chains (e.g., less than four
carbons in the main chain)
comprising double bonds. While thioesterases have been identified that
hydrolyze saturated
short carbon chains, it would not have been expected that the identified
thioesterases would act
upon an unsaturated carbon chain. Thioesterases would be expected to exhibit a
high degree of
substrate specificity with respect to short carbon chains to avoid hydrolysis
of acetyl-CoA, which
is critical to fatty synthesis. Unexpectedly, thioesterases that hydrolyze CoA
intermediates
attached to short, unsaturated carbon chains were identified and successfully
produced (or
overproduced) in host cells.
Exemplary thioesterases include, but are not limited to, TesB from E. coli and
homologs
thereof from different organisms. In this regard, the host cell optionally
comprises a
polynucleotide comprising a nucleic acid sequence encoding an amino acid
sequence at least
80% identical (e.g., 85%, 90%, 95%, 99%, or 100% identical) to the amino acid
sequence set
forth in SEQ ID NO: 90 (TesB), and encoding a polypeptide having thioesterase
activity ( i.e.,
the polypeptide hydrolyzes thioesters bonds). An exemplary DNA sequence
encoding the TesB
amino acid sequence is set out in SEQ ID NO: 89. The amino acid sequences of
other known
thioesterases are set out in SEQ ID NO: 96, 98, 100, 102, 104, 106 and 108.
Exemplary codon-
optimized (for E.coli) DNA sequences encoding the thioesterases are
respectively set out in SEQ
ID NOs: 95, 97, 99, 101, 103, 105 and 107.
Engineered thioesterases also are appropriate for use in the invention. For
example,
mutation(s) within the active site of a CoA transferase confers thioesterase
activity to the enzyme
while substantially reducing (if not eliminating) transferase activity. Use of
a thioesterase is, in
various aspects, superior to use of a CoA transferase by releasing energy
associated with the CoA
bond. The energy release drives the acrylic acid or crotonic acid pathway to
completion. An
exemplary method of modifying a CoA transferase to obtain thioesterase
activity comprises
substituting the amino acid serving as the catalytic carboxylate with an
alternate amino acid.
CoA transferases suitable for modification and use in the context of the
invention include, but are
not limited to, acetyl-CoA transferases, propionyl-CoA transferases, and
butyryl-CoA
transferases. In one aspect, the thioesterase of the invention comprises the
amino acid sequence
of a propionyl-CoA transferase wherein the catalytic glutamate residue is
replaced with an
alternate amino acid, such as aspartate. Exemplary propionyl-CoA transferases
suitable for
mutation include propionyl-CoA transferases from C. propionicum and M.
elsdenii. Glutamate
residue 324 and glutamate residue 325 are the catalytic carboxylates in C.
propionicum
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propionyl-CoA transferase and M. elsdenii propionyl-CoA transferase,
respectively. As the
catalytic carboxylate is conserved among CoA transferases, the catalytic amino
acid residue in
propionate CoA transferases from other sources is identified by sequence
alignment with, e.g.,
the amino acid sequence of C. propionicum propionyl-CoA transferase.
Similarly, the catalytic
amino acid residue in other CoA transferases (e.g., acetyl-CoA transferase or
butyryl-CoA
transferases) is identified by sequence alignment with, e.g., the amino acid
sequence of C.
propionicum propionyl-CoA transferase. C. propionicum propionyl-CoA
transferase is an
example of a sequence suitable for comparison with other CoA transferases; it
will be
appreciated that sequences of other CoA transferase sequences can be compared
to identify the
conserved glutamate catalytic residue for mutation. It will also be
appreciated that mutated CoA
transferase having thioesterase activity can be generated by altering the
nucleic acid sequence of
an existing CoA transferase-encoding polynucleotide, or by generating a new
polynucleotide
based on the coding sequence of a CoA transferase. Thus, in these embodiments,
the host cell of
the invention comprises a polynucleotide comprising a nucleic acid sequence
encoding an amino
acid sequence at least 80% identical (e.g., 85%, 90%, 95%, 99%, or 100%
identical) to the amino
acid sequence set forth in SEQ ID NO: 92 (C. propionicum-derived thioesterase
including an
E324D substitution) or SEQ ID NO: 94 (M. elsdenii-derived thioesterase
including an E325D
substitution) and encoding a polypeptide having thioesterase activity.
Exemplary codon-
optimized (for E. coli) DNA sequences encoding the two thioesterases are
respectively set out in
SEQ ID NOs: 91 and 93. Amino acid sequences of other engineered thioesterases
are set out in
SEQ ID NOs: 109, 110, 111 and 112.
Isolated Enzymes
In some embodiments, isolated enzymes can be used to catalyze one or more
steps
described in the aspects of the invention. Advantages may include higher
product yields, easier
product recovery from a more concentrated solution without cell related
impurities, a greater
range of possible reaction conditions the use of less expensive reactors.
BRIEF DESCRIPTION OF THE DRAWING
Figure 1 shows steps in the conversion of glucose to homoserine.
Figure 2 shows steps in methods of the invention for producing acrylate, 3-
hydroxypropionyl-CoA, 3-hydroxypropionate and poly-3-hydroxypropionate from
homoserine.
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Figure 3 shows steps in methods of the invention for producing acrylate, 3-
hydroxypropionate, 1,3-propanediol and 3-hydroxypropionyl-CoA from homoserine.
Figure 4 shows single ion monitoring (SIM) LC-MS chromatograms of 2-keto-4-
hydroxybutyrate and glutamate, after incubation of L-homoserine and
a¨ketoglutarate with
(reaction) or without (control) Pf AT aminotransferase.
Figure 5 show initial rates of deamination as a function of L-homoserine
concentration by
Pf AT aminotransferase.
Figure 6 shows the production of 3-hydroxypropionyl-CoA from L-homoserine
catalyzed
by D-amino acid oxidase and 2-ketoglutarate dehydrogenase or D-amino acid
oxidase, KdcA
decarboxylase, and PduP dehydrogenase.
Figure 7 shows HPLC chromatograms of samples of acryloyl-CoA after incubation
with
(top) or without (bottom) a dehydratase, evidencing the formation of 3-
hydroxypropionyl-CoA
only when the enzyme was present.
Figure 8 shows the production 3-hydroxypropionyl-CoA from acryloyl-CoA
catalyzed by
a dehydratase.
Figure 9 shows the consumption of 3-hydroxypropionyl-CoA after incubation with
PHA
synthase suggesting the formation of the poly(3-hydroxypropionate).
Figure 10 shows thioesterase activity against an acryloyl-CoA substrate.
Activity is
monitored by optical density (OD) at 412 nm.
Figure 11 shows thioesterase activity against an octanoyl-CoA substrate.
Activity is
monitored by optical density (OD) at 412 nm.
Figure 12 shows thioesterase activity against an acryloyl-CoA substrate.
Activity is
monitored by optical density (OD) at 412 nm.
Figure 13 shows thioesterase activity against an acryloyl-CoA substrate.
Activity is
monitored by optical density (OD) at 412 nm.
Figure 14 shows thioesterase activity against an octanoyl-CoA substrate.
Activity is
monitored by optical density (OD) at 412 nm.
Figure 15 shows thioesterase activity against an octanoyl-CoA substrate.
Activity is
monitored by optical density (OD) at 412 nm.
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DETAILED DESCRIPTION OF THE INVENTION
Definitions
The invention provides the products acrylic acid and acrylate. As is
understood in the
5 art, acrylate is the carboxylate anion (i.e., conjugate base) of acrylic
acid. The pH of the product
solution determines the relative amount of acrylate versus acrylic in a
preparation according to
the Henderson-Hasselbalch equation lpH=pKa + log(lA-NHAll, where pKa is
¨log(Ka). Ka is
the acid dissociation constant of acrylic acid. The pKa of acrylic acid in
water is about 4.35.
Thus, at or near neutral pH, acrylic acid will exist primarily as the
carboxylate anion. As used
10 herein, "acrylic acid" and "acrylate" are both meant to encompass the
other.
As used herein, "amplify," "amplified," or "amplification" refers to any
process or
protocol for copying a polynucleotide sequence into a larger number of
polynucleotide
molecules, e.g., by reverse transcription, polymerase chain reaction, and
ligase chain reaction.
As used herein, an "antisense sequence" refers to a sequence that specifically
15 hybridizes with a second polynucleotide sequence. For instance, an
antisense sequence is a DNA
sequence that is inverted relative to its normal orientation for
transcription. Antisense sequences
can express an RNA transcript that is complementary to a target mRNA molecule
expressed
within the host cell (e.g., it can hybridize to target mRNA molecule through
Watson-Crick base
pairing).
20 As used herein, "cDNA" refers to a DNA that is complementary or
identical to an
mRNA, in either single stranded or double stranded form.
As used herein, "complementary" refers to a polynucleotide that base pairs
with a
second polynucleotide. Put another way, "complementary" describes the
relationship between
two single-stranded nucleic acid sequences that anneal by base-pairing. For
example, a
polynucleotide having the sequence 5'-GTCCGA-3' is complementary to a
polynucleotide with
the sequence 5'-TCGGAC-3'.
As used herein, a "conservative substitution" refers to the substitution in a
polypeptide
of an amino acid with a functionally similar amino acid. Put another way, a
conservative
substitution involves replacement of an amino acid residue with an amino acid
residue having a
similar side chain. Families of amino acid residues having similar side chains
have been defined
within the art, and include amino acids with basic side chains (e.g., lysine,
arginine, and
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histidine), acidic side chains (e.g., aspartic acid and glutamic acid),
uncharged polar side chains
(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and
cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, and
tryptophan), beta-branched side chains (e.g., threonine, valine, and
isoleucine) and aromatic side
chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).
As used herein, a "corresponding wild-type microorganism" is the naturally-
occurring
microorganism that would be the same as the microorganism of the invention
except that the
naturally-occurring microorganism has not been genetically engineered to
express any
recombinant genes.
As used herein, "encoding" refers to the inherent property of nucleotides to
serve as
templates for synthesis of other polymers and macromolecules. Unless otherwise
specified, a
"nucleotide sequence encoding an amino acid sequence" includes all nucleotide
sequences that
are degenerate versions of each other and that encode the same amino acid
sequence.
As used herein, "endogenous" refers to polynucleotides, polypeptides, or other
compounds that are expressed naturally or originate within an organism or
cell. That is,
endogenous polynucleotides, polypeptides, or other compounds are not
exogenous. For instance,
an "endogenous" polynucleotide or peptide is present in the cell when the cell
was originally
isolated from nature.
As used herein, "expression vector" refers to a vector comprising a
recombinant
polynucleotide comprising expression control sequences operatively linked to a
nucleotide
sequence to be expressed. For example, suitable expression vectors can be an
autonomously
replicating plasmid or integrated into the chromosome.
As used herein, "exogenous" refers to any polynucleotide or polypeptide that
is not
naturally found or expressed in the particular cell or organism where
expression is desired.
Exogenous polynucleotides, polypeptides, or other compounds are not
endogenous.
As used herein "homoserine" includes enantiomers such as L-homoserine and D-
homoserine.
As used herein, "hybridization" includes any process by which a strand of a
nucleic
acid joins with a complementary nucleic acid strand through base-pairing.
Thus, the term refers
to the ability of the complement of the target sequence to bind to a test
(i.e., target) sequence, or
vice-versa.
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As used herein, "hybridization conditions" are typically classified by degree
of
"stringency" of the conditions under which hybridization is measured. The
degree of stringency
can be based, for example, on the melting temperature (Tm) of the nucleic acid
binding complex
or probe. For example, "maximum stringency" typically occurs at about Tm -5
C. (5 below the
Tm of the probe); "high stringency" at about 5-10 below the Tm; "intermediate
stringency" at
about 10-20 below the Tm of the probe; and "low stringency" at about 20-25
below the Tm.
Alternatively, or in addition, hybridization conditions can be based upon the
salt or ionic strength
conditions of hybridization and/or one or more stringency washes. For example,
6x8SC=very
low stringency; 3x8SC=low to medium stringency; lx8SC=medium stringency; and
0.5x8SC=high stringency. Functionally, maximum stringency conditions may be
used to identify
nucleic acid sequences having strict (i. e., about 100%) identity or near-
strict identity with the
hybridization probe; while high stringency conditions are used to identify
nucleic acid sequences
having about 80% or more sequence identity with the probe.
As used herein, "identical" or percent "identity," in the context of two or
more
polynucleotide or polypeptide sequences, refers to two or more sequences that
are the same or
have a specified percentage of nucleotides or amino acid residues that are the
same, when
compared and aligned for maximum correspondence, as measured using sequence
comparison
algorithms or by visual inspection.
"Microorganisms" of the invention expressing recombinant genes are not
naturally-
occurring. In other words, the microorganisms are man-made and have been
genetically
engineered to express recombinant genes. The microorganisms of the invention
have been
genetically engineered to express the recombinant genes encoding the enzymes
necessary to
carry out the conversion of homoserine to the desired product. Microorganisms
of the invention
are bacteria, yeast, fungi or algae. Bacteria include, but not limited to, E.
coli strains K, B or C.
Microorganisms that are more resistant to toxicity of the products of the
invention are preferred.
Plant cells that are not naturally-occurring (are man-made) and have been
genetically engineered
to express recombinant genes carrying out the conversions detailed herein are
contemplated by
the invention to be alternative cells to microorganisms, for example in the
production of poly-3-
hydroxypropionate.
As used herein, "naturally-occurring" refers to an object that can be found in
nature.
For example, a polypeptide or polynucleotide sequence that is present in an
organism (including
viruses) that can be isolated from a source in nature and which has not been
intentionally
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23
modified by man in the laboratory is naturally-occurring. As used herein,
"naturally-occurring"
and "wild-type" are synonyms.
As used herein, "operably linked," when describing the relationship between
two DNA
regions or two polypeptide regions, means that the regions are functionally
related to each other.
For example, a promoter is operably linked to a coding sequence if it controls
the transcription of
the sequence; a ribosome binding site is operably linked to a coding sequence
if it is positioned
so as to permit translation; and a sequence is operably linked to a peptide if
it functions as a
signal sequence, such as by participating in the secretion of the mature form
of the protein.
As used herein, a recombinant gene that is "over-expressed" produces more RNA
and/or protein than a corresponding naturally-occurring gene in the
microorganism. Methods of
measuring amounts of RNA and protein are known in the art. Over-expression can
also be
determined by measuring protein activity such as enzyme activity. Depending on
the
embodiment of the invention, "over-expression" is an amount at least 3%, at
least 5%, at least
10%, at least 20%, at least 25%, or at least 50% more. An over-expressed
polynucleotide is
generally a polynucleotide native to the host cell, the product of which is
generated in a greater
amount than that normally found in the host cell. Over-expression is achieved
by, for instance
and without limitation, operably linking the polynucleotide to a different
promoter than the
polynucleotide's native promoter or introducing additional copies of the
polynucleotide into the
host cell.
As used herein, "polynucleotide" refers to a polymer composed of nucleotides.
The
polynucleotide may be in the form of a separate fragment or as a component of
a larger
nucleotide sequence construct, which has been derived from a nucleotide
sequence isolated at
least once in a quantity or concentration enabling identification,
manipulation, and recovery of
the sequence and its component nucleotide sequences by standard molecular
biology methods,
for example, using a cloning vector. When a nucleotide sequence is represented
by a DNA
sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U,
G, C) in which "U"
replaces T. Put another way, "polynucleotide" refers to a polymer of
nucleotides removed from
other nucleotides (a separate fragment or entity) or can be a component or
element of a larger
nucleotide construct, such as an expression vector or a polycistronic
sequence. Polynucleotides
include DNA, RNA and cDNA sequences.
As used herein, "polypeptide" refers to a polymer composed of amino acid
residues
which may or may not contain modifications such as phosphates and formyl
groups.
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As used herein, "primer" refers to a polynucleotide that is capable of
specifically
hybridizing to a designated polynucleotide template and providing a point of
initiation for
synthesis of a complementary polynucleotide when the polynucleotide primer is
placed under
conditions in which synthesis is induced.
As used herein, "recombinant polynucleotide" refers to a polynucleotide having
sequences that are not joined together in nature. A recombinant polynucleotide
may be included
in a suitable vector, and the vector can be used to transform a suitable host
cell. A host cell that
comprises the recombinant polynucleotide is referred to as a "recombinant host
cell." The
polynucleotide is then expressed in the recombinant host cell to produce,
e.g., a "recombinant
polypeptide."
As used herein, "recombinant expression vector" refers to a DNA construct used
to
express a polynucleotide that, e.g., encodes a desired polypeptide. A
recombinant expression
vector can include, for example, a transcriptional subunit comprising (i) an
assembly of genetic
elements having a regulatory role in gene expression, for example, promoters
and enhancers, (ii)
a structural or coding sequence which is transcribed into mRNA and translated
into protein, and
(iii) appropriate transcription and translation initiation and termination
sequences. Recombinant
expression vectors are constructed in any suitable manner. The nature of the
vector is not critical,
and any vector may be used, including plasmid, virus, bacteriophage, and
transposon. Possible
vectors for use in the invention include, but are not limited to, chromosomal,
nonchromosomal
and synthetic DNA sequences, e.g., bacterial plasmids; phage DNA; yeast
plasmids; and vectors
derived from combinations of plasmids and phage DNA, DNA from viruses such as
vaccinia,
adenovirus, fowl pox, baculovirus, SV40, and pseudorabies.
As used herein, a "recombinant gene" is not a naturally-occurring gene. A
recombinant gene is man-made. A recombinant gene includes a protein coding
sequence
operably linked to expression control sequences. Embodiments include, but are
not limited to, an
exogenous gene introduced into a microorganism, an endogenous protein coding
sequence
operably linked to a heterologous promoter (i.e., a promoter not naturally
linked to the protein
coding sequence) and a gene with a modified protein coding sequence (e.g., a
protein coding
sequence encoding an amino acid change or a protein coding sequence optimized
for expression
in the microorganism). The recombinant gene is maintained in the genome of the
microorganism, on a plasmid in the microorganism or on a phage in the
microorganism.
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As used herein, "reduced" expression is expression of less RNA or protein than
the
corresponding natural level of expression. Methods of measuring amounts of RNA
and protein
are known in the art. Reduced expression can also be determined by measuring
protein activity
such as enzyme activity. Depending on the embodiment of the invention,
"reduced" is an
5 amount at least 3%, at least 5%, at least 10%, at least 20%, at least
25%, or at least 50% less.
As used herein, "specific hybridization" refers to the binding, duplexing, or
hybridizing of a polynucleotide preferentially to a particular nucleotide
sequence under stringent
conditions.
As used herein, "stringent conditions" refers to conditions under which a
probe will
10 hybridize preferentially to its target subsequence, and to a lesser
extent to, or not at all to, other
sequences.
As used herein, "substantially homologous" or "substantially identical" in the
context
of two nucleic acids or polypeptides, generally refers to two or more
sequences or subsequences
that have at least 40%, 60%, 80%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide or
amino acid
15 residue identity, when compared and aligned for maximum correspondence,
as measured using
sequence comparison algorithms or by visual inspection. The substantial
identity can exist over
any suitable region of the sequences, such as, for example, a region that is
at least about 50
residues in length, a region that is at least about 100 residues, or a region
that is at least about 150
residues. In certain embodiments, the sequences are substantially identical
over the entire length
20 of either or both comparison biopolymers.
Polynucleotides
The polynucleotide(s) encoding one or more enzyme activities for steps in the
pathways of the invention may be derived from any source. Depending on the
embodiment of
25 the invention, the polynucleotide is isolated from a natural source such
as bacteria, algae, fungi,
plants, or animals; produced via a semi-synthetic route (e.g., the nucleic
acid sequence of a
polynucleotide is codon optimized for expression in a particular host cell,
such as E. coli); or
synthesized de novo. In certain embodiments, it is advantageous to select an
enzyme from a
particular source based on, e.g., the substrate specificity of the enzyme or
the level of enzyme
activity in a given host cell. In some embodiments of the invention, the
enzyme and
corresponding polynucleotide are naturally found in the host cell and over-
expression of the
polynucleotide is desired. In this regard, in some embodiments, additional
copies of the
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polynucleotide are introduced in the host cell to increase the amount of
enzyme. In some
embodiments, over-expression of an endogenous polynucleotide may be achieved
by
upregulating endogenous promoter activity, or operably linking the
polynucleotide to a more
robust heterologous promoter.
Exogenous enzymes and their corresponding polynucleotides also are suitable
for use
in the context of the invention, and the features of the biosynthesis pathway
or end product can
be tailored depending on the particular enzyme used.
The invention contemplates that polynucleotides of the invention may be
engineered to
include alternative degenerate codons to optimize expression of the
polynucleotide in a particular
microorganism. For example, a polynucleotide may be engineered to include
codons preferred in
E. coli if the DNA sequence will be expressed in E. coli. Methods for codon-
optimization are
known in the art.
Enzyme variants
In certain embodiments, the microorganism produces an analog or variant of the
polypeptide encoding an enzyme activity. Amino acid sequence variants of the
polypeptide
include substitution, insertion, or deletion variants, and variants may be
substantially homologous
or substantially identical to the unmodified polypeptides. In certain
embodiments, the variants
retain at least some of the biological activity, e.g., catalytic activity, of
the polypeptide. Other
variants include variants of the polypeptide that retain at least about 50%,
preferably at least
about 75%, more preferably at least about 90%, of the biological activity.
Substitutional variants typically exchange one amino acid for another at one
or more
sites within the protein. Substitutions of this kind can be conservative, that
is, one amino acid is
replaced with one of similar shape and charge. Conservative substitutions
include, for example,
the changes of: alanine to serine; arginine to lysine; asparagine to
glutamine; aspartate to
glutamate; cysteine to serine; glutamine to asparagine; glutamate to
aspartate; isoleucine to
leucine or valine; leucine to valine or isoleucine; lysine to arginine;
methionine to leucine or
isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to
threonine; threonine to
serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and
valine to isoleucine
or leucine. An example of the nomenclature used herein to indicate a amino
acid substitution is
"S345F ThrA" wherein the naturally occurring serine occurring at position 345
of the naturally
occurring ThrA enzyme which has been substituted with a phenylalanine.
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In some instances, the microorganism comprises an analog or variant of the
exogenous
or over-expressed polynucleotide(s) described herein. Nucleic acid sequence
variants include
one or more substitutions, insertions, or deletions, and variants may be
substantially homologous
or substantially identical to the unmodified polynucleotide. Polynucleotide
variants or analogs
encode mutant enzymes having at least partial activity of the unmodified
enzyme. Alternatively,
polynucleotide variants or analogs encode the same amino acid sequence as the
unmodified
polynucleotide. Codon optimized sequences, for example, generally encode the
same amino acid
sequence as the parent/native sequence but contain codons that are
preferentially expressed in a
particular host organism.
A polypeptide or polynucleotide "derived from" an organism contains one or
more
modifications to the naturally-occurring amino acid sequence or nucleotide
sequence and exhibits
similar, if not better, activity compared to the native enzyme (e.g., at least
70%, at least 80%, at
least 90%, at least 95%, at least 100%, or at least 110% the level of activity
of the native
enzyme). For example, enzyme activity is improved in some contexts by directed
evolution of a
parent/naturally-occurring sequence. Additionally or alternatively, an enzyme
coding sequence
is mutated to achieve feedback resistance.
Expression vectors/Transfer into microorganisms
Expression vectors for recombinant genes can be produced in any suitable
manner to
establish expression of the genes in a microorganism. Expression vectors
include, but are not
limited to, plasmids and phage. The expression vector can include the
exogenous polynucleotide
operably linked to expression elements, such as, for example, promoters,
enhancers, ribosome
binding sites, operators and activating sequences. Such expression elements
may be regulatable,
for example, inducible (via the addition of an inducer). Alternatively or in
addition, the
expression vector can include additional copies of a polynucleotide encoding a
native gene
product operably linked to expression elements. Representative examples of
useful heterologous
promoters include, but are not limited to: the LTR (long terminal 35 repeat
from a retrovirus) or
SV40 promoter, the E. coli lac, tet, or trp promoter, the phage Lambda PL
promoter, and other
promoters known to control expression of genes in prokaryotic or eukaryotic
cells or their
viruses. In one aspect, the expression vector also includes appropriate
sequences for amplifying
expression. The expression vector can comprise elements to facilitate
incorporation of
polynucleotides into the cellular genome.
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Introduction of the expression vector or other polynucleotides into cells can
be
performed using any suitable method, such as, for example, transformation,
electroporation,
microinjection, microprojectile bombardment, calcium phosphate precipitation,
modified calcium
phosphate precipitation, cationic lipid treatment, photoporation, fusion
methodologies, receptor
mediated transfer, or polybrene precipitation. Alternatively, the expression
vector or other
polynucleotides can be introduced by infection with a viral vector, by
conjugation, by
transduction, or by other suitable methods.
Culture
Microorganisms of the invention comprising recombinant genes are cultured
under
conditions appropriate for growth of the cells and expression of the gene(s).
Microorganisms
expressing the polypeptide(s) can be identified by any suitable methods, such
as, for example, by
PCR screening, screening by Southern blot analysis, or screening for the
expression of the
protein. In some embodiments, microorganisms that contain the polynucleotide
can be selected
by including a selectable marker in the DNA construct, with subsequent
culturing of
microorganisms containing a selectable marker gene, under conditions
appropriate for survival of
only those cells that express the selectable marker gene. The introduced DNA
construct can be
further amplified by culturing genetically modified microorganisms under
appropriate conditions
(e.g., culturing genetically modified microorganisms containing an amplifiable
marker gene in
the presence of a concentration of a drug at which only microorganisms
containing multiple
copies of the amplifiable marker gene can survive).
In some embodiments, the microorganisms (such as genetically modified
bacterial
cells) have an optimal temperature for growth, such as, for example, a lower
temperature than
normally encountered for growth and/or fermentation. In addition, in certain
embodiments, cells
of the invention exhibit a decline in growth at higher temperatures as
compared to normal growth
and/or fermentation temperatures as typically found in cells of the type.
Any cell culture condition appropriate for growing a microorganism and
synthesizing
a product of interest is suitable for use in the inventive method.
Recovery
The methods of the invention optionally comprise a step of product recovery.
Recovery of acrylate, 3-hydroxypropionyl-CoA, 3-hydroxypropionate, poly-3-
hydroxypropionate
or 1,3-propanediol can be carried out by methods known in the art. For
example, acrylate can be
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recovered by distillation methods, extraction methods, crystallization
methods, or combinations
thereof; 3-hydroxypropionate can be recovered as described in U.S. Published
Patent Application
No. 2011/038364 or International Publication No. WO 2011/0125118;
polyhydroxyalkanoates
can be recovered as described in Yu and Chen, Biotechnol Prog, 22(2): 547-553
(2006); and 1,3
propanediol can be recovered as described in U.S. Patent No. 6428992 or Cho et
al., Process
Biotechnology, 41(3): 739-744 (2006).
Examples
The following examples further describe and demonstrate embodiments within the
scope
of the present invention. The examples are given solely for the purpose of
illustration and are not
to be construed as limiting the present invention. Examples 1 to 6 describe
the construction of
different plasmids for heterologous expression of proteins in E. coli;
Examples 7 and 8 describe
the transformation and culture of E. coli strains; Examples 9 and 10 describe
the purification of
several proteins; Example 12 describes a method for quantification of acyl-CoA
molecules;
Examples 11 and 13 to 16 describe the in vitro reconstitution of the enzymatic
activity of several
proteins described in the present invention; Example 17 describe the
production of 3-
hydroxypropionic acid in engineered E. coli.
Example 1
Expression vectors for aminotransferase genes
E. coli expression vectors were constructed for production of recombinant
aminotransferases. A common cloning strategy was established utilizing the
pET30a vector
(Novagen, EMD Chemicals, Gibbstown, NJ, catalog #69909-30) for expression of
proteins
linked to an N-terminal hexahistidine tag under the T7 promoter. Modifications
to the pET30a
vector were made by replacing the DNA sequence between the SphI and XhoI sites
with a
synthesized DNA sequence (SEQ ID NO: 117) (GenScript, Piscataway, NJ). In this
resulting
vector, designated pET30a-BB, the XbaI site in the lac operator was removed
and the region
encoding for the thrombin, S-tag and enterokinase sites was replaced for a
sequence encoding for
a Factor Xa recognition site. Furthermore, the multiple cloning site was
modified to include
EcoRV, EcoRI, BamHI, Sad, and PstI sites.
Several aminotransferase genes were codon-optimized for expression in E. coli.
To
facilitate cloning, the common restriction sites: AvrII; BamHI; BglII; BstBI;
EagI; EcoRI;
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EcoRV; HindIII; KpnI; NcoI; NheI; NotI; NspV; PstI; PvuII; Sad; Sall; SapI;
SfuI; SpeI; XbaI;
XhoI were also removed from the gene sequences. In addition, the 5' prefix
sequence (SEQ ID
NO: 118) was added immediately upstream of the start codon and a SpeI, Nod and
PstI
restriction site 3' suffix sequence (SEQ ID NO: 119) was added immediately
downstream of the
5 stop
codon. The optimized sequences were synthesized (GenScript, Piscataway, NJ)
and cloned
into the pET30a-BB vector at the KpnI and PstI sites. The resulting plasmids
and the encoded
proteins are described in Table 1.
Table 1. List of plasmids encoding for different aminotransferases
Accession#
Enzym
Plasmid Species and Protein (DNA SEQ ID NO:)
(Amino Acid
e Key
SEQ ID NO: )
Pseudomonas fluorescens branched-chain
YP_002873519.1
pET30a-BB Pf AT Pf AT amino acid aminotransferase (SEQ ID NO:
(SEQ ID NO: 8)
122)
E. coli valine-pyruvate aminotransferase (SEQ NP_416793.1
pET30a-BB Ec AT Ec AT
ID NO: 123) (SEQ ID NO: 9)
Rattus norvegicus Alanine aminotransferase
BAA01185.1
pET30a-BB Rn AT Rn AT
(SEQ ID NO: 121) (SEQ ID NO: 4)
Sus scrofa aspartate aminotransferase, NP
999092.1
pET30a-BB Ss AT Ss AT
cytoplasmic (SEQ ID NO: 120) (SEQ ID NO: 2)
Example 2
Expression vector for branched-chain 2-keto acid decarboxylase (KdcA)
An E. coli expression vector was constructed for production of a recombinant
branched-
chain 2-keto acid decarboxylase (KdcA). A Lactococcus lactis branched-chain 2-
keto acid
decarboxylase gene was codon-optimized for expression in E. coli, and the
common restriction
sites: AvrII; BamHI; BglII; BstBI; EagI; EcoRI; EcoRV; HindIII; KpnI; NcoI;
NheI; Nod; NspV;
PstI; PvuII; Sad; Sall; SapI; SfuI; SpeI; XbaI; XhoI were removed to
facilitate cloning.
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Furthermore, additional EcoRI, Nod, XbaI restriction sites and a ribosomal
binding site (RBS) 5'
to the ATG start codon, and SpeI, NotI and PstI restriction sites 3' to the
stop codon were
included into the sequence. The optimized sequence (SEQ ID NO: 124) was
synthesized
(GenScript, Piscataway, NJ) and cloned into the pET30a-BB vector at the EcoRI
and PstI sites.
The resulting expression vector encoding N-terminal histidine tagged KdcA (SEQ
ID NO: 54)
was designated pET30a-BB Ll KDCA.
Example 3
Expression vector for coenzyme-A acylating propionaldehyde dehydrogenase
(PduP)
An E. coli expression vector was constructed for production of a recombinant
coenzyme-
A acylating propionaldehyde dehydrogenase (PduP). A Salmonella enterica
coenzyme-A
acylating propionaldehyde dehydrogenase gene was codon-optimized for
expression in E. coli,
and the common restriction sites: AvrII; BamHI; BglII; BstBI; EagI; EcoRI;
EcoRV; HindIII;
KpnI; NcoI; NheI; Nod; NspV; PstI; PvuII; Sad; Sall; SapI; SfuI; SpeI; XbaI;
XhoI were
removed to facilitate cloning. Furthermore, additional EcoRI, Nod, XbaI
restriction sites and a
ribosomal binding site (RBS) 5' to the ATG start codon, and SpeI, Nod and PstI
restriction sites
3' to the stop codon were included into the sequence. The optimized sequence
(SEQ ID NO:
125) was synthesized (GenScript, Piscataway, NJ) and cloned into the pET30a-BB
vector at the
EcoRI and PstI sites. The resulting expression vector, designated pET30a-BB Se
PDUP, encodes
N-terminal histidine tagged version of PduP (SEQ ID NO: 60).
Example 4
Expression vector for poly(3-hydroxybutyrate) polymerase (PhaC or PHA
synthase)
An E. coli expression vector was constructed for production of a recombinant
poly(3-
hydroxybutyrate) polymerase. A Cupriavidus necator poly (3-hydroxybutyrate)
polymerase
(phaC) gene was codon-optimized for expression in E. coli, and the common
restriction sites:
AvrII; BamHI; BglII; BstBI; EagI; EcoRI; EcoRV; HindIII; KpnI; NcoI; NheI;
Nod; NspV; PstI;
Pvull; Sad; Sall; SapI; SfuI; SpeI; XbaI; XhoI were removed to facilitate
cloning. Furthermore,
additional EcoRI, Nod, XbaI restriction sites and a ribosomal binding site
(RBS) 5' to the ATG
start codon, and SpeI, NotI and PstI restriction sites 3' to the stop codon
were included into the
sequence. The optimized sequence (SEQ ID NO: 126) was synthesized (GenScript,
Piscataway,
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NJ) and cloned into the pET30a-BB vector at the EcoRI and PstI sites. The
resulting expression
vector, designated pET30a-BB Cn PHAS, encodes N-terminal histidine tagged
version of PHA
synthase (SEQ ID NO: 42).
Example 5
Expression vector for 3-hydroxypropionyl-CoA dehydratase
An E. coli expression vector was constructed for production of a recombinant 3-
hydroxypropionyl-CoA dehydratase. A Metallosphaera sedula 3-hydroxypropionyl-
CoA
dehydratase gene was codon-optimized for expression in E. coli, and the common
restriction
sites: AvrII; BamHI; BglII; BstBI; EagI; EcoRI; EcoRV; HindIII; KpnI; NcoI;
NheI; Nod; NspV;
PstI; PvuII; Sad; Sall; SapI; SfuI; SpeI; XbaI; XhoI were removed to
facilitate cloning.
Furthermore, additional EcoRI, Nod, XbaI restriction sites and a ribosomal
binding site (RBS) 5'
to the ATG start codon, and SpeI, Nod and PstI restriction sites 3' to the
stop codon were
included into the sequence. The optimized sequence (SEQ ID NO: 127) was
synthesized
(GenScript, Piscataway, NJ) and cloned into the pET30a-BB vector at the EcoRI
and PstI sites.
The resulting expression vector, designated pET30a-BB Ms 3HP-CD, encodes N-
terminal
histidine tagged version of the dehydratase (SEQ ID NO: 48).
Example 6
Expression vectors for acyl-CoA thioesterase
E. coli expression vectors were constructed for production of recombinant
short to
medium-chain acyl-CoA thioesterases. Thioesterase genes from different
organisms were codon-
optimized for expression in E. coli, and the common restriction sites: BamHI,
BglII, BstBI,
EcoRI, HindIII, KpnI, PstI, NcoI, Nod, Sad, Sall, XbaI, and XhoI were removed
to facilitate
cloning. Furthermore, additional BamHI and XbaI restriction sites 5' to the
ATG start codon, and
Sad and HindIII restriction sites 3' to the stop codon were included into the
sequence. The
optimized sequences were synthesized (GenScript, Piscataway, NJ or GeneArt,
Invitrogen,
Carlsbad, CA) and cloned into the pET30a vector at the BamHI and Sad sites.
The resulting
plasmids and the encoded proteins are described in Table 2.
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Table 2. List of plasmids encoding for different thioesterases
Plasmid Enzyme Key Species / Protein
Accession #
pET30a-Sc Acot8 ScACOT8 Saccharomyces cerevisiae NP_012553
peroxisomal acyl-CoA thioesterase
(SEQ ID NO: 96)
pET30a-Mus Acot8 MusACOT8 Mus muscu/us acyl-CoA thioesterase AAL35333
8 (SEQ ID NO: 98)
pET30a-Rn Acot12 RnACOT12 R. norvegicus acyl-CoA thioesterase NP_570103
12 (SEQ ID NO: 100)
pET30a-Ec TesB EcTesB E. coli acyl-CoA thioesterase II NP_286194
(TesB) (SEQ ID NO: 90)
pET30a-Bs SrfD BsSrfD Bacillus subtilis surfactin synthetase NP_388234
(SrfAD) (SEQ ID NO: 102)
pET30a-Cp T CpT C. propionicum propionate CoA- CAB77207
transferase
pET30a-Cp TT CpTT C. propionicum propionate CoA- Similar to
transferase (with E324D mutation) CAB77207
(SEQ ID NO: 92)
pET30a-Me T MeT M. elsdenii coenzyme A-transferase Similar to
CCC72964
except for
T271A and
K517R
pET30a-Me TT MeTT M. elsdenii coenzyme A-transferase Similar to
(with E325D mutation) (SEQ ID CCC72964
NO: 94) except for
T271A,
K517R, and
E325D
pET30a-Hi YbgC HiYbgC Haemophilus influenzae thioesterase YP_248101
(YbgC) (SEQ ID NO: 108)
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Example7
Transformation of E. coli
The recombinant plasmids were then used to transform chemically competent One
Shot
BL21 (DE3) pLysS E. coli cells (Invitrogen, Carlsbad, CA). Individual vials of
cells were
thawed on ice and gently mixed with 10 ng of plasmid DNA. The vials were
incubated on ice for
30 min. The vials were briefly incubated at 42 C for 30 sec and quickly
replaced back on ice for
an additional 2 min. An aliquot of 250 L of 37 C SOC medium was added and the
vials were
secured horizontally on a shaking incubator platform and incubated for 1 h at
37 C, 225 rpm.
Aliquots of 20 L and 200 L cells were plated onto LB agar plates
supplemented with the
appropriate antiobiotics (50 ng/mL kanamycin; 34 ng/mL chloramphenicol) to
select for cells
carrying the recombinant and pLysS plasmids respectively, followed by
incubation overnight at
37 C. Single colony isolates were isolated, cultured in 5 mL of selective LB
broth and
recombinant plasmids were isolated using a QIAPrep Spin Miniprep kit (Qiagen,
Valencia,
CA) spin plasmid miniprep kit. Plasmid DNAs were characterized by gel
electrophoresis of
restriction digests with MM.
Example 8
Culture of E. coli strains and expression of recombinant proteins
Aliquots of LB broth (15 mL), supplemented with the appropriate antibiotics
(34 p g/mL
chloramphenicol; 50 p g/mL kanamycin) were inoculated with different E. coli
strains from
frozen glycerol stocks. Cultures were incubated overnight at 25 C with 250 rpm
shaking. LB
broth (150 ml, containing 34 p g/mL chloramphenicol, 50 p g/mL kanamycin;
equilibrated to 25
C) in 1 to 2.8 L fluted Erlenmeyer flasks was inoculated from the overnight
cultures at an optical
density (OD) at 600 nm of ¨0.1. Cultures were continued at 25 C with 250 rpm
shaking and
optical density was monitored until A600 of ¨0.4. Production of recombinant
proteins was
induced by addition of 1M IPTG (Teknova, Hollister, CA; 1 mM final
concentration). Cultures
were further incubated for 24 h at 25 C with 250 rpm shaking before harvesting
by
centrifugation. The cell pellets were stored at -80 C until used.
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Example 9
Recombinant protein isolation
His-tagged recombinant proteins were isolated by immobilized metal affinity
chromatography (IMAC) utilizing nickel-nitrilotriacetic acid coupled Sepharose
CL-6B resin
5 (Ni-NTA, Qiagen, Valencia, CA) as follows. Cell pellets were thawed on
ice and suspended in
20 mL of binding buffer (20 mM sodium phosphate, 500 mM NaC1, 20 mM imidazole,
pH 7.4)
supplemented with 1 mg/mL lysozyme and 1 pellet of Complete EDTA-free protease
inhibitor
(Roche Applied Science, Indianapolis, IN) . Samples were incubated at 4 C with
30 rpm rotation
for 30 min followed by French-pressing (1000 psi). Cell debris was pelleted by
centrifugation for
10 1 h at 15,000 x g and 4 C. The supernatant was transferred to a 5 mL
column bed of Ni-NTA
resin equilibrated with binding buffer. The Ni-NTA resin was resuspended in
the supernatant
and incubated for 60 min with slow rocker mixing at 4 C. The unbound material
was removed
by gravity flow and the resin was washed by gravity flow with 20 column volume
(CV, 100 mL)
of binding buffer followed by 10 CV (50 mL) of rinse buffer (20 mM sodium
phosphate, 500
15 mM NaC1, 100 mM imidazole, pH 7.4). Bound proteins were eluted by
gravity-flow in 10 CV
(50 mL) of elution buffer (20 mM sodium phosphate, 500 mM NaC1, 500 mM
imidazole, pH 7.4)
and collected in fractions. Elution aliquots were assayed for protein content
by SDS-PAGE
analysis, pooled, and concentrated with Amicon Ultra-15 centrifugal filter
devices (EMD
Millipore, Billerica, MA) with 30 kDa nominal molecular weight cut-off. The
concentrated
20 protein isolates were desalted and eluted into 3.5 mL of storage buffer
(50 mM HEPES, 300 mM
NaC1, 20% glycerol, pH 7.3) using PD-10 desalting columns (GE Healthcare
Biosciences,
Pittsburgh, PA).
Example 10
25 Recombinant thioesterases isolation
His-tagged recombinant thioesterases were isolated by IMAC utilizing sepharose
based
magnetic beads with nickel ions (His Mag Sepharose Ni) as follows. Cell
pellets were thawed at
room temperature and suspended in 1.7 mL of 1X BugBuster (primary amine free;
with lpl/mL
Benzonase nuclease; Novagen # 70923-3 and 70750-3 respectively). Samples were
incubated at
30 room temperature with 60 rpm rotation for 30 min. Cell debris was
pelleted by centrifugation for
10 min at 14,000 rpm. The supernatants were transferred to His Mag Sepharose
Ni (GE
Healthcare Biosciences, Piscataway, NJ # 28-9799-17) beads equilibrated
according to kit
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instructions in binding buffer (20 mM sodium phosphate, 500 mM NaC1, 20 mM
imidazole, pH
7.4). The beads were suspended in the supernatant and incubated for 60 min
with slow end-over-
end mixing. The beads were then washed for a total of 5 times in 800 pl of
binding buffer and
slow end-over-end mixing for ¨3-5 min with each wash. The recombinant
thioesterases were
eluted from the beads in 300 p L of elution buffer (20 mM sodium phosphate,
500 mM NaC1, 500
mM imidazole, pH 7.4) by slow end-over-end mixing for 5 min.
Example 11
In vitro reconsitution of aminotransferases and liquid chromatography coupled
to mass
spectrometry (LC-MS) analysis
The activities of purified recombinant aminotransferases (Example 9) were
tested by LC-
MS analysis of expected products. In separate reactions, each enzyme was added
at 0.27 mg/mL
final concentration to reaction buffer (20 mM potassium phosphate, 500 mM
sodium chloride,
pH 8). L-Homoserine (Sigma, St. Louis, MO; catalog # H6515) or a different
amino acid
substrate (Sigma, St. Louis, MO) was added at 1 mM final concentration.
Secondary substrates
were either a-ketoglutaric acid (disodium salt, dehydrate; Sigma, St. Louis,
MO; catalog #
75892) or pyruvate (Sigma, St. Louis, MO; catalog # P2256), each at 1mM final
concentration.
Pyroxidal 5'-phosphate hydrate (Sigma, St. Louis, MO; catalog # P9255) was
added at 50 p.M
final concentration. The reactions were incubated overnight at room
temperature. After
incubation, each solution was filtered using Amicon Ultra centrifugal filter
devices (EMD
Millipore, Billerica, MA) with 3 kDa nominal molecular weight cut-off that had
been prewashed
with ultra pure water. The filtrates were collected and stored at -20 C until
LC-MS analysis.
Aliquots of reaction mixture were diluted 50-100 x and analyzed by high
performance
liquid chromatography coupled to mass spectrometry (LC-MS) in negative mode,
using an
electrospray ionization (ESI) Fourier transform orbital trapping MS (Exactive
Model; Thermo
Fisher, San Jose,CA) at 50,000 resolution. Separations were performed using a
ZIC-pHILIC
column (2.1 x 100 mm, 5 p m polymer, Sequant, EMD Millipore, Catalog #
1504620001;
Darmstadt, Germany) and a mobile phase of 2 mM ammonium formate in 85%
acetonitrile /
15% water at a flow rate of 200 p L/min. The LC-MS analysis indicated that
every tested enzyme
(Table 1) produced the expected product when combined with its ideal substrate
and all enzymes
produced 2-keto-4-hydroxybutyrate when combined with L-homoserine (Figure 4).
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Spectrophotometric assays with aminotransferases
To further confirm the enzymatic activity of the aminotransferases, the
purified
recombinant proteins were assayed spectrophotometrically in a series of
coupled enzyme
reactions. In separate reactions, Pf AT aminotransferase was added at 0.27
mg/m1 final
concentration to 100 mM potassium phosphate buffer (pH 8.0; Sigma, St. Louis,
MO). L-
Homoserine (Sigma, St. Louis, MO; catalog # H6515) or L-Valine (Sigma, St.
Louis, MO;
catalog # V0500) was added as a substrate at 10 - 25 mM final concentration.
The
aminotransferase reaction was coupled with a dehdrogenase reaction in order to
generate reduced
[3-nicotinamide adenine dinucleotide (NADH) which can be detected
spectrophotometrically. [3-
Nicotinamide adenine dinucleotide (NAD ; Sigma, St. Louis, MO; catalog #
N8410) was added
at 3 mM final concentration. Pyroxidal 5'-phosphate hydrate (Sigma, St. Louis,
MO; catalog #
P9255) was added at 50 pM final concentration. a-Ketoglutaric acid, disodium
salt, dehydrate
(Sigma, St. Louis, MO; catalog # 75892) was added as a secondary substrate at
1 mM final
concentration. L-Glutamic dehydrogenase from bovine liver (Sigma, St. Louis,
MO; catalog #
G2626) was added at 10 U/mL. Each reaction was added to a 1 mL quartz cuvette
and the
formation of NADH was followed over time at 340 nm in a spectrophotometer. As
expected, the
initial rate of conversion of L-homoserine was dependent on its concentration
(Figure 5).
Saturation of the enzyme with L-homoserine was not achieved even when high
concentrations
were used.
Example 12
Acyl-CoA levels as a measurement of enzymatic activities: Liquid
chromatography coupled to
mass spectrometry (LC-MS).
E. coli culture sample preparation for acyl-CoA levels analysis
A stable-labeled (deuterium) internal standard-containing master mix is
prepared,
comprising d3-3-hydroxymethylglutaryl-CoA (200 p L of 50 p g/mL stock in 10 mL
of 15%
trichloroacetic acid). An aliquot (500 pl) of the master mix is added to a 2-
mL tube. Silicone oil
(AR200; Sigma, St. Louis, MO; catalog # 85419; 800 pl) is layered onto the
master mix. An
aliquot of E. coli culture (800 pl) is layered gently on top of the silicone
oil. The sample is
subject to centrifugation at 20,000 x g for 5 min at 4 C in an Eppendorf 5417C
centrifuge. An
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38
aliquot (300 p L) of the master mix-containing layer is transferred to an
empty tube and frozen on
dry ice for 30 min.
Measurement of acyl-CoA levels
The acyl-CoA content of samples was determined using LC-MS/MS. Individual acyl-
CoA standards were purchased from Sigma (St. Louis, MO) and prepared as 500 p
g/mL stocks in
methanol. Acryloyl-CoA was synthesized and prepared similarly. The analytes
were pooled,
and standards with all of the analytes were prepared by dilution with 15%
trichloroacetic acid.
Standards for regression were prepared by transferring 500 p L of the working
standards to an
autosampler vial containing 10 p L of the 50 p g/mL internal standard. Sample
peak areas (or
heights) were normalized to the stable-labeled internal standard (d3-3-
hydroxymethylglutaryl-
CoA). Samples were assayed by LC-MS/MS on a Sciex API5000 mass spectrometer in
positive
ion Turbo Ion Spray. Separation was carried out by reversed-phase high
performance liquid
chromatography (HPLC) using a Phenomenex Onyx Monolithic C18 column (2 x 100
mm) and
mobile phases A (5 mM ammonium acetate, 5 mM dimethylbutylamine, and 6.5 mM
acetic acid)
and B (0.1% formic acid in acetonitrile), with the gradient described in table
3 at a flow rate of
0.6 mL/min.
Table 3. Composition of mobile phase during LC-MS/MS analysis
Time Mobile Phase A (%) Mobile Phase B (%)
0 min 97.5 2.5
1.0 min 97.5 2.5
2.5 min 91.0 9.0
5.5 min 45 55
6.0 min 45 55
6.1 min 97.5 2.5
7.5 min
9.5 min End Run
The conditions on the mass spectrometer were: DP 160, CUR 30, GS1 65, G52 65,
IS 4500,
CAD 7, TEMP 650 C. The transitions used for the multiple reaction monitoring
(MRM) are described in table 4.
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Table 4. Description of parameters for quantification of different acyl-CoA
molecules
Compound Precursor Product
Collision Energy CXP
I
Ioni on1
3-Hydroxypropionyl-CoA2 840.3 333.2 45 13
n-Propionyl-CoA 824.3 317.2 41 32
Succinyl-CoA 868.2 361.1 49 38
Isobutyryl-CoA 838.3 331.2 43 21
Lactoyl-CoA 840.3 333.2 45 38
Acryloyl-CoA 822.4 315.4 45 36
Coenzyme A 768.3 261.2 45 34
Isovaleryl-CoA 852.2 345.2 45 34
Malonyl-CoA 854.2 347.2 41 36
Acetyl-CoA 810.3 303.2 43 30
d3-3-Hydroxymethylglutaryl-CoA 915.2 408.2 49 13
lEnergies, in volts, for the MS/MS analysis
2Quantified based on n-propionyl-CoA response
Example 13
In vitro production of 3-hydroxypropionyl-CoA with 2-keto acid decarboxylases
or
dehydrogenases
In a first assay, D-homoserine (2 mM; Acros, Geel, Belgium; catalog #
348362500) was
incubated with D-amino acid oxidase (1 U/mL; Sigma, St. Louis, MO; catalog #
A5222) and
bovine liver catalase (600 U/mL; Sigma, St. Louis, MO; catalog # C40) in the
presence of
HEPES buffer (50 mM, pH 7.3). After incubation at room temperature for 2 ¨ 4
h, coenzyme A
(2 mM), [3-NAD (2 mM), thiamine pyrophosphate (0.2 mM), and MgC12 (2 mM) were
added to
the solution and the components were further incubated with or without
commercial porcine
heart a-ketoglutarate dehydrogenase (1.0 mg/mL; Sigma, St. Louis, MO; catalog
# K1502).
In a second assay, D-homoserine (2 mM; Acros, Geel, Belgium; catalog #
348362500)
was incubated with D-amino acid oxidase (1 U/mL; Sigma, St. Louis, MO; catalog
# A5222) and
bovine liver catalase (600 U/mL; Sigma, St. Louis, MO; catalog # C40) in the
presence of
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HEPES buffer (50 mM, pH 7.3). After incubation at room temperature for 2 ¨ 4
h, coenzyme A
(2 mM), [3-NAD (2 mM), thiamine pyrophosphate (0.2 mM), and MgC12 (2 mM) were
added to
the solution and the components were further incubated with or without
purified 2-keto acid
decarboxylase KdcA (1.8 p m) and propionaldehyde dehydrogenase PduP (1.8 pm).
5 The samples were incubated at room temperature overnight, followed by LC-
MS analysis
to determine concentrations of 3-hydroxypropionyl-CoA as described in example
12. Only when
the dehydrogenases (and decarboxylase) were present, the product was detected
in significant
amounts (Figure 6).
10 Example 14
In vitro production of 3-hydroxypropionyl-CoA from acryloyl-CoA with 3-
hydroxypropionyl-
CoA dehydratase
Acryloyl-CoA (1 mM) was incubated with or without 3-hydroxypropionyl-CoA
15 dehydratase (20 p M) in the presence of HEPES buffer (50 mM, pH 7.3).
After incubation at
room temperature for 2 ¨ 4 h, aliquots were analyzed by high performance
liquid
chromatography (HPLC) using an Agilent 1100 system (Agilent, Santa Clara, CA)
monitoring
absorbance at 254 nm and a Waters Atlantis T3 column (Waters, Milford, MA;
catalog #
186003748). Mobile phases were 0.1% phosphoric acid in water (A) and 0.1%
phosphoric acid in
20 80% acetonitrile / 20% water (B). Analytes were eluted isocratically at
2% B in A over 12 min,
followed by a linear gradient from 2% to 35% B in A over 18 min. The HPLC
analysis indicates
consumption of acryloyl-CoA and formation of a different absorbing molecule
(Figure 7). The
identity of the reaction product, 3-hydroxypropionyl-CoA, was confirmed by LC-
MS analysis as
described in example 12 (Figure 8).
Example 15
In vitro reconstitution of PHA synthase
A solution of 3-hydroxypropionic acid (5 mM; Aldrich, St. Louis, MO; catalog #
AM5000335), coenzyme A (2 mM), ATP (6 mM), MgC12 (2 mM), and HEPES buffer (50
mM,
pH 7.3) was incubated with acetyl-CoA synthetase (5 U/mL; Sigma, St. Louis,
MO; catalog #
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A1765 ) and with or without purified PHA synthase (1 p M). After incubation at
room
temperature for 2 ¨ 4 h, aliquots were analyzed by LC-MS as described in
example 12 to
determine concentrations of 3-hydroxypropionyl-CoA. When PHA synthase was
present, the
concentration of 3-hydroxypropionyl-CoA considerably decreased compared with a
sample with
no enzyme (Figure 9).
Example 16
Thioesterase activity assay: Ellman's reagent
To measure relative thioesterase enzyme activity, Ellman's reagent, also known
as
DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)), was used. The assay buffer was 50
mM KC1, 10
mM HEPES (pH 7.4). A 10mM Ellman's reagent stock solution was prepared in
ethanol. An
acryloyl-CoA substrate stock solution was prepared to 10 mM in assay buffer.
For each enzyme and substrate tested, the reaction was as follows: a 10mM
Ellman's
reagent stock solution was diluted to a 50 uM final concentration in assay
buffer. Acryloyl-CoA
stock solution was added to provide a 90 uM final concentration. The Ellman's
reagent/acryloyl-
CoA mixture (95 L per well) was added to a 96-well polystyrene untreated
microtiter plate.
Equivalent reactions with no substrate were prepared as controls. Purified
enzyme was serially
diluted 1:3 in assay buffer in a separate plate, and 5 L was added to a
reaction well.
Thioesterase activity was assessed at 60 min by measuring the optical density
(OD) at 412 nm on
a plate reader. Relative enzyme activities were calculated by subtracting OD
(412 nm) of
substrate-free controls from OD (412 nm) of substrate-containing samples.
Two thioesterases, EcTesB and CpTT, each showed hydrolysis activity against
the
acryloyl-CoA substrate, with the activity increasing with increasing amounts
of thioesterase
(Figure 10). EcTesB was also active against other substrates (Figure 11).
EcTesB hydrolyzed
octanoyl-CoA, even at relatively low amounts of EcTesB. In contrast, CpTT only
showed an
increase in octanyol-CoA hydrolysis with the highest amounts of thioesterase
(Figure 11). The
other thioesterases showed little or no thioesterase activity against acryloyl-
CoA (Figures 10 and
11), yet their apparent hydrolysis of octanoyl-CoA suggested that the
recombinant enyzmes were
active (Figures 12 and 13). To confirm that the thioesterases were active on
the coenzyme A
substrate tested, samples were analyzed using liquid chromatography coupled to
mass
spectrometry (LC-MS) as described in example 12.
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Monitoring of substrate and product by LC-MS
EcTesB and CpTT showed acryloyl-CoA thioesterase activity in the assay based
on
generation of a free sulfhydryl from the acryloyl-CoA. As a further test of
this thioesterase
activity, it is useful to observe the disappearance of substrate and
appearance of product.
Therefore, LC-MS was used to monitor substrate and product amounts in assays
with these
enzymes as described in Example 12. The amount of EcTesB correlates with the
increase in
acryloyl-CoA hydrolysis, as indicated by both the detection of Coenzyme A by
Ellman's reagent
and by the disappearance of acryloyl-CoA (Table 1). As the enzyme is diluted,
the thioesterase
activity levels decline, as indicated by each assay. These results support
EcTesB's role as a
thioesterase that is active on acryloyl-CoA.
Table 5. Relative enzyme activity and acryloyl-CoA quantitation of TesB
thioesterase samples. The
activity (OD at 412 nm) refers to the assay based on color change in the
presence of Ellman's reagent.
The acryloyl-CoA measurements were based on LC/MS.
TesB Activity Acryloyl CoA
Dilution OD(412nm) (ng)**
Neat 0.140 <200
1:3 0.113 508
1:9 0.101 14600
1:27 0.058 39400
** Values above 5000 are extrapolated estimates.
EcTesB and CpTT each show acryloyl-CoA hydrolysis activity by two different
assays
(Table 6). Each enzyme causes in increase in coenzyme A, as detected with
Ellman's reagent.
Each enzyme also causes a changing profile in the LC-MS analysis, with the
thioesterases
causing a decrease in acryloyl-CoA and an increase in coenzyme A (Table 6).
Table 6. Coenzyme A and acryloyl-CoA quantitation of thioesterase samples. The
activity (OD
at 412 nm) refers to the assay based on color change in the presence of
Ellman's reagent. The
acryloyl-CoA and coenzyme A measurements were based on LC-MS analysis.
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Sample Activity Acryloyl CoA
Name OD(412nm) CoA (ng) (ng)**
EcTesB 0.2157 28100 756
CpTT 0.0992 13600 1430
no enzyme 0.051 259 79000
Example 17
Production of 3-hydroxypropionic acid in engineered E. coli
This example demonstrates increased production of 3-hydroxypropionic acid in
E. coli
host cells which can then be converted to poly-3-hydroxypropionic acid or
acylic acid. E. coli
strains were established to overexpress P. fluorescens branched-chain amino
acid
aminotransferase (Pf AT) set out in SEQ ID NO: 8, L. lactis branched-chain 2-
keto acid
decarboxylase (KdcA) set out in SEQ ID NO: 54, S. enterica coenzyme-A
acylating
propionaldehyde dehydrogenase (PduP) set out in SEQ ID NO: 60, and in some
instances C.
necator Poly(3-hydroxybutyrate) polymerase (PhaC) set out in SEQ ID NO: 42.
In this example, P. fluorescens branched-chain amino acid aminotransferase
(SEQ ID
NO: 8) promoted the conversion of L-homoserine to 2-keto-4-hydroxybutyrate.
The L. lactis
branched-chain 2-keto acid decarboxylase (KdcA, set out in SEQ ID NO: 540
catalyzed the
conversion of 2-keto-4-hydroxybutyrate to 3-hydroxy-propionaldehyde. The S.
enterica
coenzyme-A acylating propionaldehyde dehydrogenase (PduP, set out in SEQ ID
NO: 60)
catalyzed the conversion of 3-hydroxy-propionaldehyde to 3-hydroxypropionyl-
CoA. A
thioesterase catalyzed the conversion of 3-hydroxypropionyl-CoA to 3-
hydroxypropionate.
Alternative, the C. necator Poly (3-hydroxybutyrate) polymerase (PhaC, set out
in SEQ ID NO:
42) can catalyze the conversion of 3-hydroxypropionyl-CoA to poly-3-
hydroxypropionate.
Plasmid construction
An E. coli expression vector was constructed for overexpression of a
recombinant
P. fluorescens branched-chain amino acid aminotransferase (Pf AT) and C.
necator Poly (3-
hydroxybutyrate) polymerase (PhaC). The codon optimized C. necator Poly (3-
hydroxybutyrate)
polymerase (phaC) from pET30a-BB Cn PHAS (Example 4) was cloned into pET30a-BB
Pf AT
(Example 1) by double digestion of pET30a-BB Cn PHAS with restriction enzymes
XbaI and
PstI. The Cn PHAS fragment was band isolated, purified using a QIAquick Gel
Extraction Kit
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(Qiagen, Carlsbad, CA) and ligated (Fast-Link Epicentre Biotechnologies,
Madison, WI) with
SpeI/PstI-digested pET30a-BB Pf AT vector. The ligation mix was used to
transform OneShot
ToplOTm E. coli cells (Invitrogen, Carlsbad, CA).
Individual vials of cells were thawed on ice and gently mixed with 2 L of
ligation mix.
The vials were incubated on ice for 30 min. The vials were briefly incubated
at 42 C for 30 sec
and quickly replaced back on ice for an additional 2 min. 250 L of 37 C SOC
medium was
added and the vials were secured horizontally on a shaking incubator platform
and incubated for
1 h at 37 C and 225 rpm. Aliquots of 20 L and 200 L cells were plated onto
LB agar
supplemented with kanamycin (50 p g/mL). Single colony isolates were isolated
and cultured in
5 mL of LB broth with kanamycin (50 p g/mL). The recombinant plasmid was
isolated using a
Qiagen Plasmid Mini Kit and characterized by gel electrophoresis of
restriction digests with
MM. The resulting plasmid was designated pET30a-BB Pf AT_Cn PHAS.
An E. coli expression vector was constructed for overexpression of a
recombinant S.
enterica coenzyme-A acylating propionaldehyde dehydrogenase (PduP) and L.
lactis branched-
chain 2-keto acid decarboxylase (KdcA). The codon optimized L. lactis branched-
chain 2-keto
acid decarboxylase (kdcA) from pET30a-BB Ll KDCA (Example 2) was cloned into
pET30a-BB
Se PDUP (Example 3) by double digestion of pET30a-BB Ll KDCA with restriction
enzymes
XbaI and PstI. The Ll KDCA fragment was band isolated, purified using a
QIAquick Gel
Extraction Kit (Qiagen, Carlsbad, CA) and ligated (Fast-Link Epicentre
Biotechnologies,
Madison, WI) with SpeI/PstI-digested pET30a-BB Se PDUP vector. The ligation
mix was used
to transform OneShot ToplOTm E. coli cells (Invitrogen, Carlsbad, CA).
Individual vials of cells
were thawed on ice and gently mixed with 2 L of ligation mix. The vials were
incubated on ice
for 30 min. The vials were briefly incubated at 42 C for 30 sec and quickly
replaced back on ice
for an additional 2 min. 250 L of 37 C SOC medium was added and the vials
were secured
horizontally on a shaking incubator platform and incubated for 1 h at 37 C and
225 rpm.
Aliquots of 20 L and 200 L cells were plated onto LB agar supplemented with
kanamycin (50
p g/mL). Single colony isolates were isolated and cultured in 5 mL of LB broth
with kanamycin
(50 p g/mL) and the recombinant plasmid was isolated using a Qiagen Plasmid
Mini Kit and
characterized by gel electrophoresis of restriction digests with AflIII. The
resulting plasmid was
designated pET30a-BB Se PDUP_L/ KDCA.
To facilitate cotransformation with pET30a-BB Pf AT or pET30a-BB Pf AT_Cn
PHAS,
the codon optimized S. enterica coenzyme-A acylating propionaldehyde
dehydrogenase (pduP)
and L. lactis Branched-chain 2-keto acid decarboxylase (kdcA) gene pair was
subcloned from
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pET30a-BB Se PDUP_L/ KDCA into the pCDFDuet-1 vector (Novagen, EMD Chemicals,
Gibbstown, NJ; catalog # 71340-3) by double digestion of pET30a-BB Se PDUP_L/
KDCA with
restriction enzymes EcoRI and PstI. The Se PDUP_L/ KDCA fragment was band
isolated,
purified using a QIAquick Gel Extraction Kit (Qiagen, Carlsbad, CA) and
ligated (Fast-Link
5 Epicentre Biotechnologies, Madison, WI) with EcoRI/PstI-digested pCDEDuet-
1. The ligation
mix was used to transform OneShot ToplOTm E. coli cells (Invitrogen, Carlsbad,
CA). Individual
vials of cells were thawed on ice and gently mixed with 2 L of ligation mix.
The vials were
incubated on ice for 30 min. The vials were briefly incubated at 42 C for 30
sec and quickly
replaced back on ice for an additional 2 min. 250 L of 37 C SOC medium was
added and the
10 vials were secured horizontally on a shaking incubator platform and
incubated for 1 h at 37 C
and 225 rpm. Aliquots of 20 L and 200 L cells were plated onto LB agar
supplemented with
spectinomycin (50 p g/mL). Single colony isolates were isolated, cultured in 5
mL of LB broth
with spectinomycin (50 p g/mL) and the recombinant plasmid was isolated using
a Qiagen
Plasmid Mini kit and characterized by gel electrophoresis of restriction
digests with MM. The
15 resulting plasmid was designated pCDFDuet-1 Se PDUP_L/ KDCA.
Co-transformation of E. coli
The recombinant plasmids and empty parent vectors were used to co-transform
chemically competent BL21 (DE3) pLysS E. coli cells (Invitrogen, Carlsbad, CA)
in the
20 following combinations:
pET30a-BB Pf AT_Cn PHAS and pCDFDuet-1 Se PDUP_L/ KDCA
pET30a-BB Pf AT and pCDFDuet-1 Se PDUP_L/ KDCA
pET30a-BB and pCDFDuet-1
Individual vials of cells were thawed on ice and gently mixed with 50 ng of
plasmid
25 DNA. The vials were incubated on ice for 30 min. The vials were briefly
incubated at 42 C for
30 sec and quickly replaced back on ice for an additional 2 min. 250 L of 37
C SOC medium
was added and the vials were secured horizontally on a shaking incubator
platform and incubated
for 1 h at 37 C and 225 rpm. Aliquots of 20 L and 200 L cells were plated
onto LB agar
supplemented with the appropriate antibiotics (50 ng/mL kanamycin; 50 ng/mL
spectinomycin;
30 34 ng/mL chloramphenicol) to select for cells carrying the recombinant
pET30a-BB, pCDFDuet-
1 and pLysS plasmids respectively and incubated overnight at 37 C. Single
colony isolates were
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isolated, cultured in 5 mL of selective LB broth and the recombinant plasmids
were isolated
using a QIAPrep Spin Miniprep Kit (Qiagen, Valencia, CA) and characterized by
gel
electrophoresis of restriction digests with NMI.
Strain culture
Single colony forming units of E. coli BL21 (DE3) pLysS cells co-transformed
with the
described plasmids were used to inoculated aliquots of minimal M9 broth (25
mL) supplemented
with the appropriate antibiotics (34 p g/mL chloramphenicol, 50 p g/mL
kanamycin, and 50
p g/mL spectinomycin). The cultures were incubated overnight at 37 C with
shaking at 250 rpm
and used to inoculated fresh minimal M9 media (50 mL) supplemented with the
same
antibiotics. After overnight incubation under similar conditions, aliquots of
cultures were used to
inoculate a new set of M9 broths (50 mL) with antibiotics (34 p g/mL
chloramphenicol, 50
p g/mL kanamycin, and 50 p g/mL spectinomycin) and supplemented with or
without
L-homoserine (1 g/L; Sigma, St. Louis, MO), followed by incubation at 25 C
with shaking at 250
rpm. When 0D600 of about 0.2 was reached, protein expression was induced by
addition of 50
p L of 1M IPTG (1 mM final concentration; Teknova, Hollister, CA), followed by
incubation for
17 h at 25 C with 250 rpm shaking. Cells were harvested by centrifugation and
supernatants were
filtered through Acrodisc Syringe Filters (0.2 p.m HT Tuffryn membrane; low
protein binding;
Pall Corporation, Ann Arbor, MI) and frozen on dry ice prior to storage at -80
C until analysis.
Minimal M9 Media
1X Base Recipe
Component
Na2HPO4 6 g/L
KH2PO4 3 g/L
NaC1 0.5 g/L
NH4C1 1 g/L
CaC12* 2H20 0.1 mM
MgSO4 1 mM
Dextrose 80 mM
Thiamine 1 mg/L
Chloramphenicol 34 p g/mL
Kanamycin 50 ug/mL
Spectinomycin 50 ug/mL
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47
Detection of 3-hydroxypropionic acid by engineered E. coli
An internal standard solution of 100 p g/mL of 13C3-labelled lactic acid in
1:1 MeOH:H20
was prepared. External standard solutions were prepared at 3-hydroxypropanoic
acid
concentrations of 1 p g/mL, 2.5 p g/mL, 5 p g/mL, 10 p g/mL and 25 pg/mL in
1:1 MeOH:H20.
900 p L of filtered supernatant or external standard was added to 100 p L of
the internal standard
solution. These solutions were subjected to ion exclusion liquid
chromatography (LC)
separations and mass spectrometry (MS) detection.
The LC separation conditions were as follows: 10 p L of sample/standard were
injected
onto a Thermo Fisher Dionex ICE-AS1 (4x250 mm) column (with guard) running an
isocratic
mobile phase of 1 mM heptafluorbutyric acid at a flow rate of 0.15 mL/min. 20
mM NH4OH in
MeCN at 0.15 mL/min was teed into the column effluent.
The MS detection conditions were as follows: A Sciex API-4000 MS was run in
negative
ion mode and monitored the m/z 89 to 59 (unit resolution) transition of 3-
hydroxypropanoic acid
and the m/z 92 to 45 (unit resolution) transition of 13C3-labelled lactic
acid. The dwell time used
was 300 ms, the declustering potential was set at -38, the entrance potential
was set at -10, the
collision gas was set at 12, the curtain gas was set at 15, the ion source gas
1 was set at 55, the
ion source gas 2 was set at 55, the ionspray voltage was set at -3500, the
temperature was set at
650, the interface heater was on. For 3-hydroxypropanoic acid, the collision
energy was set at -16
and the collision set exit potential was set at -9. For 13C3-labelled lactic
acid, the collision energy
was set at -18 and the collision set exit potential was set at -16.
The results of the analysis are shown in Table 7. The data evidenced that
increased levels
of 3-hydroxypropanoic acid were produced when Pf AT, KdcA, and PduP were
overexpressed
and L-homoserine was supplemented to the culture media. Endogenous L-
homoserine and E. coli
proteins likely supported production of small amounts of 3-hydroxypropionic
acid when no
exogenous homoserine was added to the culture medium and/or when empty pET30a-
BB and
pCDF Duet-1 vectors were present.
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48
Table 7. Production of 3-hydroxypropionic acid by engineered E. coli
Concentration of
Addition of 3-hydroxypropionic
Plasmids L-homoserine? acid (it' g/mL)
pET30a-BB Pf AT::Cn_PHAS and
pCDF Duet-1 Se_PDUP:D_KDCA No 0.08
pET30a-BB Pf AT and
pCDF Duet-1 Se_PDUP:D_KDCA No 0.08
pET30a-BB and
pCDF Duet-1 No 0.15
pET30a-BB Pf AT::Cn_PHAS and
pCDF Duet-1 Se_PDUP:D_KDCA Yes 2.00
pET30a-BB Pf AT and
pCDF Duet-1 Se_PDUP:D_KDCA Yes 4.13
pET30a-BB and
pCDF Duet-1 Yes 0.73
While the present invention has been described in terms of specific
embodiments, it is
understood that variations and modifications will occur to those skilled in
the art. Accordingly,
only such limitations as appear in the claims should be placed on the
invention.
All documents referred to in this application are hereby incorporated by
reference in
their entirety.
