Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TITLE
IN SITU EXPRESSION OF LIPASE FOR ENZYMATIC PRODUCTION OF
ALCOHOL ESTERS DURING FERMENTATION
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is related to and claims the benefit of priority of
U.S. Provisional Patent Application No. 61/466712, filed March 23, 2011
and U.S. Provisional Patent Application No. 61/498,292, filed June 17,
2011, the contents of which are herein incorporated by reference in their
entireties.
REFERENCE TO SEQUENCE LISTING SUBMITTTED
ELECTRONICALLY
The content of the electronically submitted sequence listing in
ASCII text file (Name: 20120322_CL5145USNA_SeqList_5T25.txt, Size:
656,901 bytes, and Date of Creation: March 22, 2012 ) filed with the
application is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to the fermentative production of
alcohols, including ethanol and butanol, and processes for improving
alcohol fermentation employing in situ product removal methods.
BACKGROUND OF THE INVENTION
Alcohols have a variety of applications in industry and science. For
example, alcohols can be used as a beverage (i.e, ethanol), fuel,
reagents, solvents, and antiseptics. For example, butanol is an alcohol
that is an important industrial chemical with a variety of applications,
including use as a fuel additive, as a feedstock chemical in the plastics
industry, and as a food-grade extractant in the food and flavor industry.
Accordingly, there is a high demand for alcohols, such as butanol, as well
as for efficient production methods which do not rely on non-renewable
resources.
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Production of alcohol utilizing fermentation by microorganisms is
one such production method which utilizes substrates from renewable
feedstocks. In the
production of butanol in particular, some
microorganisms that produce butanol in high yields also have low butanol
toxicity thresholds, such that butanol needs to be removed from the
fermentation vessel as it is being produced. Thus, there is a continuing
need to develop efficient methods and systems for producing butanol in
high yields despite low butanol toxicity thresholds of the butanol-producing
microorganisms in the fermentation medium. In situ product removal
(ISPR) (also referred to as extractive fermentation) can be used to remove
butanol (or other fermentative alcohol) from the fermentation vessel as it is
produced, thereby allowing the microorganism to produce butanol at high
yields. One method for ISPR for removing fermentative alcohol that has
been described in the art is liquid-liquid extraction (U.S. Patent Appl. Pub.
No. 20090305370). In general, with regard to butanol fermentation, for
example, the fermentation medium, which includes the microorganism, is
contacted with an organic extractant. The organic extractant and the
fermentation medium form a biphasic mixture. The butanol partitions into
the organic extractant phase, decreasing the concentration in the aqueous
phase containing the microorganism, thereby limiting the exposure of the
microorganism to the inhibitory butanol. Liquid-liquid extraction results
from contact between the extractant and the fermentation broth for transfer
of the product alcohol into the extractant; separation of the extractant
phase from the aqueous phase; and, preferably, recycle of the extractant
with minimal degradation of the partition coefficient of the extractant over a
long-term operation.
The extractant can become contaminated over time with each
recycle by, for example, the build-up of lipids present in the biomass that is
fed to the fermentation vessel as feedstock of hydrolysable starch. As an
example, a liquified corn mash loaded to a fermentation vessel can result
in a fermentation broth that contains corn oil during conversion of glucose
to butanol by simultaneous saccharification and fermentation (with
saccharification of the liquified mash occurring during fermentation by the
addition of glucoamylase to produce glucose). The dissolution of the corn
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oil lipids into an extractant during ISPR can result in build-up of lipid
concentration with each extractant recycle, decreasing the partition
coefficient for the product alcohol in extractant as the lipid concentration
in
extractant increases with each recycle.
Converting the lipids present in a liquefied mash into an extractant
that can be used in ISPR is a method of decreasing the amount of lipids
that are fed to the fermentation vessel, as is esterifying the product alcohol
as it is produced during the fermentation with a fatty acid by adding lipase
as an esterification catalyst to the fermentation. Such methods are
described for example in US Appl. Pub. Nos. 20110312044 and
20110312043, and PCT Appl. Pub. No. W02011/159998
There is a continuing need for alternative extractive fermentation
methods which can also reduce costs associated with adding lipase to the
fermentation.
SUMMARY OF THE INVENTION
Provided herein are methods comprising: a) providing a
fermentation medium comprising fermentable carbon substrate derived
from a biomass feedstock, alcohol produced from a fermentable carbon
substrate derived from a biomass feedstock, and an alcohol producing
microorganism wherein the alcohol producing microorganism comprises a
polynucleotide encoding a polypeptide having lipase activity and the
microorganism expresses and displays or secretes said polypeptide such
that the lipase activity is present in the fermentation medium; b) contacting
the fermentation medium with a carboxylic acid; wherein the lipase activity
is present in the fermentation medium in sufficient amount to convert at
least a portion of the alcohol produced by the microorganism to alcohol
esters extracellularly. In
embodiments, the alcohol producing
microorganism is yeast. In embodiments, the polynucleotide encoding a
polypeptide having lipase activity is engineered. In embodiments, the
methods further comprise contacting the fermentation medium with an
extractant to form a two-phase mixture comprising an aqueous phase and
an organic phase. In
embodiments, the extractant comprises the
carboxylic acid. In embodiments, the product alcohol is a C2 to C8 alkyl
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alcohol. In embodiments, the product alcohol is ethanol. In
embodiments, the alcohol esters comprise fatty acid ethyl esters. In
embodiments, the product alcohol is butanol. In embodiments, the
alcohol esters comprise fatty acid butyl esters. In embodiments, the
alcohol esters further comprise fatty acid ethyl esters.
In embodiments, polypeptides provided herein having lipase activity
are displayed on the surface of the microorganism. In embodiments,
polypeptides having lipase activity are secreted. In embodiments, the
polypeptide having lipase activity comprises a sequence having at least
about 70% identity, at least about 80% identity, at least about 90%
identity, or at least about 95% identity to any one of SEQ ID NOs: 249,
250, 251, 252, 253 or a fragment thereof. In
embodiments, the
polynucleotide encoding a polypeptide having lipase activity comprises a
sequence with at least about 70% identity to a polynucleotide having SEQ
ID NO: 1, 3, 5, 7, 8, 9, 46, 48, 50, 52, 54, 255, 271 or 273. In
embodiments, the polypeptide having lipase activity comprises a
sequence with at least about 70% identity, at least about 80% identity, at
least about 90% identity, or at least about 95% identity to a polypeptide
having SEQ ID NO: 2,4, 6, 256, 47, 49, 51, 53, 55, 241, 242, 243, 244,
245, 246, 247, 248, 272, or 274 or an active fragment thereof. In
embodiments, the polypeptide having lipase activity does not contain a
glycosylation motif. In embodiments, the polypeptide having lipase activity
is not glycosylated.
In embodiments, the carboxylic acid comprises free fatty acids
derived from corn oil, canola oil, palm oil, linseed oil, jatropha oil, or
soybean oil. In embodiments, the carboxylic acid is derived from the
same biomass feedstock as the fermentable carbon substrate. In
embodiments, the carboxylic acid comprises carboxylic acids having 012 to
022 linear or branched aliphatic chains. In embodiments, the contacting
with extractant and the contacting with carboxylic acid occur
contemporaneously. In embodiments, at least about 60% of the effective
titer of alcohol produced by the microorganism is converted to alcohol
esters. In
embodiments, the fermentation medium further comprises
triglycerides, diglycerides, monoglycerides, and phospholipids, or
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combinations thereof and the lipase activity hydrolyzes at least a portion of
the triglycerides, diglycerides, monoglycerides, and phospholipids, or
combinations thereof to form free fatty acids.
In embodiments, the effective titer of alcohol produced during a
fermentation is greater than that produced during a fermentation by an
alcohol-producing microorganism that does not comprise a polynucleotide
encoding a polypeptide having lipase activity and the microorganism
expresses and secretes or displays said polypeptide such that the lipase
activity is present in the fermentation medium. In embodiments, the
effective rate of alcohol produced during a fermentation is greater than the
rate of alcohol production during a fermentation by an alcohol-producing
microorganism that does not comprise a polynucleotide encoding a
polypeptide having lipase activity and the microorganism expresses and
secretes or displays said polypeptide such that the lipase activity is
present in the fermentation medium.
Also provided herein are recombinant host cells comprising an
engineered alcohol production pathway; and an engineered polynucleotide
encoding a polypeptide having lipase activity. In embodiments, the
polypeptide having lipase activity comprises a sequence having at least
about 70% identity, at least about 80% identity, at least about 90%
identity, or at least about 95% identity to SEQ ID NO: 2, 4, 6, 256, 47, 49,
51, 53, 55, 241, 242, 243, 244, 245, 246, 247, 248, 272, or 274 or an
active fragment thereof. In embodiments, the polypeptide having lipase
activity comprises a sequence having at least about 70% identity, at least
about 80% identity, at least about 90% identity, or at least about 95%
identity to any one of SEQ ID NOs: 249, 250, 251, 252, 253 or a fragment
thereof. In embodiments, the polypeptide having lipase activity does not
contain a glycosylation motif. In embodiments, the polypeptide having
lipase activity is not glycosylated. In embodiments, the engineered
polynucleotide encoding a polypeptide having lipase activity comprises a
sequence having at least about 70% identity, at least about 80% identity,
at least about 90% identity, or at least about 95% identity to SEQ ID NO:
1, 3, 5, 7, 8, 9, 46, 48, 50, 52, 54, 255, 271 or 273.
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Also provided herein are recombinant host cells comprising an
alcohol production pathway; and an engineered polynucleotide encoding a
polypeptide having lipase activity wherein the polypeptide having lipase
activity comprises a sequence having at least about 70% identity, at least
about 80% identity, at least about 90% identity, or at least about 95%
identity to SEQ ID NO: 2, 4, 6, 256, 47, 49, 51, 53, 55, 241, 242, 243, 244,
245, 246, 247, 248, 272, or 274 or an active fragment thereof. In
embodiments, the polypeptide having lipase activity further comprises a
sequence having at least about 70% identity, at least about 80% identity,
at least about 90% identity, or at least about 95% identity to any one of
SEQ ID NOs: 249, 250, 251, 252, 253 or a fragment thereof. In
embodiments, the alcohol production pathway is a butanol production
pathway. In embodiments, the butanol production pathway is an
isobutanol production pathway. In embodiments, the host cell further
comprises reduced or eliminated pyruvate decarboxylase activity.
Also provided herein are methods of increasing tolerance of an
alcohol-producing microorganism to the produced alcohol, the methods
comprising: engineering a microorganism to express and secrete or
display a polypeptide having lipase activity; contacting the engineered
microorganism with trig lycerides, diglycerides,
monoglycerides,
phospholipids, free fatty acids, or a mixture thereof and a carbon substrate
under conditions whereby the microorganism produces an alcohol. In
embodiments, the engineered microorganism is contacted with
triglycerides, diglycerides, monoglycerides, and phospholipids, or
combinations thereof and wherein the secreted or displayed lipase
converts at least a portion of the trigylcerides, diglycerides,
monoglycerides, and phospholipids, or combinations thereof into free fatty
acids. In embodiments, the lipase catalyzes the formation of alcohol
esters. In embodiments, the microorganism produces alcohol at an
effective titer greater than that produced by a microorganism that has not
been engineered to express and secrete a polypeptide with lipase activity.
In embodiments, the microorganism further comprises an engineered
alcohol biosynthetic pathway. In embodiments, the engineered alcohol
biosynthetic pathway is a 1-butanol, a 2-butanol, or an isobutanol
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biosynthetic pathway. In
embodiments, the isobutanol biosynthetic
pathway comprises the following substrate to product conversions:
pyruvate to acetolactate, acetolactate to 2,3-dihydroxyisovalerate, 2,3-
dihydroxyisovalerate to 2-ketoisovalerate, 2-ketoisovalerate to
isobutyraldehyde; and, isobutyraldehyde to isobutanol.
Provided herein are methods of producing butyl esters during a
fermentation comprising providing a fermentation medium comprising a
carbon substrate and triglycerides, diglycerides, monoglycerides, and
phospholipids, or a mixture thereof; and contacting the fermentation
medium with an alcohol-producing microorganism comprising a butanol
biosynthetic pathway wherein said microorganism further comprises an
engineered polynucleotide encoding a polypeptide having lipase activity
and which expresses and secretes or displays the polypeptide such that
the lipase activity is present in the fermentation medium. In embodiments,
the fermentation medium further comprises one or more carboxylic acids.
In embodiments, the carbon substrate is derived from biomass. In
embodiments, the biomass is corn or sugar cane. In embodiments, the
carbon substrate and the triglycerides diglycerides, monoglycerides, and
phospholipids are derived from the same biomass.
Provided herein are fermentation media comprising an alcohol-
producing microorganism comprising a butanol biosynthetic pathway and
further comprising an engineered polynucleotide encoding a polypeptide
having lipase activity which is expressed and secreted or displayed, butyl
esters, and butanol.
Also provided are animal feed products comprising a
microorganisms described herein.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES
The accompanying drawings and sequence listing, which are
incorporated herein and form a part of the specification, illustrate the
present invention and, together with the description, further serve to
explain the principles of the invention and to enable a person skilled in the
pertinent art to make and use the invention.
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FIG. 1 schematically illustrates an exemplary method and system of
the present invention, in which a microorganism is supplied to a
fermentation vessel along with carboxylic acid and/or native oil.
FIG. 2 depicts example biosynthetic pathways for biosynthesis of
isobutanol from pyruvate.
FIG. 3 is a map of plasmid pRS423::TEF1(M4)-CdLIP1 ("pNAK10";
SEQ ID NO: 45; see Example 1), bearing the Candida deformans LIP1
lipase under transcriptional control of the constitutive TEF1(M4) promoter
(Nevoigt E, Kohnke J, Fischer CR, Alper H, Stahl U, & Stephanopoulos G
(2006), Engineering of promoter replacement cassettes for fine-tuning of
gene expression in Saccharomyces cerevisiae. Appl Environ Microbiol
72:5266-5273) and the CYC1 transcriptional terminator, in a yeast-E. coli
shuttle vector.
FIG. 4 is a map of plasmid pRS423::TEF1(M4)-THlip ("pTVAN2";
SEQ ID NO: 100; see Example 2), bearing the Thermomyces lanuginosus
Tlan lipase under transcriptional control of the constitutive TEF1(M4)
promoter (Nevoigt E, et al.) and the CYC1 transcriptional terminator, in a
yeast-E. coli shuttle vector.
FIG. 5 is a map of plasmid pRS423::TEF1(M4)-CalB ("pTVAN3";
SEQ ID NO: ; See Example 7), bearing the Candida antarctica CalB lipase
under transcriptional control of the constitutive TEF1(M4) promoter
(Nevoigt E, et al.) and the CYC1 transcriptional terminator, in a yeast-E.
coli shuttle vector.
FIG. 6 is a map of plasmid pYZ090AalsS (SEQ ID NO: 43; see
Examples), which bears the ketol-acid reductoisomerase (KARI) enzyme
ORF in a yeast-E. coli shuttle vector.
FIG 7. Map of plasmid pBP915 (SEQ ID NO: 44; see Examples 9
and 10), which bears the ORFs encoding the dihydroxyacid dehydratase
enzyme and the alcohol dehydrogenase enzyme in a yeast-E. coil shuttle
vector.
SEQ ID NOs: 1 and 2 are nucleic acid and amino acid sequences
for lipase B ("CalB") from Candida antarctica.
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SEQ ID NOs: 3 and 4 are nucleic acid and amino acid sequences
for lipase 1 ("LIP1") from Candida deformans.
SEQ ID NOs: 5 and 6 are nucleic acid and amino acid sequences
for Tlan lipase ("Tlan") from Thermomyces lanuginosus.
SEQ ID NOs: 255 and 256 are nucleic acid and amino acid
sequences for lipase 3 ("lip3") from Aspergillus tubingensis.
SEQ ID NOs: 7, 8, 9, and 257 are coding sequences for CalB, LIP1,
Tlan, and lip3 lipases from Candida antarctica, Candida deformans,
Thermomyces lanuginosus, and Aspergillus tubingensis, codon -optimized
for expression in S. cerevisiae.
SEQ ID NOs: 46 and 47 are nucleic acid and amino acid
sequences for a CalB variant with the modification N99A.
SEQ ID NOs: 48 and 49 are nucleic acid and amino acid
sequences for a LIP1 variant with the modification N146A.
SEQ ID NOs: 50 and 51 are nucleic acid and amino acid
sequences for a LIP1 variant with the modification N167A.
SEQ ID NOs: 52 and 53 are nucleic acid and amino acid
sequences for a LIP1 variant with the modifications N146A and N167A.
SEQ ID NOs: 54 and 55 are nucleic acid and amino acid
sequences for a Tlan variant with the modification N55A.
SEQ ID NOs: 271 and 272 are nucleic acid and amino acid
sequences for a lip3 variant with the modification N59A.
SEQ ID NOs: 273 and 274 are nucleic acid and amino acid
sequences for a lip3 variant with the modification N269A.
SEQ ID NOs: 275 and 276 are nucleic acid and amino acid
sequences for a lip3 variant with the modifications N59A and N269A.
SEQ ID NOs: 241 and 248 are amino acid sequences for lipases
from Aspergillus kawachii, Aspergillus niger, Yarrowia lipolytica,
Talaromyces thermophilus.
SEQ ID NOs: 249 and 254 are amino acid sequences of cell
surface anchor domains of S. cerevisiae.
SEQ ID NOs: 258 and 259 are the amino acid sequences of alcohol
dehydrogenase enzymes from Achromobacter xylosoxidans and
Beijerinkia indica.
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SEQ ID NOs: 260 and 261 are the amino acid sequences of keto-
acid decarboxylases from Lactococcus lactis and Listeria grayi.
SEQ ID NOs: 262 and 263 are the amino acid sequences of
dihydroxyacid dehdratases from Streptococcus mutans and Lactococcus
lactis.
SEQ ID NOs: 10-45, 56-144, 153-238, 240, 264-270, 277 and 278
are sequences of synthetic constructs and primers described in the
Examples.
DETAILED DESCRIPTION
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. In case of
conflict,
the present application including the definitions will control. Also, unless
otherwise required by context, singular terms shall include pluralities and
plural terms shall include the singular. All publications, patents and other
references mentioned herein are incorporated by reference in their
entireties for all purposes.
In order to further define this invention, the following terms and
definitions are herein provided.
As used herein, the terms "comprises," "comprising," "includes,"
"including," "has," "having," "contains" or "containing," or any other
variation thereof, will be understood to imply the inclusion of a stated
integer or group of integers but not the exclusion of any other integer or
group of integers. For example, a composition, a mixture, a process, a
method, an article, or an apparatus that comprises a list of elements is not
necessarily limited to only those elements but can include other elements
not expressly listed or inherent to such composition, mixture, process,
method, article, or apparatus. Further, unless expressly stated to the
contrary, "or" refers to an inclusive or and not to an exclusive or. For
example, a condition A or B is satisfied by any one of the following: A is
true (or present) and B is false (or not present), A is false (or not present)
and B is true (or present), and both A and B are true (or present).
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Also, the indefinite articles "a" and "an" preceding an element or
component of the invention are intended to be nonrestrictive regarding the
number of instances, i.e., occurrences of the element or component.
Therefore "a" or "an" should be read to include one or at least one, and the
singular word form of the element or component also includes the plural
unless the number is obviously meant to be singular.
The term "invention" or "present invention" as used herein is a non-
limiting term and is not intended to refer to any single embodiment of the
particular invention but encompasses all possible embodiments as
described in the application.
As used herein, the term "about" modifying the quantity of an
ingredient or reactant of the invention employed refers to variation in the
numerical quantity that can occur, for example, through typical measuring
and liquid handling procedures used for making concentrates or solutions
in the real world; through inadvertent error in these procedures; through
differences in the manufacture, source, or purity of the ingredients
employed to make the compositions or to carry out the methods; and the
like. The term "about" also encompasses amounts that differ due to
different equilibrium conditions for a composition resulting from a particular
initial mixture. Whether or not modified by the term "about", the claims
include equivalents to the quantities. In one embodiment, the term "about"
means within 10% of the reported numerical value, alternatively within 5%
of the reported numerical value.
"Biomass" as used herein refers to a natural product containing
hydrolysable polysaccharides that provide fermentable sugars, including
any sugars and starch derived from natural resources such as corn, sugar
cane, wheat, cellulosic or lignocellulosic material and materials comprising
cellulose, hemicellulose, lignin, starch, oligosaccharides, disaccharides
and/or monosaccharides, and mixtures thereof. Biomass may also
comprise additional components, such as protein and/or lipids. Biomass
may be derived from a single source, or biomass can comprise a mixture
derived from more than one source; for example, biomass may comprise a
mixture of corn cobs and corn stover, or a mixture of grass and leaves.
Biomass includes, but is not limited to, bioenergy crops, agricultural
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residues, municipal solid waste, industrial solid waste, sludge from paper
manufacture, yard waste, wood and forestry waste. Examples of biomass
include, but are not limited to, corn grain, corn cobs, crop residues such as
corn husks, corn stover, grasses, wheat, rye, wheat straw, barley, barley
straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse,
sorghum, soy, components obtained from milling of grains, trees,
branches, roots, leaves, wood chips, sawdust, shrubs and bushes,
vegetables, fruits, flowers, animal manure, and mixtures thereof. For
example, mash or juice or molasses or hydrolysate may be formed from
biomass by any processing known in the art for processing the biomass
for purposes of fermentation, such as by milling, treating and/or liquefying
and comprises fermentable sugar and may comprise an amount of water.
For example, cellulosic and/or lignocellulosic biomass may be processed
to obtain a hydrolysate containing fermentable sugars by any method
known to one skilled in the art. A low ammonia pretreatment is disclosed
in US Patent Application Publication US20070031918A1, which is herein
incorporated by reference. Enzymatic saccharification of cellulosic and/or
lignocellulosic biomass typically makes use of an enzyme consortium for
breaking down cellulose and hemicellulose to produce a hydrolysate
containing sugars including glucose, xylose, and arabinose.
(Saccharification enzymes suitable for cellulosic and/or lignocellulosic
biomass are reviewed in Lynd, L. R., et al. (Microbiol. Mol. Biol. Rev.,
66:506-577, 2002).
Mash or juice or molasses or hydrolysate may include feedstock 12
and feedstock slurry 16 as described herein. An aqueous feedstream may
be derived or formed from biomass by any processing known in the art for
processing the biomass for purposes of fermentation, such as by milling,
treating and/or liquefying and comprises fermentable carbon substrate (eg.
sugar) and water. An aqueous feedstream may include feedstock 12 and
feedstock slurry 16 as described herein.
"Product alcohol" as used herein refers to any alcohol that can be
produced by a microorganism in a fermentation process that utilizes
biomass as a source of fermentable carbon substrate. Product alcohols
include, but are not limited to, Ci to 08 alkyl alcohols. In embodiments, the
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product alcohols are 02 to 08 alkyl alcohols. In additional embodiments,
the product alcohols are 02 to 05 alkyl alcohols. It will be appreciated that
Ci to 08 alkyl alcohols include, but are not limited to, methanol, ethanol,
propanol, butanol, and pentanol. Likewise 02 to 08 alkyl alcohols include,
but are not limited to, ethanol, propanol, butanol, and pentanol . "Alcohol"
is also used herein with reference to a product alcohol.
"Butanol" as used herein refers with specificity to the butanol
isomers 1-butanol (1-BuOH), 2-butanol (2-BuOH) and/or isobutanol
(iBuOH or i-BuOH or I-BUOH, also known as 2-methyl-1-propanol), either
individually or as mixtures thereof.
"Propanol" as used herein refers to the propanol isomers
isopropanol or 1-propanol.
"Pentanol" as used herein refers to the pentanol isomers 1-
pentanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 2,2-dimethy1-1-propanol,
3-pentanol, 2-pentanol, 3-methyl-2-butanol, or 2-methyl-2-butanol.
"In Situ Product Removal (ISPR)" as used herein means the
selective removal of a specific fermentation product from a biological
process such as fermentation to control the product concentration in the
biological process as the product is produced.
"Fermentable carbon source" or "fermentable carbon substrate" as
used herein means a carbon source capable of being metabolized by the
microorganisms disclosed herein for the production of fermentative
alcohol. Suitable fermentable carbon sources include, but are not limited
to, monosaccharides, such as glucose or fructose; disaccharides, such as
lactose or sucrose; oligosaccharides; polysaccharides, such as starch or
cellulose; one carbon substrates including methane; and mixtures thereof.
"Feedstock" as used herein means a feed in a fermentation
process, the feed containing a fermentable carbon source with or without
undissolved solids, and where applicable, the feed containing the
fermentable carbon source before or after the fermentable carbon source
has been liberated from starch or obtained from the breakdown of complex
sugars by further processing, such as by liquefaction, saccharification, or
other process. Feedstock includes or is derived from a biomass. Suitable
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feedstocks include, but are not limited to, rye, wheat, corn, cane and
mixtures thereof.
"Undissolved solids" as used herein means non-fermentable
portions of feedstock, for example germ, fiber, and gluten.
"Fermentation broth" as used herein means the mixture of water,
sugars, dissolved solids, microorganisms producing alcohol, product
alcohol and all other constituents of the material held in the fermentation
vessel in which product alcohol is being made by the reaction of sugars to
alcohol, water and carbon dioxide (002) by the microorganisms present.
From time to time, as used herein the term "fermentation medium" and
"fermented mixture" can be used synonymously with "fermentation broth".
"Fermentation vessel" as used herein means the vessel in which
the fermentation reaction by which product alcohol such as butanol is
made from sugars is carried out.
The term "effective titer" as used herein, refers to the total amount
of a particular alcohol (e.g., butanol) produced by fermentation or alcohol
equivalent of the alcohol ester produced by alcohol esterification per liter
of fermentation medium. For example, the effective titer of butanol in a
unit volume of a fermentation includes: (i) the amount of butanol in the
fermentation medium; (ii) the amount of butanol recovered from the
organic extractant; (iii) the amount of butanol recovered from the gas
phase, if gas stripping is used, and (iv) the alcohol equivalent of the
butanol ester in either the organic or aqueous phase.
"Saccharification " as used herein means the break down of
oligosaccharides into monosaccharides. "Simultaneous saccharification
and fermentation" means fermentation and saccharification occur
concurrently in the same vessel.
As used herein, "saccharification enzyme" means one or more
enzymes that are capable of hydrolyzing polysaccharides and/or
ologosaccharides, e.g, alpha-1,4-glucosidic bonds of glycogen, starch.
Saccharification enzymes may include enzymes capable of hydrolyzing
cellulosic or lignocellulosic materials as well.
As used herein, "lipase activity" means the enzymatic activity of
catalyzing the hydrolysis of ester chemical bonds in water-insoluble or
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poorly water soluble lipid substrates. Lipases are a subclass of the
esterases, and as such, "lipase activity" also means the enzymatic activity
of catalyzing the hydrolysis of an ester into a carboxylic acid and an
alcohol, and, as used herein, "lipase activity" also means the enzymatic
activity of esterifying alcohol and carboxylic acid into an alcohol ester of a
carboxylic acid.
As used herein, "glycosylation" is the enzymatic addition of
carbohydrate molecules to biological macromolecules such as proteins,
which can occur when proteins are targeted for secretion out of the cell. In
0-glycosylation of proteins, the carbohydrates are attached to the hydroxyl
groups of serine, threonine, or tyrosine residues. In N-glycosylation of
proteins, the carbohydrates are attached to the amide side chain of
asparagine (N) residues in the consensus sequence NXS/T, where X is
any amino acid and SIT is serine or threonine. "Glycosylated" as used
herein refers to a protein molecule with carbohydrates covalently attached.
"Liquefaction vessel" as used herein means the vessel in which
liquefaction is carried out. Liquefaction is the process in which
oligosaccharides are liberated from the feedstock. In embodiments where
the feedstock is corn, oligosaccharides are liberated from the corn starch
content during liquefaction.
The term "separation" as used herein is synonymous with
"recovery" and refers to removing a chemical compound from an initial
mixture to obtain the compound in greater purity or at a higher
concentration than the purity or concentration of the compound in the
initial mixture.
The terms "water-immiscible" or "insoluble" refer to a chemical
component, such as an extractant or solvent, which is incapable of mixing
with an aqueous solution, such as a fermentation broth, in such a manner
as to form one liquid phase.
"Extractant" or "ISPR extractant" as used herein means an organic
solvent used to extract any product alcohol such as butanol, or used to
extract any product alcohol ester produced by a catalyst from a product
alcohol and a carboxylic acid or lipid. From time to time, as used herein
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the term "solvent" may be used synonymously with "extractant". For the
processes described herein, extractants are water-immiscible.
"Native oil" as used herein refers to lipids obtained from plants
(e.g., biomass) or animals. "Plant-derived oil" as used herein refers to
lipids obtained from plants in particular. From time to time, "lipids" may be
used synonymously with "oil" and "acyl glycerides." Native oils include, but
are not limited to, tallow, corn, canola, capric/caprylic triglycerides,
castor,
coconut, cottonseed, fish, jojoba, lard, linseed, neetsfoot, oiticica, palm,
peanut, rapeseed, rice, safflower, soya, sunflower, tung, jatropha and
vegetable oil blends.
The term "organic phase", as used herein, refers to the non-
aqueous phase of a biphasic mixture obtained by contacting a
fermentation broth with a water-immiscible organic extractant.
The term "fatty acid" as used herein refers to a carboxylic acid (e.g.,
aliphatic monocarboxylic acid) having 04 to 028 carbon atoms (most
commonly 012 to 024 carbon atoms), which is either saturated or
unsaturated. Fatty acids may also be branched or unbranched. Fatty
acids may be derived from, or contained in esterified form, in an animal or
vegetable fat, oil, or wax. Fatty acids may occur naturally in the form of
glycerides in fats and fatty oils or may be obtained by hydrolysis of fats or
by synthesis. The term fatty acid may describe a single chemical species
or a mixture of fatty acids. Fatty acids may comprise a mixture of both
protonated and unprotonated fatty acids, wherein the unprotonated fatty
acids are salts (e.g., sodium, potassium, ammonium or calcium ion salts)
of unprotonated fatty acids. In addition,
the term fatty acid also
encompasses free fatty acids.
The term "fatty alcohol" as used herein refers to an alcohol having
an aliphatic chain of 04 to 022 carbon atoms, which is either saturated or
unsaturated.
The term "fatty aldehyde" as used herein refers to an aldehyde
having an aliphatic chain of 04 to 022 carbon atoms, which is either
saturated or unsaturated.
The term "carboxylic acid" as used herein refers to any organic
compound with the general chemical formula -COOH in which a carbon
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atom is bonded to an oxygen atom by a double bond to make a carbonyl
group (-0=0) and to a hydroxyl group (-OH) by a single bond. A
carboxylic acid may be in the form of the protonated carboxylic acid, or in
the form of a salt of a carboxylic acid (for example, an ammonium, sodium
or potassium salt), or as a mixture of protonated carboxylic acid and salt of
a carboxylic acid. The term carboxylic acid may describe a single chemical
species (e.g., oleic acid), or a mixture of carboxylic acids as can be
produced, for example, by the hydrolysis of biomass-derived fatty-acid
esters or triglycerides, diglycerides, monoglyerides and phopholipids.
The term "butanol biosynthetic pathway" or "butanol production
pathway" as used herein refers to an enzyme pathway to produce 1-
butanol, 2-butanol, or isobutanol.
The term "1-butanol biosynthetic pathway" or "1-butanol production
pathway" as used herein refers to an enzyme pathway to produce 1-
butanol from acetyl-coenzyme A (acetyl-CoA).
The term "2-butanol biosynthetic pathway" or "2-butanol production
pathway" as used herein refers to an enzyme pathway to produce 2-
butanol from pyruvate.
The term "isobutanol biosynthetic pathway" or "isobutanol
production pathway" as used herein refers to an enzyme pathway to
produce isobutanol from pyruvate.
The term "alcohol biosynthetic pathway" or "alcohol production
pathway" as used herein refers to an enzymatic pathway to convert a
carbon substrate to an alcohol. A recombinant host cell comprising an
"engineered alcohol production pathway" refers to a host cell containing a
modified pathway that produces alcohol in a manner different than that
normally present in the host cell. Such differences include production of
an alcohol not typically produced by the host cell, or increased or more
efficient production.
The term "gene" refers to a nucleic acid fragment that is capable of
being expressed as a specific protein, optionally including regulatory
sequences preceding (5' non-coding sequences) and following (3' non-
coding sequences) the coding sequence. "Native gene" refers to a gene
as found in nature with its own regulatory sequences. "Chimeric gene"
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refers to any gene that is not a native gene (i.e, it is modified from its
native state or is from another source), comprising regulatory and coding
sequences that are not found together in nature. Accordingly, a chimeric
gene may comprise regulatory sequences and coding sequences that are
derived from different sources, or regulatory sequences and coding
sequences derived from the same source, but arranged in a manner
different than that found in nature. "Endogenous gene" refers to a native
gene in its natural location in the genome of an organism. A "foreign
gene" or "heterologous gene" refers to a gene not normally found as a
native gene in the host organism, but that is introduced into the host
organism by gene transfer. Foreign genes can comprise native genes
inserted into a non-native organism, or chimeric genes.
As used herein the term "coding region" refers to a DNA sequence
that codes for a specific amino acid sequence. "Suitable regulatory
sequences" refer to nucleotide sequences located upstream (5' non-
coding sequences), within, or downstream (3' non-coding sequences) of a
coding sequence, and which influence the transcription, RNA processing
or stability, or translation of the associated coding sequence. Regulatory
sequences may include promoters, translation leader sequences, introns,
polyadenylation recognition sequences, RNA processing sites, effector
binding sites and stem-loop structures.
The term "polynucleotide" is intended to encompass a singular
nucleic acid as well as plural nucleic acids, and refers to a nucleic acid
molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA
(pDNA). As used herein, a "gene" is a polynucleotide. A polynucleotide
can contain the nucleotide sequence of the full-length gene or cDNA
sequence, or a fragment thereof, including the untranslated 5' and 3'
sequences and the coding sequences. The polynucleotide can be
composed of any polyribonucleotide or polydeoxyribonucleotide, which
may be unmodified RNA or DNA or modified RNA or DNA (e.g.
heterologous DNA). For example, polynucleotides can be composed of
single- and double-stranded DNA, DNA that is a mixture of single- and
double-stranded regions, single- and double-stranded RNA, and RNA that
is mixture of single- and double-stranded regions, hybrid molecules
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comprising DNA and RNA that may be single-stranded or, more typically,
double-stranded or a mixture of single- and double-stranded regions.
"Polynucleotide" embraces chemically, enzymatically, or metabolically
modified forms.
"Engineered polynucleotide" as used herein refers to a
polynucleotide that has been modified from a form found in nature or that
is introduced into a host organism by gene transfer such as by
transformation. Such modification includes, for example, linking two
sequences not found linked in nature, such as operably linking a coding
sequence with a promoter not found operably linked with the coding
sequence in nature, or linking two coding sequences together to create a
chimeric coding sequence. Such modification also includes creating one
or more nucleotide changes, including base substitutions, insertions, or
deletions, to a polynucleotide found in nature.
A polynucleotide sequence may be referred to as "isolated," in
which it has been removed from its native environment. For example, a
heterologous polynucleotide encoding a polypeptide or polypeptide
fragment having dihydroxy-acid dehydratase activity contained in a vector
is considered isolated for the purposes of the present invention. Further
examples of an isolated polynucleotide include recombinant
polynucleotides maintained in heterologous host cells or purified (partially
or substantially) polynucleotides in solution. Isolated polynucleotides or
nucleic acids according to the present invention further include such
molecules produced synthetically. An isolated polynucleotide fragment in
the form of a polymer of DNA may be comprised of one or more segments
of cDNA, genomic DNA or synthetic DNA.
As used herein, the term "polypeptide" is intended to encompass a
singular "polypeptide" as well as plural "polypeptides," and refers to a
molecule composed of monomers (amino acids) linearly linked by amide
bonds (also known as peptide bonds). The term "polypeptide" refers to
any chain or chains of two or more amino acids, and does not refer to a
specific length of the product. Thus, peptides, dipeptides, tripeptides,
oligopeptides, "protein, " "amino acid chain," or any other term used to
refer to a chain or chains of two or more amino acids, are included within
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the definition of "polypeptide," and the term "polypeptide" may be used
instead of, or interchangeably with any of these terms. A polypeptide may
be derived from a natural biological source or produced by recombinant
technology, but is not necessarily translated from a designated nucleic
acid sequence. It may be generated in any manner, including by chemical
synthesis.
By an "isolated" polypeptide or a fragment, variant, or derivative
thereof is intended a polypeptide that is not in its natural milieu. No
particular level of purification is required. For
example, an isolated
polypeptide can be removed from its native or natural environment.
Recombinantly produced polypeptides and proteins expressed in host
cells are considered isolated for purposed of the invention, as are native or
recombinant polypeptides which have been separated, fractionated, or
partially or substantially purified by any suitable technique.
As used herein, "recombinant microorganism" refers to
microorganisms, such as bacteria or yeast, that are modified by use of
recombinant DNA techniques, such as by engineering a host cell to
comprise a biosynthetic pathway such as butanol.
As used herein the term "codon degeneracy" refers to the nature in
the genetic code permitting variation of the nucleotide sequence without
effecting the amino acid sequence of an encoded polypeptide. The skilled
artisan is well aware of the "codon-bias" exhibited by a specific host cell in
usage of nucleotide codons to specify a given amino acid. Therefore,
when synthesizing a gene for improved expression in a host cell, it is
desirable to design the gene such that its frequency of codon usage
approaches the frequency of preferred codon usage of the host cell.
The term "codon-optimized" as it refers to genes or coding regions
of nucleic acid molecules for transformation of various hosts, refers to the
alteration of codons in the gene or coding regions of the nucleic acid
molecules to reflect the typical codon usage of the host organism without
altering the polypeptide encoded by the DNA. Such optimization includes
replacing at least one, or more than one, or a significant number, of
codons with one or more codons that are more frequently used in the
genes of that organism.
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Deviations in the nucleotide sequence that comprise the codons
encoding the amino acids of any polypeptide chain allow for variations in
the sequence coding for the gene. Since each codon consists of three
nucleotides, and the nucleotides comprising DNA are restricted to four
specific bases, there are 64 possible combinations of nucleotides, 61 of
which encode amino acids (the remaining three codons encode signals
ending translation). The "genetic code" which shows which codons
encode which amino acids is reproduced herein as Table 1. As a result,
many amino acids are designated by more than one codon. For example,
the amino acids alanine and proline are coded for by four triplets, serine
and arginine by six, whereas tryptophan and methionine are coded by just
one triplet. This degeneracy allows for DNA base composition to vary
over a wide range without altering the amino acid sequence of the proteins
encoded by the DNA.
Table 1. The Standard Genetic Code
1 ic IA G ............ 1
1
'TTT Phe (F) fl'CT Ser (S) ',TAT Tyr (Y) sTGT
Cys (C)
'TTC " i-'cc " ..TAC " sTGC 1
T '[TA Leu (L) -1-CA" ..[AA Stop sTGA Stop 1
1
1
'TTG " -1-CG" ..TAG Stop sTGG Trp (W) 1
1
3 ........................ I i
is
. ,=
TT Leu (L) ;CCT Pro (P) AT His (H) GT Arg
(R) 1
1
TC " CCC " AC" GC"
1
C TA" !CCA" AA Gin (Q) GA" 1
1
1
TG" CCG " AG" GG " 1
,
1
1
1
1
,
;
= TT Ile (I) = CT Thr (T) = = T Asn
(N) = GT Ser (S) 1
i
'TC" 'CC" "C" 'GC" 1
1
1
A = TA" = CA" = = = Lys (K) = GA Arg
(R) 1
1
1
= TG Met (M) = CG" = = G"
= GG " 1
1
1
1
,
___________________________________________________________________ i
CTT Val (V) !CT Ala (A) iGAT Asp (D) 4GGT Gly (G)
CTC " !QCC " iGAC " 4GGC " i
G CTA " !GCA " iGAA Glu (E) 4GGA " i
i
CTG " CG"!Q iGAG " 4GGG " 1
1
1
1 1
1 1
Many organisms display a bias for use of particular codons to code
for insertion of a particular amino acid in a growing peptide chain. Codon
preference, or codon bias, differences in codon usage between
organisms, is afforded by degeneracy of the genetic code, and is well
documented among many organisms. Codon bias often correlates with
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the efficiency of translation of messenger RNA (mRNA), which is in turn
believed to be dependent on, inter alia, the properties of the codons being
translated and the availability of particular transfer RNA (tRNA) molecules.
The predominance of selected tRNAs in a cell is generally a reflection of
the codons used most frequently in peptide synthesis. Accordingly, genes
can be tailored for optimal gene expression in a given organism based on
codon optimization.
Given the large number of gene sequences available for a wide
variety of animal, plant and microbial species, it is possible to calculate
the
relative frequencies of codon usage. Codon usage tables are readily
available, for example, at the "Codon Usage Database" available at
http://www.kazusa.or.jp/codon/ (visited March 20, 2008), and these tables
can be adapted in a number of ways. See Nakamura, Y., et al. Nucl.
Acids Res. 28:292 (2000). Codon usage tables for yeast, calculated from
GenBank Release 128.0 [15 February 2002], are reproduced below as
Table 2. This table uses mRNA nomenclature, and so instead of thymine
(T) which is found in DNA, the tables use uracil (U) which is found in RNA.
Table 2 has been adapted so that frequencies are calculated for each
amino acid, rather than for all 64 codons.
Table 2. Codon Usage Table for Saccharomyces cerevisiae
Genes
Amino Acid Codon Number Frequency per
thousand
Phe UUU 170666 26.1
Phe UUC 120510 18.4
Leu UUA 170884 26.2
Leu UUG 177573 27.2
Leu CUU 80076 12.3
Leu CUC 35545 5.4
Leu CUA 87619 13.4
Leu CUG 68494 10.5
Ile AUU 196893 30.1
Ile AUC 112176 17.2
Ile AUA 116254 17.8
Met AUG 136805 20.9
Val GUU 144243 22.1
Val GUC 76947 11.8
Val GUA 76927 11.8
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Amino Acid Codon Number Frequency per
thousand
Val GUG 70337 10.8
Ser UCU 153557 23.5
Ser UCC 92923 14.2
Ser UCA 122028 18.7
Ser UCG 55951 8.6
Ser AGU 92466 14.2
Ser AGC 63726 9.8
Pro CCU 88263 13.5
Pro CCC 44309 6.8
Pro CCA 119641 18.3
Pro CCG 34597 5.3
Thr ACU 132522 20.3
Thr ACC 83207 12.7
Thr ACA 116084 17.8
Thr ACG 52045 8.0
Ala GCU 138358 21.2
Ala GCC 82357 12.6
Ala GCA 105910 16.2
Ala GCG 40358 6.2
Tyr UAU 122728 18.8
Tyr UAC 96596 14.8
His CAU 89007 13.6
His CAC 50785 7.8
Gln CAA 178251 27.3
Gln CAG 79121 12.1
Asn AAU 233124 35.7
Asn AAC 162199 24.8
Lys AAA 273618 41.9
Lys AAG 201361 30.8
Asp GAU 245641 37.6
Asp GAC 132048 20.2
Glu GAA 297944 45.6
Glu GAG 125717 19.2
Cys UGU 52903 8.1
Cys UGC 31095 4.8
Trp UGG 67789 10.4
Arg CGU 41791 6.4
Arg CGC 16993 2.6
Arg CGA 19562 3.0
Arg CGG 11351 1.7
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Amino Acid Codon Number Frequency per
thousand
Arg AGA 139081 21.3
Arg AGG 60289 9.2
Gly GGU 156109 23.9
Gly GGC 63903 9.8
Gly GGA 71216 10.9
Gly GGG 39359 6.0
Stop UAA 6913 1.1
Stop UAG 3312 0.5
Stop UGA 4447 0.7
By utilizing this or similar tables, one of ordinary skill in the art can
apply the frequencies to any given polypeptide sequence, and produce a
nucleic acid fragment of a codon-optimized coding region which encodes
the polypeptide, but which uses codons optimal for a given species.
Randomly assigning codons at an optimized frequency to encode a
given polypeptide sequence, can be done manually by calculating codon
frequencies for each amino acid, and then assigning the codons to the
polypeptide sequence randomly. Additionally, various algorithms and
computer software programs are readily available to those of ordinary skill
in the art. For example, the "EditSeq" function in the Lasergene Package,
available from DNAstar, Inc., Madison, WI, the backtranslation function in
the VectorNTI Suite, available from InforMax, Inc., Bethesda, MD, and the
"backtranslate" function in the GCG-Wisconsin Package, available from
Accelrys, Inc., San Diego, CA. In addition, various resources are publicly
available to codon-optimize coding region sequences, e.g., the
"backtranslation" function at
http://www.entelechon.com/bioinformatics/backtranslation.php?lang=eng
(visited April 15, 2008) and the "backtranseq" function available at
http://bioinfo.pbi.nrc.ca:8090/EMBOSS/index.html (visited July 9, 2002).
Constructing a rudimentary algorithm to assign codons based on a given
frequency can also easily be accomplished with basic mathematical
functions by one of ordinary skill in the art.
Codon-optimized coding regions can be designed by various
methods known to those skilled in the art including software packages
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such as "synthetic gene
designer"
(userpages.umbc.edu/¨wug1/codon/sgd/, accessed March 19, 2012_).
A polynucleotide or nucleic acid fragment is "hybridizable" to
another nucleic acid fragment, such as a cDNA, genomic DNA, or RNA
molecule, when a single-stranded form of the nucleic acid fragment can
anneal to the other nucleic acid fragment under the appropriate conditions
of temperature and solution ionic strength. Hybridization and washing
conditions are well known and exemplified in Sambrook, J., Fritsch, E. F.
and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold
Spring Harbor Laboratory: Cold Spring Harbor, NY (1989), particularly
Chapter 11 and Table 11.1 therein. The conditions of temperature and
ionic strength determine the "stringency" of the hybridization. Stringency
conditions can be adjusted to screen for moderately similar fragments
(such as homologous sequences from distantly related organisms), to
highly similar fragments (such as genes that duplicate functional enzymes
from closely related organisms). Post-hybridization washes determine
stringency conditions. One set of preferred conditions uses a series of
washes starting with 6X SSC, 0.5% SDS at room temperature for 15 min,
then repeated with 2X SSC, 0.5% SDS at 45 C for 30 min, and then
repeated twice with 0.2X SSC, 0.5% SDS at 50 C for 30 min. A more
preferred set of stringent conditions uses higher temperatures in which the
washes are identical to those above except for the temperature of the final
two 30 min washes in 0.2X SSC, 0.5% SDS was increased to 60 C.
Another preferred set of highly stringent conditions uses two final washes
in 0.1X SSC, 0.1% SDS at 65 C. An additional set of stringent conditions
include hybridization at 0.1X SSC, 0.1% SDS, 65 C and washes with 2X
SSC, 0.1% SDS followed by 0.1X SSC, 0.1% SDS, for example.
Hybridization requires that the two nucleic acids contain
complementary sequences, although depending on the stringency of the
hybridization, mismatches between bases are possible. The appropriate
stringency for hybridizing nucleic acids depends on the length of the
nucleic acids and the degree of complementation, variables well known in
the art. The greater the degree of similarity or homology between
two nucleotide sequences, the greater the value of T, for hybrids of
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nucleic acids having those sequences. The relative stability
(corresponding to higher Tm) of nucleic acid hybridizations decreases in
the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of
greater than 100 nucleotides in length, equations for calculating Tm have
been derived (see Sambrook et al., supra, 9.50-9.51). For hybridizations
with shorter nucleic acids, i.e., oligonucleotides, the position of
mismatches becomes more important, and the length of the
oligonucleotide determines its specificity (see Sambrook et al., supra,
11.7-11.8). In one embodiment the length for a hybridizable nucleic acid is
at least about 10 nucleotides. Preferably a
minimum length for a
hybridizable nucleic acid is at least about 15 nucleotides; more preferably
at least about 20 nucleotides; and most preferably the length is at least
about 30 nucleotides. Furthermore, the skilled artisan will recognize that
the temperature and wash solution salt concentration may be adjusted as
necessary according to factors such as length of the probe.
A "substantial portion" of an amino acid or nucleotide sequence is
that portion comprising enough of the amino acid sequence of a
polypeptide or the nucleotide sequence of a gene to putatively identify that
polypeptide or gene, either by manual evaluation of the sequence by one
skilled in the art, or by computer-automated sequence comparison and
identification using algorithms such as Basic Local Alignment Search Tool
("BLAST"; Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)). In
general, a sequence of ten or more contiguous amino acids or thirty or
more nucleotides is necessary in order to putatively identify a polypeptide
or nucleic acid sequence as homologous to a known protein or gene.
Moreover, with respect to nucleotide sequences, gene specific
oligonucleotide probes comprising 20-30 contiguous nucleotides may be
used in sequence-dependent methods of gene identification (e.g.,
Southern hybridization) and isolation (e.g., in situ hybridization of
bacterial
colonies or bacteriophage plaques). In addition, short oligonucleotides of
12-15 bases may be used as amplification primers in PCR in order to
obtain a particular nucleic acid fragment comprising the primers.
Accordingly, a "substantial portion" of a nucleotide sequence comprises
enough of the sequence to specifically identify and/or isolate a nucleic acid
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fragment comprising the sequence. The instant specification teaches the
complete amino acid and nucleotide sequence encoding particular
proteins. The skilled artisan, having the benefit of the sequences as
reported herein, may now use all or a substantial portion of the disclosed
sequences for purposes known to those skilled in this art. Accordingly, the
instant invention comprises the complete sequences as provided herein,
as well as substantial portions of those sequences as defined above.
The term "complementary" is used to describe the relationship
between nucleotide bases that are capable of hybridizing to one another.
For example, with respect to DNA, adenosine is complementary to
thymine and cytosine is complementary to guanine.
The term "percent identity", as known in the art, is a relationship
between two or more polypeptide sequences or two or more
polynucleotide sequences, as determined by comparing the sequences.
In the art, "identity" also means the degree of sequence relatedness
between polypeptide or polynucleotide sequences, as the case may be, as
determined by the match between strings of such sequences. "Identity"
and "similarity" can be readily calculated by known methods, including but
not limited to those disclosed in: 1.) Computational Molecular Biology
(Lesk, A. M., Ed.) Oxford University: NY (1988); 2.) Biocomputing:
Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY
(1993); 3.) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and
Griffin, H. G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in
Molecular Biology (von Heinje, G., Ed.) Academic (1987); and
5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.)
Stockton: NY (1991).
Preferred methods to determine identity are designed to give the
best match between the sequences tested. Methods to determine identity
and similarity are codified in publicly available computer programs.
Sequence alignments and percent identity calculations may be performed
using the MegAlignTM program of the LASERGENE bioinformatics
computing suite (DNASTAR Inc., Madison, WI). Multiple alignment of the
sequences is performed using the "Clustal method of alignment" which
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encompasses several varieties of the algorithm including the "Clustal V
method of alignment" corresponding to the alignment method labeled
Clustal V (disclosed by Higgins and Sharp, CAB/OS. 5:151-153 (1989);
Higgins, D.G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in
the MegAlignTM program of the LASERGENE bioinformatics computing
suite (DNASTAR Inc.). For
multiple alignments, the default values
correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10.
Default parameters for pairwise alignments and calculation of percent
identity of protein sequences using the Clustal method are KTUPLE=1,
GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic
acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4
and DIAGONALS SAVED=4. After alignment of the sequences using the
Clustal V program, it is possible to obtain a "percent identity" by viewing
the "sequence distances" table in the same program. Additionally the
"Clustal W method of alignment" is available and corresponds to the
alignment method labeled Clustal W (described by Higgins and Sharp,
CAB/OS. 5:151-153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci.
8:189-191(1992)) and found in the MegAlignTM v6.1 program of the
LASERGENE bioinformatics computing suite (DNASTAR Inc.). Default
parameters for multiple alignment (GAP PENALTY=10, GAP LENGTH
PENALTY=0.2, Delay Divergen Seqs(`)/0)=30, DNA Transition Weight=0.5,
Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB ). After
alignment of the sequences using the Clustal W program, it is possible to
obtain a "percent identity" by viewing the "sequence distances" table in the
same program.
It is well understood by one skilled in the art that many levels of
sequence identity are useful in identifying polypeptides, including variants
or polypeptides from other species, wherein such polypeptides have the
same or similar function or activity. Useful examples of percent identities
include, but are not limited to: 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, or 95%, or any integer percentage from 55% to 100% may be useful
in describing the present invention, such as 55%, 56%, 57%, 58%, 59%,
60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,
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73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%
or 99%. Suitable nucleic acid fragments not only have the above
homologies but typically encode a polypeptide having at least 50 amino
acids, preferably at least 100 amino acids, more preferably at least
150 amino acids, still more preferably at least 200 amino acids, and most
preferably at least 250 amino acids.
The term "sequence analysis software" refers to any computer
algorithm or software program that is useful for the analysis of nucleotide
or amino acid sequences. "Sequence
analysis software" may be
commercially available or independently developed. Typical sequence
analysis software will include, but is not limited to: 1.) the GCG suite of
programs (Wisconsin Package Version 9.0, Genetics Computer Group
(GCG), Madison, WI); 2.) BLASTP, BLASTN, BLASTX (Altschul et al.,
J. Mol. Biol., 215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc.
Madison, WI); 4.) Sequencher (Gene Codes Corporation, Ann Arbor, MI);
and 5.) the FASTA program incorporating the Smith-Waterman algorithm
(W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.]
(1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Plenum:
New York, NY). Within the context of this application it will be understood
that where sequence analysis software is used for analysis, that the
results of the analysis will be based on the "default values" of the program
referenced, unless otherwise specified. As used herein "default values"
will mean any set of values or parameters that originally load with the
software when first initialized.
Standard recombinant DNA and molecular cloning techniques used
here are well known in the art and are described by Sambrook, J., Fritsch,
E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
(1989) (hereinafter "Maniatis"); and by Silhavy, T. J., Bennan, M. L. and
Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY (1984); and by Ausubel, F. M.
et al., Current Protocols in Molecular Biology, published by Greene
Publishing Assoc. and Wiley-Interscience (1987). Additional methods
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used here are in Methods in Enzymology, Volume 194, Guide to Yeast
Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie
and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, CA).
The genetic manipulations of a recombinant host cell disclosed
herein can be performed using standard genetic techniques and screening
and can be made in any host cell that is suitable to genetic manipulation
(Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY, pp. 201-202). In embodiments, the recombinant
host cell is E. coli. In embodiments, a recombinant host cell disclosed
herein can be any yeast or fungal host useful for genetic modification and
recombinant gene expression. In other embodiments, a recombinant host
cell can be a member of the genera Zygosaccharomyces,
Schizosaccharomyces, Dekkera, lssatchenkia, Torulopsis, Brettanomyces,
Torulaspora, Hanseniaspora, Kluyveromyces, and some species of
Candida. In another embodiment, a recombinant host cell can be
Saccharomyces cerevisiae.
The Applicants have discovered that recombinant host cells which
are able to express and secrete lipase enzymes into a fermentation
medium produce a catalyst that will catalyze the esterification of alcohol
and carboxylic acid. Such host cells represent an improvement to host
cells used in fermentative production of alcohols because the esterification
of the alcohol may allow the cells to produce alcohol with greater
efficiency, or to produce an amount of alcohol in excess of the amount of
alcohol that would exert a toxic effect on the host cells. Also, use of such
recombinant microorganisms can reduce or eliminate the need to add
purified lipase enzyme to a fermentation medium to carry out the
processes described herein, which may provide cost and operational
advantages.
Furthermore, fermentative production of alcohols typically utilizes a
renewable biomass feedstock to supply the carbon substrate which a
recombinant microorganism converts to product alcohol. Such feedstocks
can contain an amount of triglycerides. When extractive fermentation is
practiced to remove the product alcohol from the fermentation, the
triglycerides may build up over time, decreasing the partition coefficient
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and recyclability of the extractant. The
lipases secreted by the
recombinant host cells provided herein can advantageously hydrolyze the
triglycerides into free fatty acids which may be substrates for esterification
and which may also have less effect on the partition coefficient of an
extractant for product alcohols.
Polypeptides having lipase activity
Recombinant host cells disclosed herein comprise polynucleotides
having polypeptides having lipase activity. Examples
of lipase
polynucleotides and polypeptides and the organisms from which they are
derived are provided in Table 3.
Table 3. Example lipase polynucleotides and polypeptides
Species and Nucleic acid Amino acid Nucleic Acid
Accession Number SEQ ID NO: SEQ ID NO: Sequence,
or Reference Codon-
optimized for
expression in
S. cerevisiae
Candida antarctica 1 2 7
Z30645
Candida deformans 3 4 8
AJ428393
The rmomyces 5 6 9
lanuginosus
AF054513
Aspergillus 255 256 257
tubingensis lip3
US Patent
7,371,42362;
PCT App. Pub. No.
W098/45453
BLAST analysis of the non-redundant protein sequence database
at the National Center for Biotechnology Information was performed using,
as query sequences, lipases described in Table 3, in order to identify
proteins with high (>90%) sequence similarity. Results are shown in Table
4 (information retrieved from the non-redundant protein sequence
database online at the National Center for Biotechnology Information on
January 22, 2012). While proteins with sequence similarity greater than
90% are considered to be lipases that are predicted to perform similarly to
lipases described herein, sequences with similarity as low as ¨30% to the
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query sequences are annotated as lipases and are contemplated for use
with the methods and compositions described herein.
Table 4. Additional example lipase polypeptides
Source Organism GenBank Identity to lipase Amino
Accession acid SEQ
Number ID NO:
Aspergillus kawachii GAA84811 99% to Aspergillus tubingensis 241
Aspergillus niger BAL22280 98% to Aspergillus tubingensis 242
Aspergillus niger XP_001397501 93% to Aspergillus tubingensis 243
Aspergillus niger ABG73613 93% to Aspergillus tubingensis 244
Aspergillus niger ABG37906 93% to Aspergillus tubingensis 245
Yarrowia lipolytica XP 500282 92% to Candida deformans 246
Yarrowia lipolytica ADL57415 91% to Candida deformans 247
Talaromyces the rmophiles AEE61324 90% to Thermomyces
lanuginosus 248
In addition to the lipases described above and in Tables 3 and 4,
suitable lipase sequences may be derived from any source, including, for
example, Absidia, Achromobacter, Aeromonas, Alcaligenes, Altemaria,
Aspergillus, Achromobacter, Aureobasidium, Bacillus, Beauveria,
Brochothrix, Candida, Chromobacter, Coprinus, Fusarium, Geotricum,
Hansenula, Humicola, Hyphozyma, Lactobacillus, Metarhizium, Mucor,
Nectria, Neurospora, Paecilomyces, Penicillium, Pseudomonas,
Rhizoctonia, Rhizomucor, Rhizopus, Rhodosporidium, Rhodotorula,
Saccharomyces, Sus, Sporobolomyces, Thermomyces, Thiarosporella,
Trichoderma, Verticillium, and/or a strain of Yarrowia. In embodiments,
the source of the lipase is selected from the group consisting of Absidia
blakesleena, Absidia corymbifera, Achromobacter iophagus, Alcaligenes
sp., Altemaria brassiciola, Aspergillus flavus, Aspergillus niger, Aspergillus
kawachii, Aspergillus tubingensis, Aureobasidium pullulans, Bacillus
pumilus, Bacillus strearothermophilus, Bacillus subtilis, Brochothrix
thermosohata, Candida cylindracea (Candida rugosa), Candida
paralipolytica, Candida antarctica lipase A, Candida antarctica lipase B,
Candida emobii, Candida deformans, Candida thermophila, Chromobacter
viscosum, Coprinus cinerius, Fusarium oxysporum, Fusarium solani,
Fusarium solani pisi, Fusarium roseum culmorum, Geotrichum candidum,
Geotricum penicilla turn, Hansen ula anomala, Humicola brevispora,
Humicola brevis var. thermoidea, Humicola insolens, Lactobacillus
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curvatus, Rhizopus niveus, Rhizopus oryzae, Penicillium cyclopium,
Penicillium crustosum, Penicillium expansum, Penicillium sp. I, Penicillium
sp. II, Pseudomonas aeruginosa, Pseudomonas alcaligenes,
Pseudomonas cepacia (syn. Burkholderia cepacia), Pseudomonas
fluorescens, Pseudomonas fragi, Pseudomonas maltophilia,
Pseudomonas men docina, Pseudomonas mephitica lipolytica,
Pseudomonas alcaligenes, Pseudomonas plantar, Pseudomonas
pseudoalcaligenes, Pseudomonas putida, Pseudomonas stutzeri, and
Pseudomonas wisconsinensis, Rhizoctonia solani, Rhizomucor miehei,
Rhizopus japonicus, Rhizopus microsporus, Rhizopus nodosus,
Rhodosporidium toruloides, Rhodotorula glutinis, Saccharomyces
cerevisiae, Sporobolomyces shibatanus, Sus scrofa, Talaromyces
the rmophiles, The rmomyces lanuginosus (formerly Humicola lanuginose),
Thiarosporella phaseolina, Trichoderma harzianum, Trichoderma reesei,
and Yarrowia lipolytica. In embodiments, the lipase is selected from the
group consisting of Thermomyces lanuginosus lipase, Aspergillus sp.
lipase, Aspergillus niger lipase, Aspergillus tubingensis lip3, Candida
antarctica lipase B, Pseudomonas sp. lipase, Penicillium roqueforti lipase,
Penicillium camembertii lipase, Mucor javanicus lipase, Burkholderia
cepacia lipase, Alcaligenes sp. lipase, Candida rugosa lipase, Candida
parapsilosis lipase, Candida deformans lipases, lipases A and B from
Geotrichum can didum, Neurospora crassa lipase, Nectria haematococca
lipase, Fusarium heterosporum lipase Rhizopus delemar lipase,
Rhizomucor miehei lipase, Rhizopus arrhizus lipase, and Rhizopus oryzae
lipase.
One of skill in the art will appreciate that polynucleotide sequences
that encode polypeptides with lipase activity such as the polynucleotide
sequences in the table above or derived from the indicated sources can be
codon-optimized for the recombinant host cell. Further, one of skill in the
art will appreciate that truncations and conservative substitutions can be
made to the polypeptide sequences given without eliminating the lipase
activity of the polypeptide. Accordingly, provided herein are polypeptides
having at least about 75%, at least about 80%, at least about 90%, at least
about 95%, at least about 97%, or at least about 99% identity to the
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sequences provided and active fragments thereof. Also provided are
polynucleotides encoding such polypeptides.
For embodiments of the methods and host cells described herein,
that the polypeptide having lipase activity may be expressed and secreted
by the microorganism such that the lipase has activity in the fermentation
medium during the production of a product alcohol. One of skill in the art
will appreciate that polypeptides expressed on the surface of a
microorganism, such as cell wall proteins which are processed through the
secretory pathway, will be considered to be secreted since the activity of a
polypeptide expressed on the cell surface can be available external to the
cell. Thus, in embodiments, the secreted lipase is expressed on the
surface of the microorganism. Surface expression of proteins is known in
the art, as is modification of polypeptides to target them for surface
expression. (Washida, M., S. Takahashi, M. Ueda and A. Tanaka (2001).
"Spacer-mediated display of active lipase on the yeast cell surface." Appl
Microbiol Biotechnol 56(5-6): 681-686, Matsumoto, T., H. Fukuda, M.
Ueda, A. Tanaka and A. Kondo (2002). "Construction of yeast strains with
high cell surface lipase activity by using novel display systems based on
the Flo1p flocculation functional domain." Appl Environ Microbiol 68(9):
4517-4522, Mormeneo, M., I. Andres, C. Bofill, P. Diaz and J. Zueco
(2008). "Efficient secretion of Bacillus subtilis lipase A in Saccharomyces
cerevisiae by translational fusion to the Pir4 cell wall protein." Appl.
Microbiol. Biotechnol. 80(3): 437-445, Liu, W., H. Zhao, B. Jia, L. Xu and
Y. Yan (2010). "Surface display of active lipase in Saccharomyces
cerevisiae using Cwp2 as an anchor protein." Biotechnology Letters 32(2):
255-260, Su, G.-d., X. Zhang and Y. Lin (2010). "Surface display of active
lipase in Pichia pastoris using Sed1 as an anchor protein." Biotechnology
Letters 32(8): 1131-1136, Kuroda, K. and M. Ueda (2011). "Cell surface
engineering of yeast for applications in white biotechnology."
Biotechnology Letters 33(1): 1-9). In
embodiments, a polypeptide
provided herein is fused to a domain of a protein which targets the
polypeptide to the cell surface In embodiments, polypeptides provided
herein are fused to a domain of Flo1p, Pir4, Sed1, Sag1p, Cwp2, or Aga2.
In embodiments, polypeptides provided herein are fused to a protein, or a
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fragment of a protein, having a GPI anchor motif. GPI anchor motifs are
known to those of skill in the art and can be predicted by bioinformatics,
for example by using prediction engines (for example, the prediction
engine online at mendel.imp.ac.at/gpi/fungi_server.html, accessed March
19, 2012). (Eisenhaber B., et al.
"A sensitive predictor for potential GPI lipid modification sites in fungal
protein sequences and its application to genome-wide studies for
Aspergillus nidulans, Candida albicans, Neurospora crassa,
Saccharomyces cerevisiae, and Schizosaccharomyces pombe"
J Mol Biol. 2004 Mar 19;337(2):243-53.) Example polypeptide sequence
domains which may be used target a polypeptide to the cell surface of
Saccharomyces cerevisiae are shown in Table 5. Systematic names of
the proteins in Table 5 are according to the Saccharomyces Genome
Database ("SGD"; online at www.yeastgenome.orgi; information retrieved
March 13, 2012). One of skill in the art, equipped with this disclosure, will
be able to use the example polypeptide sequences and other such
sequences known in the art to construct polypeptides which target lipase
activity to the cell surface of a recombinant microorganism.
Table 5. Polypeptide sequences of cell surface anchor domains of
S. cerevisiae proteins for surface display.
Protein name SGD systematic Codons of nucleic Amino acid
name of protein acid sequence sequence of
corresponding to domain
protein domain SEQ ID NO:
Sag1 YJR004C 331-650 249
Aga2 YGL032C 1-871 250
Flo1 YARO5OW 1-1099 251
Cwp2 YKL096W 1-92 252
Sed1 YDR077W 2-338 253
ICo-express Aga2 domain with Aga1 (SGD systematic name: YNR044W;
SEQ ID NO: 254)
In embodiments, the lipase polypeptide sequences provided herein
may be modified such that glycosylation, including, but not limited to, N-
glycosylation, is reduced or eliminated. Such modification can be carried
out by mutating the polynucleotide encoding the polypeptide such that one
or more glycosylation motifs is removed. In
embodiments, the
glycosylation motif is an N-glycosylation motif. In embodiments, the
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glycosylation motif is NXS/T. In embodiments, the polypeptide having
lipase activity does not contain the glycosylation motif NXS/T.
Glycosylation can be reduced or eliminated by any means known in
the art. For example, inhibitors of glycosylation such as tunicamycin may
be employed or the glycosylation mechanism in a host cell may be altered.
Also, glycosylation motifs can be removed by site-directed mutagenesis
using techniques known in the art. For
example, site-directed
mutagenesis can be carried out using commercially available kits (for
example, the QuikChange II XL site directed mutagenesis kit, Catalog #
200524, Stratagene, La Jolla, CA). Site-direct mutagenesis can be carried
out by the method of Kunkel, involving incorporation of uracil into the
template to be mutated (Kunkel TA (1985) Rapid and efficient site-specific
mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA
82:488-492), or by the method of phosphorothioate incorporation (Taylor
JW, Ott J, & Eckstein F (1985), The rapid generation of oligonucleotide-
directed mutations at high frequency using phosphorothioate-modified
DNA. Nucleic Acids Res 13:8765-8785), or by other methods, in vitro and
in vivo, known in the art. Primer design for target sites for mutagenesis is
well-known in the art, and sequence analysis such as multiple sequence
alignment to identify target sites for mutagenesis is likewise well-known.
In embodiments, mutagenesis is carried out such that the N of the
motif is substituted with any other naturally occurring amino acid (A, R, D,
C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V; see Table 1). In
embodiments, the N of the motif is substituted with A. In embodiments,
mutagenesis is carried out such that the S/T of the motif is replaced with
any other naturally occurring amino acid (A, R, N, D, C, E, Q, G, H, I, L, K,
M, F, P, W, Y, or V; see Table 1). In embodiments, both the N and the S/T
are replaced with any other naturally occurring amino acid (A, R, J, D, C,
E, Q, G, H, I, L, K, M, F, P, W, Y, or V, or S or T at the N residue; A, R, J,
D, C, E, Q, G, H, I, L, K, M, F, P, W, Y, or V,S or N at a T residue; A, R, J,
D, C, E, Q, G, H, I, L, K, M, F, P, W, Y, or V, or T or N at an S residue). In
embodiments, the glycosylation motif NXS/T is replaced with the motif
AXS/T.
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In one non-limiting example, C. deformans contains two
glycosylation sequences, NIS at codon 146 and NNT at 167. In
embodiments, one or both of those glycosylation sites is targeted for
substitution and the indicated glycosylation sites are replaced with AIS and
ANT, respectively. C. antarctica has NDT at 99, and T. lanuginosus has
NIT at 55. In embodiments, the indicated glycosylation sites are mutated
such that the sequences are ADT and AIT at the indicated positions.
Given in Table 6 are predicted glycosylation sites lipase open
reading frames from C. deformans, C. antarctica, and T. lanuginosus, and
examples of mutations that abolish those sites. The first column lists the
position in the polypeptide at which the glycosylation site occurs. The
second column gives the glycosylation sequence at that position, and the
DNA sequence encoding it in the codon-optimized polynucleotide. The
third column gives the polypeptide sequence at that position after
mutagenesis, and the DNA sequence required to effect that amino acid
change.
Table 6. Predicted glycosylation sites
Yeast species and Native Modified SEQ ID SEQ ID
glycosylation site NO: of NO: of
position nucleic amino
acid acid
sequence sequence
C. deformans 146 N I S A I S 48 49
AATATCAGT GCTATCAGT
C. deformans 167 N N T A N T 50 51
ACAATACAT GC TATACAT
C. antarctica 99 N D T A D T 46 47
AATGATACT GCTGATACT
T. lanuginosus 55 N I T A I T 54 55
AACATTACA GC TAT TACA
A. tubingensis 59 N L T A L T 271 272
AACTTAACA GCTTTAACA
A. tubingensis 269 N S T A S T 273 274
AATTCTACA GCTTCTACA
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In addition, the nucleic acid and amino acid sequences of Candida
deformans lipase with both of the modifications listed in Table 6 (N146A
and N167A) are given as SEQ ID NOs: 52 and 53. The nucleic acid and
amino acid sequences of an A. tubingensis lipase with both of the
modifications listed in Table 6 (N59A and N269A) are given as SEQ ID
NOs: 275 and 276.
As shown in the Examples, using techniques known in the art
and/or provided herein, one of skill in the art can readily modify
glycosylation motifs in lipases and determine the activity of such lipases in
methods and compositions provided herein.
One of skill in the art will appreciate that provided herein are
polypeptides having at least about 75%, at least about 80%, at least about
90%, at least about 95%, at least about 97%, or at least about 99%
identity to the sequences provided and active fragments thereof. Also
provided are polynucleotides encoding such polypeptides. One of skill in
the art will also appreciate that active variants of the sequences provided
herein can be created using techniques known in the art and or described
herein for use in the methods and compositions described herein.
Recombinant microorganisms and butanol biosynthetic pathways
While not wishing to be bound by theory, it is believed that the
improvements and processes described herein may be useful in
conjunction with any alcohol producing microorganism, particularly
recombinant microorganisms which produce alcohol at titers above their
tolerance levels.
Alcohol-producing microorganisms are known in the art. For
example, fermentative oxidation of methane by methanotrophic bacteria
(for example, Methylosinus trichosporium) produces methanol, contacting
methanol (a Ci alkyl alcohol) with a carboxylic acid and a catalyst capable
of esterifying the carboxylic acid with methanol forms a methanol ester of
the carboxylic acid. The wild-type yeast strain CEN.PK113-7D (CBS
8340, the Centraal Buro voor Schimmelculture; van Dijken JP, et al., 2000,
An interlaboratory comparison of physiological and genetic properties of
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four Saccharomyces cerevisiae strains. Enzyme Microb. Technol. 26:706-
714) can produce ethanol; contacting ethanol with a carboxylic acid and a
catalyst capable of esterifying the carboxylic acid with the ethanol forms
ethyl ester.
Recombinant microorganisms which produce alcohol are also
known in the art (for example, Ohta et al.,1991, Appl. Environ. Microbiol.
57:893-900; Underwood et al.,2002, Appl. Environ. Microbiol. 68:1071-
1081; Shen and Liao, 2008, Metab. Eng. 10:312-320; Hahnai et al.,
2007,Appl. Environ. Microbiol. 73:7814-7818; US Patent No. 5,514,583,
US Patent No. 5,712,133; PCT Application Pub. No. W01995028476;
Feldmann et al., 1992, Appl. Microbiol. Biotechnol. 38: 354-361; Zhang et
al., 1995, Science 267:240-243; 20070031918 Al; US Patent No.
7,223,575, US Patent No. 7,741,119; US 20090203099 Al; US
Application Pub. No. 2009/0246846 Al; and PCT Application Pub. No.
W02010/075241, which are herein incorporated by reference).
Suitable recombinant microorganisms capable of producing butanol
are known in the art, and certain suitable microorganisms capable of
producing butanol are described herein. Recombinant microorganisms to
produce butanol via a biosynthetic pathway can include a member of the
genera Clostridium, Zymomonas, Escherichia, Salmonella, Serratia,
Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus,
Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus,
Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces,
Kluyveromyces, Yarrowia, Pichia, Candida, Hansenula, lssatchenkia, or
Saccharomyces. In one embodiment, recombinant microorganisms can
be selected from the group consisting of Escherichia coli, Lactobacillus
plantarum, and Saccharomyces cerevisiae. In one embodiment, the
recombinant microorganism is a yeast. In one
embodiment, the
recombinant microorganism is crabtree-positive yeast selected from
Saccharomyces, Zygosaccharomyces, Schizosaccharomyces, Dekkera,
Torulopsis, Brettanomyces, and some species of Candida. Species of
crabtree-positive yeast include, but are not limited to, Saccharomyces
cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces pombe,
Saccharomyces bayanus, Saccharomyces mikitae, Saccharomyces
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paradoxus, Zygosaccharomyces rouxii, and Candida glabrata. In some
embodiments, the host cell is Saccharomyces cerevisiae. S. cerevisiae
yeast are known in the art and are available from a variety of sources,
including, but not limited to, American Type Culture Collection (Rockville,
MD), Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity
Centre, LeSaffre, Gert Strand AB, Ferm Solutions, North American
Bioproducts, Martrex, and Lallemand. S. cerevisiae yeast include, but are
not limited to, BY4741, CEN.PK 113-7D, Ethanol Red yeast, Ferm ProTM
yeast, Bio-Ferm XR yeast, Gert Strand Prestige Batch Turbo alcohol
yeast, Gert Strand Pot Distillers yeast, Gert Strand Distillers Turbo yeast,
FerMaxTm Green yeast, FerMaxTm Gold yeast, Thermosacc0 yeast, BG-1,
PE-2, CAT-1, CB57959, CB57960, and CBS7961.
Additionally, recombinant microbial production hosts comprising a
1-butanol biosynthetic pathway (U.S. Patent Application Publication No.
US20080182308A1, herein incorporated by reference), a 2-butanol
biosynthetic pathway (U.S. Patent Publication Nos. US 20070259410A1,
herein incorporated by reference and US 20070292927, herein
incorporated by reference), and an isobutanol biosynthetic pathway (U.S.
Patent Publication No. US 20070092957, herein incorporated by
reference) have been described.
The production of butanol utilizing fermentation with a
microorganism, as well as microorganisms which produce butanol, is
disclosed, for example, in U.S. Pub. No. 2009/0305370, herein
incorporated by reference. In some
embodiments, microorganisms
comprise a butanol biosynthetic pathway. In embodiments, at least one,
at least two, at least three, or at least four polypeptides catalyzing
substrate to product conversions of a pathway are encoded by
heterologous polynucleotides in the microorganism. In embodiments, all
polypeptides catalyzing substrate to product conversions of a pathway are
encoded by heterologous polynucleotides in the microorganism. In some
embodiments, the microorganism comprises a reduction or elimination of
pyruvate decarboxylase activity. Microorganisms substantially free of
pyruvate decarboxylase activity are described in US Application
Publication No. 20090305363, herein incorporated by reference.
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Microorganisms substantially free of an enzyme having NAD-dependent
glycerol-3-phosphate dehydrogenase activity such as GPD2 are also
described therein.
Butanol Biosynthetic Pathways
Certain suitable isobutanol biosynthetic pathways are disclosed in
U.S. Patent Application Publication No. US 20070092957, which is
incorporated by reference herein. A diagram of the disclosed isobutanol
biosynthetic pathways is provided in Figure 2. As described in U.S. Patent
Application Publication No. US 20070092957 Al, which is incorporated by
reference herein, steps in an example isobutanol biosynthetic pathway
include conversion of:
- pyruvate to acetolactate (see Fig. 2, pathway step a therein), as
catalyzed for example by acetolactate synthase,
- acetolactate to 2,3-dihydroxyisovalerate (see Fig. 2, pathway step
b therein) as catalyzed for example by KARI;
- 2,3-dihydroxyisovalerate to 2-ketoisovalerate (see Fig. 2, pathway
step c therein) as catalyzed for example by acetohydroxy acid
dehydratase, also called dihydroxy-acid dehydratase (DHAD);
- 2-ketoisovalerate to isobutyraldehyde (see Fig. 2, pathway step d
therein) as catalyzed for example by branched-chain 2-keto acid
decarboxylase; and
- isobutyraldehyde to isobutanol (see Fig. 2, pathway step e
therein) as catalyzed for example by branched-chain alcohol
dehydrogenase.
The substrate to product conversions for steps f, g, h, i, j, and k of
alternative pathways are described in U.S. Patent Application Publication
No. US 2007/0092957 Al, which is incorporated by reference herein.
Genes and polypeptides that can be used for the substrate to
product conversions described above as well as those for additional
isobutanol pathways, are described in U.S. Patent Appl. Pub. No.
20070092957, incorporated by reference herein. US Appl. Pub. Nos.
20070092957 and 20100081154, describe dihydroxyacid dehydratase
(DHAD) enzymes, including a DHAD from Streptococcus mutans (SEQ ID
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NO: 262) and a DHAD from Lactococcus lactis (SEQ ID NO: 263). U.S.
Patent Appl. Publ. No. 2009/0269823 and 2011/0269199 Al, incorporated
by reference herein, describe alcohol dehydrogenases, including an
alcohol dehydrogenase from Achromobacter xylosoxidans (SEQ ID NO:
258) and an alcohol dehydrogenase from Beijerinkia indica (SEQ ID NO:
259). Ketol-acid reductoisomerase (KARI) enzymes are described in U.S.
Patent Appl. Pub. Nos. 20080261230 Al, 20090163376, 20100197519,
and PCT Appl. Pub. No. WO/2011/041415, all incorporated by reference
herein. Keto-acid decarboxylases include those from Lactococcus lactis
(SEQ ID NO: 260) and Listeria grayi (SEQ ID NO: 261)
Additionally described in U.S. Patent Nos. 7,851,188 and
7,993,889, which is incorporated by reference herein, are construction of
chimeric genes and genetic engineering of bacteria and yeast for
isobutanol production using the disclosed biosynthetic pathways.
In another embodiment, the isobutanol biosynthetic pathway
comprises the following substrate to product conversions:
¨ pyruvate to acetolactate, which may be catalyzed, for example, by
acetolactate synthase;
¨ acetolactate to 2,3-dihydroxyisovalerate, which may be catalyzed,
for example, by ketol-acid reductoisomerase;
¨ 2,3-dihydroxyisovalerate to a-ketoisovalerate, which may be
catalyzed, for example, by dihydroxyacid dehydratase;
¨ a-ketoisovalerate to valine, which may be catalyzed, for example,
by transaminase or valine dehydrogenase;
- valine to isobutylamine, which may be catalyzed, for example, by
valine decarboxylase;
¨ isobutylamine to isobutyraldehyde, which may be catalyzed by,
for example, omega transaminase; and,
¨ isobutyraldehyde to isobutanol, which may be catalyzed, for
example, by a branched-chain alcohol dehydrogenase.
In another embodiment, the isobutanol biosynthetic pathway
comprises the following substrate to product conversions:
¨ pyruvate to acetolactate, which may be catalyzed, for example, by
acetolactate synthase;
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¨ acetolactate to 2,3-dihydroxyisovalerate, which may be catalyzed,
for example, by acetohydroxy acid reductoisomerase;
¨ 2,3-dihydroxyisovalerate to a-ketoisovalerate, which may be
catalyzed, for example, by acetohydroxy acid dehydratase;
¨a-ketoisovalerate to isobutyryl-CoA, which may be catalyzed, for
example, by branched-chain keto acid dehydrogenase;
¨isobutyryl-CoA to isobutyraldehyde, which may be catalyzed, for
example, by acelylating aldehyde dehydrogenase; and,
¨ isobutyraldehyde to isobutanol, which may be catalyzed, for
example, by a branched-chain alcohol dehydrogenase.
In another embodiment, the isobutanol biosynthetic pathway
comprises the substrate to product conversions shown as steps k, g, and
e in Figure 2.
Biosynthetic pathways for the production of 1-butanol that may be
used include those described in U.S. Appl. Pub. No. 2008/0182308, which
is incorporated herein by reference. In one embodiment, the 1-butanol
biosynthetic pathway comprises the following substrate to product
conversions:
¨ a) acetyl-CoA to acetoacetyl-CoA, which may be catalyzed, for
example, by acetyl-CoA acetyl transferase;
¨ b) acetoacetyl-CoA to 3-hydroxybutyryl-00A, which may be
catalyzed, for example, by 3-hydroxybutyryl-00A dehydrogenase;
¨ c) 3-hydroxybutyryl-00A to crotonyl-CoA, which may be
catalyzed, for example, by crotonase;
- d) crotonyl-CoA to butyryl-CoA, which may be catalyzed, for
example, by butyryl-CoA dehydrogenase;
¨ e) butyryl-CoA to butyraldehyde, which may be catalyzed, for
example, by butyraldehyde dehydrogenase; and,
¨f)
butyraldehyde to 1-butanol, which may be catalyzed, for
example, by butanol dehydrogenase.
Biosynthetic pathways for the production of 2-butanol that may be
used include those described in U.S. Appl. Pub. No. 2007/0259410 and
U.S. Appl. Pub. No. 2009/0155870, which are incorporated herein by
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reference. In one
embodiment, the 2-butanol biosynthetic pathway
comprises the following substrate to product conversions:
¨ a) pyruvate to alpha-acetolactate, which may be catalyzed, for
example, by acetolactate synthase;
- b) alpha-acetolactate to acetoin, which may be catalyzed, for
example, by acetolactate decarboxylase;
¨ c) acetoin to 3-am ino-2-butanol, which may be catalyzed, for
example, acetonin aminase;
¨ d) 3-am ino-2-butanol to 3-amino-2-butanol phosphate, which
may be catalyzed, for example, by aminobutanol kinase;
¨ e) 3-amino-2-butanol phosphate to 2-butanone, which may be
catalyzed, for example, by aminobutanol phosphate phosphorylase; and,
¨f) 2-butanone to 2-butanol, which may be catalyzed, for
example, by butanol dehydrogenase.
In another embodiment, the 2-butanol biosynthetic pathway
comprises the following substrate to product conversions:
¨ a) pyruvate to alpha-acetolactate, which may be catalyzed, for
example, by acetolactate synthase;
¨ b) alpha-acetolactate to acetoin, which may be catalyzed, for
example, by acetolactate decarboxylase;
¨ c) acetoin to 2,3-butanediol, which may be catalyzed, for
example, by butanediol dehydrogenase;
¨ d) 2,3-butanediol to 2-butanone, which may be catalyzed, for
example, by dial dehydratase; and,
- e) 2-butanone to 2-butanol, which may be catalyzed, for
example, by butanol dehydrogenase.
Methods for in situ product removal
The improved micoorganisms and processes described herein may
be used in conjunction with other in situ product removal processes, such
as with those described in in PCT Appn. Pub No. W02011/159998,
incorporated by reference herein. FIG. 1 illustrates an example process
flow diagram for production of product alcohol such as ethanol or butanol
according to an embodiment of the present invention. As shown, a
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feedstock 12 can be introduced to an inlet in a liquefaction vessel 10 and
liquefied to produce a feedstock slurry 16. Feedstock 12 contains
hydrolysable polysaccharides that supplies a fermentable carbon
substrate (e.g., fermentable sugar such as glucose), and can be a
biomass such as, but not limited to rye, wheat, cane or corn, or can
otherwise be derived from a biomass. In some embodiments, feedstock
12 can be one or more components of a fractionated biomass, and in
other embodiments, feedstock 12 can be a milled, unfractionated biomass.
In some embodiments, feedstock 12 can be corn, such as dry milled,
unfractionated corn kernels, and the undissolved particles can include
germ, fiber, and gluten. The undissolved solids are non-fermentable
portions of feedstock 12. For purposes of the discussion herein with
reference to the embodiments shown in the Figures, feedstock 12 will
often be described as constituting milled, unfractionated corn, in which the
undissolved solids have not been separated therefrom. However, it
should be understood that the exemplary methods and systems described
herein can be modified for different feedstocks whether fractionated or not,
as apparent to one of skill in the art.
The process of liquefying feedstock 12 involves hydrolysis of
polysaccharides in feedstock 12 into sugars, including for example,
dextrins and oligosaccharides. Any known liquefying processes, as well
as the corresponding liquefaction vessel, normally utilized by the industry
can be used including, but not limited to, the acid process, the acid-
enzyme process, or the enzyme process. Such processes can be used
alone or in combination. In some embodiments, the enzyme process can
be utilized and an appropriate enzyme 14, for example alpha-amylase, is
introduced to an inlet in liquefaction vessel 10. Water can also be
introduced to liquefaction vessel 10. In embodiments, a saccharification
enzyme, for example glucoamylase, may also be introduced to liquefaction
vessel 10.
Feedstock slurry 16 produced from liquefying feedstock 12
comprises fermentable carbon substrate (e.g. sugar), and, optionally,
depending on the feedstock, triglycerides in the form of oil and
undissolved solids derived from the feedstock. Feedstock slurry 16 can be
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discharged from an outlet of liquefaction vessel 10. In some
embodiments, feedstock 12 is corn or corn kernels and therefore
feedstock slurry 16 is a corn mash slurry. In some embodiments,
feedstock 12 is a lignocellulosic feedstock and therefore feedstock slurry
16 may be a lignocellulosic hydrolysate. In some
embodiments,
undissolved solids are removed from feedstock slurry 16 prior to
introduction into the fermentation vessel.
Feedstock slurry 16 is introduced into a fermentation vessel 30
along with a microorganism comprising a polynucleotide encoding a
polypeptide having lipase activity provided in accordance with the present
invention 32. Fermentation vessel 30 is configured to ferment slurry 16 to
produce alcohol. In particular, microorganism 32 contacts the fermentable
carbon substrate in slurry 16 to produce product alcohol. The slurry can
include a fermentable carbon source, for example, in the form of
oligosaccharides, and water.
In some embodiments, slurry 16 is subjected to a saccharification
process in order to break the complex sugars (e.g., oligosaccharides) in
slurry 16 into monosaccharides that can be readily metabolized by
microorganism 32. Any known saccharification process, normally utilized
by the industry can be used including, but not limited to, the acid process,
the acid-enzyme process, or the enzyme process. In some embodiments,
simultaneous saccharification and fermentation (SSF) can occur inside
fermentation vessel 30, as shown in FIG. 1. In some embodiments, an
enzyme 38, such as glucoamylase, can be introduced to an inlet in
fermentation vessel 30 in order to breakdown the starch or
oligosaccharides to glucose capable of being metabolized by
microorganism 32.
Carboxylic acid 28 and/or native oil containing triglycerides 26 are
introduced into fermentation vessel 30, along with an optional catalyst 42.
Optional catalyst 42 can be introduced before, after, or
contemporaneously with enzyme 38. Thus, in some embodiments,
addition of enzyme 38 and optional catalyst 42 can be stepwise (e.g,
catalyst 42, then enzyme 38, or vice versa), or substantially simultaneous
(i.e, at exactly the same time as in the time it takes for a person or a
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machine to perform the addition in one stroke, or one enzyme/catalyst
immediately following the other catalyst/enzyme as in the time it takes for
a person or a machine to perform the addition in two strokes). Optional
catalyst 42 is capable of esterifying the product alcohol with carboxylic
acid 28 to form an alcohol ester and in embodiments is a purified lipase.
For example, in the case of butanol production, optional catalyst 42 is
capable of esterifying butanol with carboxylic acid 28 to form a butanol
ester. It is believed that catalyst 42 is optional for use in the methods
described herein because the recombinant microorganism will express
and and display or secrete into the fermentation medium a lipase to
catalyze the esterification. However, it may be desirable to add purified
lipase (optional catalyst 42) and the methods and microorganisms
provided herein allow for a reduction in the amount of optional catalyst 42
to be added.
In the instance that native oil containing triglycerides 26 is supplied
to fermentation vessel 30, at least a portion of the acyl glycerides in oil 26
can be hydrolyzed to carboxylic acid 28 by contacting oil 26 with a
polypeptide having lipase activity such as secreted or displayed by the
microorganisms provided herein and/or optional catalyst 42. In some
embodiments, the resulting acid/oil composition includes monoglycerides
and/or diglycerides from the partial hydrolysis of the acyl glycerides in the
oil. In some embodiments the resulting acid/oil composition includes
glycerol, a by-product of acyl glyceride hydrolysis.
In addition, depending on the feedstock, the acyl glycerides in the
oil derived from feedstock 12 and present in slurry 16 can also be
hydrolyzed to carboxylic acid 28. In some
embodiments, the
concentration of carboxylic acids in the broth is sufficient to form a two-
phase fermentation mixture comprising an organic phase and an aqueous
phase.
Carboxylic acid 28 can be any carboxylic acid capable of esterifying
with a product alcohol, such as butanol or ethanol, to produce an alcohol
ester of the carboxylic acid. For example, in some embodiments,
carboxylic acid 28 can be free fatty acid, and in some embodiments the
carboxylic acid or free fatty acid have a chain length of 4 to 28 carbons, 4
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to 22 carbons in other embodiments, 8 to 22 carbons in other
embodiments, 10 to 28 carbons in other embodiments, 10 to 22 carbons in
other embodiments, 12 to 22 carbons in other embodiments, 4 to 18
carbons in other embodiments, 12 to 22 carbons in other embodiments,
and 12 to 18 carbons in still other embodiments, and 16 to 22 carbons in
still other embodiments. In some embodiments, carboxylic acid 28 is one
or more of the following fatty acids: azaleic, capric, caprylic, castor,
coconut (i.e., as a naturally-occurring combination of fatty acids, including
lauric, myrisitic, plamitic, caprylic, capric, stearic, caproic, arachidic,
oleic,
and linoleic, for example), isostearic, lauric, linseed, myristic, oleic, palm
oil, palmitic, palm kernel, pelargonic, ricinoleic, sebacic, soya, stearic
acid,
tall oil, tallow, and #12 hydroxy stearic. In some embodiments, carboxylic
acid 28 is one or more of diacids, e.g., azelaic acid and sebacic acid. In
some embodiments, carboxylic acid 28 is one or more saturated, primary
carboxylic acids with defined branching of the carbon chain, where said
carboxylic acid or mixtures thereof are prepared by the oxidation of 2-
alkyl-1-alkanols well known as Guerbet alcohols, where the carboxylic
acids have a total number of carbons of from 12 to 22.
Thus, in some embodiments, carboxylic acid 28 can be a mixture of
two or more different fatty acids. In some embodiments, carboxylic acid
28 comprises free fatty acid derived from hydrolysis of acyl glycerides by
any method known in the art, including chemical or enzymatic hydrolysis.
In some embodiments as noted above, carboxylic acid 28 can be derived
from native oil 26 by enzymatic hydrolysis of the oil glycerides using an
enzyme as catalyst 42. In some embodiments, the fatty acids or mixtures
thereof comprise unsaturated fatty acids. The presence of unsaturated
fatty acids decreases the melting point, providing advantages for handling.
Of the unsaturated fatty acids, those which are monounsaturated, i.e.
possessing a single carbon-carbon double bond, may provide advantages
with respect to melting point without sacrificing suitable thermal and
oxidative stability for process considerations.
In some embodiments, native oil 26 can be tallow, corn, canola,
capric/caprylic triglycerides, castor, coconut, cottonseed, fish, jojoba,
lard,
linseed, neetsfoot, oiticica, palm, peanut, rapeseed, rice, safflower, soya,
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sunflower, tung, jatropha, pumpkin, palm, grape seed and vegetable oil
blends (or oils that can be purified into higher concentrations of different
chain length and levels of unsaturation (i.e., 18:1)). In some
embodiments, native oil 26 is a mixture of two or more native oils, such as
a mixture of palm and soybean oils, for example. In some embodiments,
native oil 26 is a plant-derived oil. In some embodiments, the plant-
derived oil can be, though not necessarily, derived from biomass that can
be used in a fermentation process. The biomass can be the same or
different source from which feedstock 12 is obtained. Thus, for example,
in some embodiments, oil 26 can be derived from corn, whereas feedstock
12 can be cane. For example, in some embodiments, oil 26 can be
derived from corn, and the biomass source of feedstock 12 is also corn.
Any possible combination of different biomass sources for oil 26 versus
feedstock 12 can be used, as should be apparent to one of skill in the art.
In some embodiments, oil 26 is derived from the biomass used in the
fermentation process. Thus, in some embodiments oil 26 is derived
directly from feedstock 12. For example, when feedstock 12 is corn, then
oil 26 is the feedstock's constituent corn oil and may be introduced into
fermentation vessel 30 along with slurry 16.
In fermentation vessel 30, alcohol produced by microorganism 32 is
esterified with carboxylic acid 28 by the polypeptide having lipase activity
secreted by the microorganism (and optionally catalyst 42) to form alcohol
esters. For example, in the case of butanol production, butanol produced
by microorganism 32 is esterified with carboxylic acid 28 to form butanol
esters. In situ product removal (ISPR) can be utilized to remove the
alcohol esters from the fermentation broth.
Utilizing a recombinant
microorganism which expresses and secretes or displays a polypeptide
having lipase activity to form esters in conjunction with ISPR can improve
the performance of the fermentation. While not wishing to be bound by
theory, it is believed that lipase activity in the fermentation medium and
esterification of the product alcohol during a fermentation may improve the
ability of the microorganism to produce the product alcohol which is
particularly desirable for product alcohols that are toxic to the production
host cells. Thus, provided herein are methods of improving tolerance of a
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microorganism to a product alcohol by engineering the microorganism to
produce and secrete a polypeptide having lipase activity.
In embodiments, using the microorganism to produce a lipase to
form esters in conjunction with ISPR (such as, for example, liquid-liquid
extraction) can increase the effective titer by at least about 10%, at least
about 20%, at least about 30%, at least about 40%, at least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least about
90%, or at least about 100% as compared to the effective titer in an
analogous fermentation using ISPR without the microorganism producing
a lipase. Similarly, in embodiments, using the microorganism to produce a
lipase to form esters in conjunction with ISPR (such as, for example,
liquid-liquid extraction) can increase the effective rate by at least about
10%, at least about 20%, at least about 30%, at least about 40%, at least
about 50%, at least about 60%, at least about 70%, at least about 80%, at
least about 90%, or at least about 100% as compared to the effective rate
in an analogous fermentation using ISPR without the microorganism
producing a lipase. In embodiments, the effective yield is increased by at
least about 10%, at least about 20%, at least about 30%, at least about
40%, or at least about 50%. In some embodiments, the resulting
fermentation broth after alcohol esterification can comprise free (i.e.,
unesterified) alcohol, and in some embodiments, the concentration of free
alcohol in the fermentation broth after alcohol esterification is not greater
than 1, 3, 6, 10, 15, 20, 25, 30 25, 40, 45, 50, 55, or 60 g/L when the
product alcohol is butanol, or, when the product alcohol is ethanol, the
concentration of free alcohol in the fermentation broth after alcohol
esterification is not greater than 15, 20, 25, 30 25, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95, or 100 g/L. In some embodiments, at least about
20%, at least about 30%, at least about 40%, at least about 50%, at least
about 60%, at least about 70%, at least about 80%, or at least about 90%
of the effective titer of alcohol is converted to alcohol ester.
In some embodiments, the fermentation broth is contacted during
fermentation with an extractant to form a two-phase mixture comprising an
aqueous phase and an organic phase. Such liquid-liquid extraction can be
performed according to the processes described in U.S. Pub. No.
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2009/0305370, the disclosure of which is hereby incorporated in its
entirety. U.S. Patent Appl. Pub. No. 2009/0305370 describes methods for
producing and recovering butanol from a fermentation broth using liquid-
liquid extraction, the methods comprising the step of contacting the
fermentation broth with a water immiscible extractant to form a two-phase
mixture comprising an aqueous phase and an organic phase. Typically,
the extractant can be an organic extractant selected from the group
consisting of saturated, mono-unsaturated, poly-unsaturated (and mixtures
thereof) 012 to 022 fatty alcohols, 012 to 022 fatty acids, esters of 012 to
022
fatty acids, 012 to 022 fatty aldehydes, 012 to 022 fatty amides, and
mixtures thereof. The extractant may also be an organic extractant
selected from the group consisting of saturated, mono-unsaturated, poly-
unsaturated (and mixtures thereof) 04 to 022 fatty alcohols, 04 to 028 fatty
acids, esters of 04 to 028 fatty acids, 04 to 022 fatty aldehydes, and
mixtures thereof. Examples of suitable extractants include an extractant
comprising at least one solvent selected from the group consisting of oleyl
alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol,
stearyl alcohol, oleic acid, lauric acid, myristic acid, stearic acid, methyl
myristate, methyl oleate, lauric aldehyde, 1-nonanol, 1-decanol, 1-
undecanol, 2-undecanol, 1-nonanal, 2-butyloctanol, 2-butyl-octanoic acid
and mixtures thereof. In embodiments, the extractant comprises oleyl
alcohol. In embodiments, the extractant comprises a branched chain
saturated alcohol, for example, 2-butyloctanol, commercially available as
ISOFAL 12 (Sasol, Houston, TX) or Jarcol 1-12 (Jarchem Industries, Inc.,
Newark, NJ). In embodiments, the extractant comprises a branched chain
carboxylic acid, for example, 2-butyl-octanoic acid, 2-hexyl-decanoic acid,
or 2-decyl-tetradecanoic acid, commercially available as ISOCARB 12,
ISOCARB 16, and ISOCARB 24, respectively (Sasol, Houston, TX).
For use with the processes described herein, the extractant(s) for ISPR
are typically non-alcohol extractants, so as to avoid consuming carboxylic
acid 28 in fermentation vessel 30 by catalytic esterification of carboxylic
acid 28 with an alcohol extractant, whereby less carboxylic acid would be
available for esterification with the product alcohol. For example, if oleyl
alcohol is used as an ISPR extractant, then oleyl alcohol esters of the
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carboxylic acid may be produced in fermentation vessel due to the
presence of lipase activity.
With reference to the embodiment of FIG. 1, the carboxylic acid 28
can also serve as an ISPR extractant 28 or a component thereof. As
earlier noted, carboxylic acid 28 can be supplied, and/or formed in situ in
the case when native oil 26 is supplied to fermentation vessel 30, and/or
formed in situ in the case when feedstock 16 includes triglycerides in the
form of oil that can be hydrolyzed. In some embodiments, ISPR extractant
28 includes free fatty acids. In some embodiments, ISPR extractant 28
includes corn oil fatty acids (COFA). In some embodiments, oil 26 is corn
oil, whereby ISPR extractant 28 is COFA. ISPR extractant (carboxylic
acid) 28 contacts the fermentation broth and forms a two-phase mixture
comprising an aqueous phase 34 and an organic phase. The product
alcohol ester formed in the fermentation broth preferentially partitions into
the organic phase to form an ester-containing organic phase 36. Any free
product alcohol in the fermentation broth also preferentially partitions into
the ester-containing organic phase. The biphasic mixture can be removed
from fermentation vessel 30 as stream 39 and introduced into a vessel 35,
in which the ester-containing organic phase 36 is separated from aqueous
phase 34. Separation of biphasic mixture 39 into ester-containing organic
phase 36 and aqueous phase 34 can be achieved using any methods
known in the art, including but not limited to, siphoning, aspiration,
decantation, centrifugation, using a gravity settler, membrane-assisted
phase splitting, and the like. All or part of aqueous phase 34 can be
recycled into fermentation vessel 30 as fermentation medium (as shown),
or otherwise discarded and replaced with fresh medium, or treated for the
removal of any remaining product
alcohol and then recycled to
fermentation vessel 30.
With reference to FIG. 1, ester-containing organic phase 36 is
introduced into vessel 50 in which the alcohol esters are reacted with one
or more substances 52 to recover product alcohol 54. Product alcohol 54
can be recovered using any method known in the art and/or described in
PCT Appn. Pub. No. W02011/159998, incorporated by reference, for
obtaining an alcohol from an alcohol ester.
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EXAMPLES
As used herein, the meaning of abbreviations used was as follows:
"L" means liter(s), "mL" means milliliter(s), "pL" means microliter(s).
General Methods
GC Analysis of Reaction Products in the Aqueous and Extra ctant Phase
Samples (ca. 5.0 g) were removed from a stirred reaction mixture or
fermentation broth containing corn oil fatty acids (COFA) as extractant,
and centrifuged to separate aqueous phase and extractant phase. A
sample of the resulting aqueous phase or extractant phase (ca. 0.50 g,
actual weight recorded) was dissolved in 4.50 mL of a solution of 5.5556
mg/mL of pentadecanoic acid methyl ester (015:0 FAME, external
standard) in isopropanol. The resulting solution was centrifuged to
remove any suspended solids, then ca. 1.25 mL of the resulting
supernatant was added to a 2.0 mL Agilent GC sample vial and the vial
capped with a PTFE septa. Samples were analyzed for isobutanol or fatty
acid butyl esters on an Agilent 6890 GC with a 7683B injector and
autosampler. The column was an Agilent DB-FFAP column (30 m x 0.25
mm ID, 0.25 pm film). The carrier gas was helium at a flow rate of 1.8
mL/min measured at 80 C with constant head pressure; injector split was
20:1 at 250 C; oven temperature was 80 C for 2.0 minutes, 80 C to
250 C at 10 C/min, then 250 C for 20 minutes. Flame ionization
detection was used at 250 C. The following GC standards (Nu-Chek
Prep; Elysian, MN) were used to confirm the identity of fatty acid isobutyl
ester products: iso-butyl palmitate, iso-butyl stearate, iso-butyl oleate, iso-
butyl linoleate, iso-butyl linolenate, iso-butyl arachidate.
Strain Constructions
Table 7
Genotypes of strains used in Examples
Strain Genotype
PNY827 MATa/MATa
PNY908 MATa MAL2-8c SUC2
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PNY931 MATa ura3A::loxP his3A pdc6A pdc1A::P[PDC1]-
DHADlilyD_Sm-PDC1t-P[FBA1]-ALSIalsS_Bs-CYC1t
pdc5L:P[PDC5]-ADHIsadB_Ax-PDC5t gpd2A::loxP
fra2A::P[TEF1(M6)]-LIPITIan-CYC1t
adh1A::UAS(PGK1)P[FBA1]-kiyD_LI(y)-ADH1t
PNY932 MATa ura3A::loxP his3A pdc6A pdc1A::P[PDC1]-
DHADlilyD_Sm-PDC1t-P[FBA1]-ALSIal5S_Bs-CYC1t
pdc5L:P[PDC5]-ADHIsadB_Ax-PDC5t gpd2A::loxP
fra2A::CYC1t-LIPITIan-P[TEF1(M6)]
adh1A::UAS(PGK1)P[FBA1]-kiyD_LI(y)-ADH1t
PNY934 Isogenic with PNY 931, transformed with pBP915 and
pYZ090AalsS
PNY935 Isogenic with PNY 932, transformed with pBP915 and
pYZ090AalsS
PNY937 Isogenic with PNY2211, transformed with pBP915 and
pYZ090AalsS
PNY1020 MATa ura3A::loxP his3A pTVAN2
PNY1022 MATa ura3A::loxP his3A pdc6A pdc1A::P[PDC1]-
DHADlilyD_Sm-PDC1t-P[FBA1]-ALSIal5S_Bs-CYC1t
pdcaL:P[PDC5]-ADHI5adB_Ax-PDC5t gpd2A::loxP
fra2A::P[PDC1]-ADHladh_HI-ADH1t
adh1A::UAS(PGK1)P[FBA1]-kiyD_Lg(y)-ADH1t
yprcL,15,6,..:P[PDC5]-ADHladh_HI-ADH1t
gpd2A::P[TEF1(M4)]-CdLip(y)-CYC1t pBP2092
PNY1023 MATa ura3A::loxP his3A pdc6A pdc1A::P[PDC1]-
DHADlilyD_Sm-PDC1t-P[FBA1]-ALSIal5S_Bs-CYC1t
pdcaL:P[PDC5]-ADHI5adB_Ax-PDC5t gpd2A::loxP
fra2A::P[PDC1]-ADHladh_HI-ADH1t
adh1A::UAS(PGK1)P[FBA1]-kiyD_Lg(y)-ADH1t
yprcL,15,6,..:P[PDC5]-ADHladh_HI-ADH1t
gpd2A::P[TEF1(M6)]-CdLip(y)-CYC1t pBP2092
PNY1024 MATa ura3A::loxP his3A pdc6A pdc1A::P[PDC1]-
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DHADlilvD_Sm-PDC1t-P[FBA1]-ALSIalsS_Bs-CYC1t
pdc5L:P[PDC5]-ADHIsadB_Ax-PDC5t gpd2A::loxP
fra2A::P[PDC1]-ADHladh_HI-ADH1t
adh1A::UAS(PGK1)P[FBA1]-kivD_Lg(y)-ADH1t
yprcL,15,6,..:P[PDC5]-ADHladh_HI-ADH1t
gpd2A::P[TEF1(M6)]-CaLip(y)-CYC1t pBP2092
PNY1052 MATa ura3A::loxP his3A pTVAN31
PNY1053 MATa ura3A::loxP his3A pTVAN32
PNY1054 MATa ura3A::loxP his3A pTVAN33
PNY1055 MATa ura3A::loxP his3A pTVAN9
PNY1056 MATa ura3A::loxP his3A pTVAN4
PNY1057 MATa ura3A::loxP his3A pTVAN10
PNY1500 MATa uraaL:loxP his3A
PNY1556 MATa ura3A::loxP his3A pdc6A pdc1A::P[PDC1]-
DHADlilvD_Sm-PDC1t-P[FBA1]-ALSIalsS_Bs-CYC1t
pdcaL:P[PDC5]-ADHIsadB_Ax-PDC5t gpd2A::loxP
fra2A::P[PDC1]-ADHladh_HI-ADH1t
adh1A::UAS(PGK1)P[FBA1]-kivD_Lg(y)-ADH1t
yprcL,15,6,..:P[PDC5]-ADHladh_HI-ADH1t
PNY2211 MATa ura3A::loxP his3A pdc6A pdc1A::P[PDC1]-
DHADlilvD_Sm-PDC1t-P[FBA1]-ALSIalsS_Bs-CYC1t
pdcaL:P[PDC5]-ADHIsadB_Ax-PDC5t gpd2A::loxP
fra2A adh1A::UAS(PGK1)P[FBA1]-kivD_LI(y)-ADH1t
PNY2242 MATa ura3A::loxP his3A pdc6A pdc1A::P[PDC1]-
DHADlilvD_Sm-PDC1t-P[FBA1]-ALSIalsS_Bs-CYC1t
pdcaL:P[PDC5]-ADHIsadB_Ax-PDC5t gpd2A::loxP
fra2A::P[PDC1]-ADHladh_HI-ADH1t
adh1A::UAS(PGK1)P[FBA1]-kivD_LI(y)-ADH1t
yprcL,15,6,..:P[PDC5]-ADHladh_HI-ADH1t ymr226cA
ald6A::loxP
Table 8
Feature information for constructs used in Examples
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Feature information for SEQ ID NO: 183
Feature Position Strand (W="Watson";
C="Crick")
AmpR 1629-2486 C
HIS3 4532-5191 W
pTEFI(M6) 1-400 C
Tian lipase ORF 6433-7308 C
CYC/ Terminator 6175-6424 C
Feature information for SEQ ID NO: 184
Feature Position Strand (W="Watson";
C="Crick")
AmpR 1629-2486 C
HIS3 4532-5191 W
pTEFI(M6) 1-400 C
Tian lipase ORF 6433-7308 C
CYC/ Terminator 6175-6424 C
N55A mutation 7144-7146 C
Feature information for SEQ ID NO: 127
Feature Position Strand (W="Watson";
C="Crick")
AmpR 4000-4860 C
Fragment A 431-931 W
Fragment B 956-1455 W
5' URA3 1464-1713 W
URA3 1714-2517 W
3' URA3 2518-2667 W
Fragment C 2676-2788 W
Feature information for SEQ ID NO: 186
AmpR 3059-3919 C
Fragment A 4550-5050 W
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pTEF1(M6) 5067-5466 W
Tian lipase ORF 5476-6350 W
CYC/ Terminator 6360-6609 W
Fragment B 15-514 W
5' URA3 523-772 W
URA3 773-1576 W
3' URA3 1577-1726 W
Fragment C 1735-1847 W
Feature information for SEQ ID NO: 187
AmpR 3059-3919 C
Fragment A 4550-5050 W
CYC/ Terminator 5067-5316 C
Tian lipase ORF 5326-6200 C
pTEF1(M6) 6210-6609 C
Fragment B 15-514 W
5' URA3 523-772 W
URA3 773-1576 W
3' URA3 1577-1726 W
Fragment C 1735-1847 W
Feature information for SEQ ID NO: 188
AmpR 4610-5470 C
Fragment A 6101-6601 W
pTEF1(M6) 7-406 W
Tian lipase N55A ORF 416-1290 W
CYC/ Terminator 1300-1549 W
Fragment B 1566-2065 W
5' URA3 2074-2323 W
URA3 2324-3127 W
3' URA3 3128-3277 W
Fragment C 3286-3398 W
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Construction of PNY1500
The strain BP857 ("PNY1500") was derived from CEN.PK 113-7D
(CBS 8340; Centraalbureau voor Schimmelcultures (CBS) Fungal
Biodiversity Centre, Netherlands) and contains deletions of the following
genes: URA3, HIS3.
URA3 Deletion
To delete the endogenous URA3 coding region, a ura3::loxP-
kanMX-IoxP cassette was PCR-amplified from pLA54 template DNA (SEQ
ID NO: 25). pLA54 contains the K. lactis TEF1 promoter and kanMX
marker, and is flanked by loxP sites to allow recombination with Cre
recombinase and removal of the marker. PCR was done using Phusion
DNA polymerase (New England BioLabs; Ipswich, MA) and primers
BK505 and BK506 (SEQ ID NOs:26 and 27). The URA3 portion of each
primer was derived from the 5' region upstream of the URA3 promoter and
3' region downstream of the coding region such that integration of the
loxP-kanMX-IoxP marker resulted in replacement of the URA3 coding
region. The PCR product was transformed into CEN.PK 113-7D using
standard genetic techniques (Methods in Yeast Genetics, 2005, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202)
and transformants were selected on YPD containing G418 (100 pg/ml) at
C. Transformants were screened to verify correct integration by PCR
using primers LA468 and LA492 (SEQ ID NOs:28 and 29) and designated
CEN.PK 113-7D Aura3::kanMX.
HIS3 Deletion
25 The four fragments for the PCR cassette for the scarless HIS3
deletion were amplified using Phusion High Fidelity PCR Master Mix (New
England BioLabs) and CEN.PK 113-7D genomic DNA as template,
prepared with a Gentra Puregene Yeast/Bact kit (Qiagen; Valencia, CA).
HIS3 Fragment A was amplified with primer oBP452 (SEQ ID NO: 30)
30 and primer 0BP453 (SEQ ID NO: 31), containing a 5' tail with
homology to
the 5' end of HIS3 Fragment B. HIS3 Fragment B was amplified with
primer oBP454 (SEQ ID NO: 32), containing a 5' tail with homology to the
3' end of HIS3 Fragment A, and primer oBP455 (SEQ ID NO: 33),
containing a 5' tail with homology to the 5' end of HIS3 Fragment U. HIS3
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Fragment U was amplified with primer 0BP456 (SEQ ID NO: 34),
containing a 5' tail with homology to the 3' end of HIS3 Fragment B, and
primer oBP457 (SEQ ID NO: 35), containing a 5' tail with homology to the
5' end of HIS3 Fragment C. HIS3 Fragment C was amplified with primer
0BP458 (SEQ ID NO: 36), containing a 5' tail with homology to the 3' end
of HIS3 Fragment U, and primer 0BP459 (SEQ ID NO: 37). PCR products
were purified with a PCR Purification kit (Qiagen). HIS3 Fragment AB was
created by overlapping PCR by mixing HI53 Fragment A and HI53
Fragment B and amplifying with primers oBP452 (SEQ ID NO: 30) and
oBP455 (SEQ ID NO: 33). HIS3 Fragment UC was created by
overlapping PCR by mixing HIS3 Fragment U and HIS3 Fragment C and
amplifying with primers 0BP456 (SEQ ID NO: 34) and 0BP459 (SEQ ID
NO: 37). The resulting PCR products were purified on an agarose gel
followed by a Gel Extraction kit (Qiagen). The HIS3 ABUC cassette was
created by overlapping PCR by mixing HIS3 Fragment AB and HIS3
Fragment UC and amplifying with primers oBP452 (SEQ ID NO: 30) and
0BP459 (SEQ ID NO: 37). The PCR product was purified with a PCR
Purification kit (Qiagen).
Competent cells of CEN.PK 113-7D Aura3::kanMX were made and
transformed with the HIS3 ABUC PCR cassette using a Frozen-EZ Yeast
Transformation II kit (Zymo Research; Orange, CA). Transformation
mixtures were plated on synthetic complete media lacking uracil
supplemented with 2% glucose at 30 C. Transformants with a his3
knockout were screened for by PCR with primers 0BP460 (SEQ ID NO:
38) and 0BP461 (SEQ ID NO: 39) using genomic DNA prepared with a
Gentra Puregene Yeast/Bact kit (Qiagen). A correct transformant was
selected as strain CEN.PK 113-7D Aura3::kanMX Ahis3::URA3.
KanMX Marker Removal from the Aura3 Site and URA3 Marker Removal
from the Ahis3 Site
The KanMX marker was removed by transforming CEN.PK 113-7D
Aura3::kanMX Ahis3::URA3 with pRS423::PGAL1-cre (SEQ ID NO: 40)
using a Frozen-EZ Yeast Transformation II kit (Zymo Research) and
plating on synthetic complete medium lacking histidine and uracil
supplemented with 2% glucose at 30 C. Transformants were grown in YP
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supplemented with 1% galactose at 30 C for ¨6 hours to induce the Ore
recombinase and KanMX marker excision and plated onto YPD (2%
glucose) plates at 30 C for recovery. An isolate was grown overnight in
YPD and plated on synthetic complete medium containing 5-fluoro-orotic
acid (0.1%) at 30 C to select for isolates that lost the URA3 marker. 5-
FOA resistant isolates were grown in and plated on YPD for removal of the
pRS423::PGAL1-cre plasmid. Isolates were checked for loss of the
KanMX marker, URA3 marker, and pRS423::PGAL1-cre plasmid by
assaying growth on YPD+G418 plates, synthetic complete medium lacking
uracil plates, and synthetic complete medium lacking histidine plates. A
correct isolate that was sensitive to G418 and auxotrophic for uracil and
histidine was selected as strain CEN.PK 113-7D Aura3::loxP Ahis3 and
designated as BP857. The deletions and marker removal were confirmed
by PCR and sequencing with primers 0BP450 (SEQ ID NO: 41) and
oBP451 (SEQ ID NO: 42) for Aura3 and primers 0BP460 (SEQ ID NO:
38) and 0BP461 (SEQ ID NO: 39) for Ahis3 using genomic DNA prepared
with a Gentra Puregene Yeast/Bact kit (Qiagen).
Construction of Strain PNY2205
PDC6 Deletion
The four fragments for the PCR cassette for the scarless PDC6
deletion were amplified using Phusion High Fidelity PCR Master Mix (New
England BioLabs) and CEN.PK 113-7D genomic DNA as template,
prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). PDC6
Fragment A was amplified with primer 0BP440 (SEQ ID NO: 18) and
primer oBP441 (SEQ ID NO: 19), containing a 5' tail with homology to the
5' end of PDC6 Fragment B. PDC6 Fragment B was amplified with primer
oBP442 (SEQ ID NO: 20), containing a 5' tail with homology to the 3" end
of PDC6 Fragment A, and primer 0BP443 (SEQ ID NO: 21), containing a
5' tail with homology to the 5' end of PDC6 Fragment U. PDC6 Fragment
U was amplified with primer oBP444 (SEQ ID NO: 22), containing a 5' tail
with homology to the 3' end of PDC6 Fragment B, and primer oBP445
(SEQ ID NO: 23), containing a 5' tail with homology to the 5' end of PDC6
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Fragment C. PDC6 Fragment C was amplified with primer 0BP446 (SEQ
ID NO: 24), containing a 5' tail with homology to the 3' end of PDC6
Fragment U, and primer oBP447 (SEQ ID NO: 56). PCR products were
purified with a PCR Purification kit (Qiagen). PDC6 Fragment AB was
created by overlapping PCR by mixing PDC6 Fragment A and PDC6
Fragment B and amplifying with primers 0BP440 (SEQ ID NO:18) and
0BP443 (SEQ ID NO: 21). PDC6 Fragment UC was created by
overlapping PCR by mixing PDC6 Fragment U and PDC6 Fragment C and
amplifying with primers oBP444 (SEQ ID NO: 22) and oBP447 (SEQ ID
NO: 56. The resulting PCR products were purified on an agarose gel
followed by a Gel Extraction kit (Qiagen). The PDC6 ABUC cassette was
created by overlapping PCR by mixing PDC6 Fragment AB and PDC6
Fragment UC and amplifying with primers 0BP440 (SEQ ID NO: 18) and
oBP447 (SEQ ID NO: 56). The PCR product was purified with a PCR
Purification kit (Qiagen).
Competent cells of CEN.PK 113-7D Aura3::loxP Ahis3 were made
and transformed with the PDC6 ABUC PCR cassette using a Frozen-EZ
Yeast Transformation II kit (Zymo Research). Transformation mixtures
were plated on synthetic complete media lacking uracil supplemented with
2% glucose at 30 C. Transformants with a pdc6 knockout were screened
for by PCR with primers 0BP448 (SEQ ID NO: 57) and 0BP449 (SEQ ID
NO: 58) using genomic DNA prepared with a Gentra Puregene Yeast/Bact
kit (Qiagen). A correct transformant was selected as strain CEN.PK 113-
7D Aura3::loxP Ahis3 Apdc6::URA3.
CEN.PK 113-7D Aura3::loxP Ahis3 Apdc6::URA3 was grown
overnight in YPD and plated on synthetic complete medium containing 5-
fluoro-orotic acid (0.1%) at 30 C to select for isolates that lost the URA3
marker. The deletion and marker removal were confirmed by PCR and
sequencing with primers 0BP448 (SEQ ID NO: 57) and 0BP449 (SEQ ID
NO: 58) using genomic DNA prepared with a Gentra Puregene
Yeast/Bact kit (Qiagen). The absence of the PDC6 gene from the isolate
was demonstrated by a negative PCR result using primers specific for the
coding sequence of PDC6, oBP554 (SEQ ID NO: 59) and oBP555 (SEQ
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ID NO: 60). The correct isolate was selected as strain CEN.PK 113-7D
Aura3::loxP Ahis3 Apdc6 and designated as BP891.
PDC1 Deletion ilvDSm Integration
The PDC1 gene was deleted and replaced with the ilvD coding
region from Streptococcus mutans ATCC #700610. The A fragment
followed by the ilvD coding region from Streptococcus mutans for the PCR
cassette for the PDC1 deletion-ilvDSm integration was amplified using
Phusion High Fidelity PCR Master Mix (New England BioLabs) and
NYLA83 genomic DNA as template, prepared with a Gentra Puregene
Yeast/Bact kit (Qiagen). NYLA83 is a strain (construction described in
U.S. App. Pub. NO. 20110124060, incorporated herein by reference in its
entirety) which carries the PDC1 deletion-ilvDSm integration described in
U.S. Patent Application Publication No. 2009/0305363 (herein
incorporated by reference in its entirety). PDC1 Fragment A-ilvDSm was
amplified with primer 0BP513 (SEQ ID NO: 61) and primer 0BP515 (SEQ
ID NO: 62), containing a 5' tail with homology to the 5' end of PDC1
Fragment B. The B, U, and C fragments for the PCR cassette for the
PDC1 deletion-ilvDSm integration were amplified using Phusion High
Fidelity PCR Master Mix (New England BioLabs) and CEN.PK 113-7D
genomic DNA as template, prepared with a Gentra Puregene Yeast/Bact
kit (Qiagen). PDC1 Fragment B was amplified with primer 0BP516 (SEQ
ID NO: 63) containing a 5' tail with homology to the 3' end of PDC1
Fragment A-ilvDSm, and primer oBP517 (SEQ ID NO: 64), containing a 5'
tail with homology to the 5' end of PDC1 Fragment U. PDC1 Fragment U
was amplified with primer 0BP518 (SEQ ID NO: 65), containing a 5' tail
with homology to the 3' end of PDC1 Fragment B, and primer 0BP519
(SEQ ID NO: 66), containing a 5' tail with homology to the 5' end of PDC1
Fragment C. PDC1 Fragment C was amplified with primer 0BP520 (SEQ
ID NO: 67), containing a 5' tail with homology to the 3' end of PDC1
Fragment U, and primer oBP521 (SEQ ID NO: 68). PCR products were
purified with a PCR Purification kit (Qiagen). PDC1 Fragment A-ilvDSm-B
was created by overlapping PCR by mixing PDC1 Fragment A-ilvDSm
(SEQ ID NO: 171) and PDC1 Fragment B and amplifying with primers
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0BP513 (SEQ ID NO: 61) and oBP517 (SEQ ID NO: 64). PDC1 Fragment
UC was created by overlapping PCR by mixing PDC1 Fragment U and
PDC1 Fragment C and amplifying with primers 0BP518 (SEQ ID NO: 65)
and oBP521 (SEQ ID NO: 68). The resulting PCR products were purified
on an agarose gel followed by a Gel Extraction kit (Qiagen). The PDC1 A-
ilvDSm-BUC cassette (SEQ ID NO: 172) was created by overlapping PCR
by mixing PDC1 Fragment A-ilvDSm-B and PDC1 Fragment UC and
amplifying with primers 0BP513 (SEQ ID NO: 61) and oBP521 (SEQ ID
NO: 68). The PCR product was purified with a PCR Purification kit
(Qiagen).
Competent cells of CEN.PK 113-7D Aura3::loxP Ahis3 Apdc6 were
made and transformed with the PDC1 A-ilvDSm-BUC PCR cassette using
a Frozen-EZ Yeast Transformation II kit (Zymo Research). Transformation
mixtures were plated on synthetic complete media lacking uracil
supplemented with 2% glucose at 30 C. Transformants with a pdc1
knockout ilvDSm integration were screened for by PCR with primers
oBP511 (SEQ ID NO: 69) and oBP512 (SEQ ID NO: 70) using genomic
DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). The
absence of the PDC1 gene from the isolate was demonstrated by a
negative PCR result using primers specific for the coding sequence of
PDC1, 0BP550 (SEQ ID NO: 71) and oBP551 (SEQ ID NO: 72). A correct
transformant was selected as strain CEN.PK 113-7D Aura3::loxP Ahis3
Apdc6 Apdc1::ilvDSm-URA3.
CEN.PK 113-7D Aura3::loxP Ahis3 Apdc6 Apdc1::ilvDSm-URA3
was grown overnight in YPD and plated on synthetic complete medium
containing 5-fluoro-orotic acid (0.1%) at 30 C to select for isolates that
lost the URA3 marker. The deletion of PDC1, integration of ilvDSm, and
marker removal were confirmed by PCR and sequencing with primers
oBP511 (SEQ ID NO: 69) and oBP512 (SEQ ID NO: 70) using genomic
DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). The
correct isolate was selected as strain CEN.PK 113-7D Aura3::loxP Ahis3
Apdc6 Apdc1::ilvDSm and designated as BP907.
PDC5 Deletion sadB Integration
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The PDC5 gene was deleted and replaced with the sadB coding
region from Achromobacter xylosoxidans (the sadB gene is described in
U.S. Patent Appl. No. 2009/0269823, which is herein incorporated by
reference in its entirety). A segment of the PCR cassette for the PDC5
deletion-sadB integration was first cloned into plasmid pUC19-URA3MCS.
pUC19-URA3MCS is pUC19 (SEQ ID NO: 94) based and contains the
sequence of the URA3 gene from S. cerevisiae situated within a multiple
cloning site (MCS). pUC19 contains the pMB1 replicon and a gene coding
for beta-lactamase for replication and selection in E. coll. In addition to
the
coding sequence for URA3, the sequences from upstream and
downstream of this gene were included for expression of the URA3 gene
in yeast. The vector can be used for cloning purposes and can be used as
a yeast integration vector.
The DNA encompassing the URA3 coding region along with 250 bp
upstream and 150 bp downstream of the URA3 coding region from
Saccharomyces cerevisiae CEN.PK 113-7D genomic DNA was amplified
with primers 0BP438 (SEQ ID NO: 89), containing BamHI, Ascl, Pmel,
and Fsel restriction sites, and 0BP439 (SEQ ID NO: 90), containing Xbal,
Pad, and Notl restriction sites, using Phusion High-Fidelity PCR Master
Mix (New England BioLabs). Genomic DNA was prepared using a Gentra
Puregene Yeast/Bact kit (Qiagen). The PCR product and pUC19 were
ligated with T4 DNA ligase after digestion with BamHI and Xbal to create
vector pUC19-URA3MCS. The vector was confirmed by PCR and
sequencing with primers 0BP264 (SEQ ID NO: 91) and 0BP265 (SEQ ID
NO: 92).
The coding sequence of sadB and PDC5 Fragment B were cloned
into pUC19-URA3MCS to create the sadB-BU portion of the PDC5 A-
sadB-BUC PCR cassette. The coding sequence of sadB was amplified
using pLH468-sadB (SEQ ID NO: 93) as template with primer 0BP530
(SEQ ID NO: 73), containing an Ascl restriction site, and primer 0BP531
(SEQ ID NO: 74), containing a 5' tail with homology to the 5' end of PDC5
Fragment B. PDC5 Fragment B was amplified with primer 0BP532 (SEQ
ID NO: 75), containing a 5' tail with homology to the 3' end of sadB, and
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primer 0BP533 (SEQ ID NO: 76), containing a Pmel restriction site. PCR
products were purified with a PCR Purification kit (Qiagen). sadB-PDC5
Fragment B was created by overlapping PCR by mixing the sadB and
PDC5 Fragment B PCR products and amplifying with primers 0BP530
(SEQ ID NO: 73) and 0BP533 (SEQ ID NO: 76). The resulting PCR
product was digested with Ascl and Pmel and ligated with T4 DNA ligase
into the corresponding sites of pUC19-URA3MCS after digestion with the
appropriate enzymes. The resulting plasmid was used as a template for
amplification of sadB-Fragment B-Fragment U using primers 0BP536
(SEQ ID NO: 77) and 0BP546 (SEQ ID NO: 78), containing a 5' tail with
homology to the 5' end of PDC5 Fragment C. PDC5 Fragment C was
amplified with primer oBP547 (SEQ ID NO: 79) containing a 5' tail with
homology to the 3' end of PDC5 sadB-Fragment B-Fragment U, and
primer 0BP539 (SEQ ID NO: 80). PCR products were purified with a PCR
Purification kit (Qiagen). PDC5 sadB-Fragment B-Fragment U-Fragment
C was created by overlapping PCR by mixing PDC5 sadB-Fragment B-
Fragment U and PDC5 Fragment C and amplifying with primers 0BP536
(SEQ ID NO: 77) and 0BP539 (SEQ ID NO: 80). The resulting PCR
product was purified on an agarose gel followed by a Gel Extraction kit
(Qiagen). The PDC5 A-sadB-BUC cassette (SEQ ID NO: 173) was
created by amplifying PDC5 sadB-Fragment B-Fragment U-Fragment C
with primers oBP542 (SEQ ID NO: 81), containing a 5' tail with homology
to the 50 nucleotides immediately upstream of the native PDC5 coding
sequence, and 0BP539 (SEQ ID NO: 80). The PCR product was purified
with a PCR Purification kit (Qiagen).
Competent cells of CEN.PK 113-7D Aura3::loxP Ahis3 Apdc6
Apdc1::ilvDSm were made and transformed with the PDC5 A-sadB-BUC
PCR cassette using a Frozen-EZ Yeast Transformation II kit (Zymo
Research). Transformation mixtures were plated on synthetic complete
media lacking uracil supplemented with 1% ethanol (no glucose) at 30 C.
Transformants with a pdc5 knockout sadB integration were screened for
by PCR with primers 0BP540 (SEQ ID NO: 82) and oBP541 (SEQ ID NO:
83) using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit
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(Qiagen). The absence of the PDC5 gene from the isolate was
demonstrated by a negative PCR result using primers specific for the
coding sequence of PDC5, oBP552 (SEQ ID NO: 84) and 0BP553 (SEQ
ID NO: 85). A correct transformant was selected as strain CEN.PK 113-7D
Aura3::loxP Ahis3 Apdc6 Apdc1::ilvDSm Apdc5::sadB-URA3.
CEN.PK 113-7D Aura3::loxP Ahis3 Apdc6 Apdc1::ilvDSm
Apdc5::sadB-URA3 was grown overnight in YPE (1% ethanol) and plated
on synthetic complete medium supplemented with ethanol (no glucose)
and containing 5-fluoro-orotic acid (0.1%) at 30 C to select for isolates
that lost the URA3 marker. The deletion of PDC5, integration of sad B, and
marker removal were confirmed by PCR with primers 0BP540 (SEQ ID
NO: 82) and oBP541 (SEQ ID NO: 83) using genomic DNA prepared
with a Gentra Puregene Yeast/Bact kit (Qiagen). The correct isolate was
selected as strain CEN.PK 113-7D Aura3::loxP Ahis3 Apdc6
Apdc1::ilvDSm Apdc5::sadB and designated as BP913.
GPD2 Deletion
To delete the endogenous GPD2 coding region, a gpd2::loxP-
URA3-loxP cassette (SEQ ID NO: 174) was PCR-amplified using loxP-
URA3-loxP PCR as template DNA. loxP-URA3-loxP (SEQ ID NO: 170)
contains the URA3 marker from pRS426 flanked by loxP recombinase
sites. PCR was done using Phusion DNA polymerase and primers LA512
(SEQ ID NO: 95) and LA513 (SEQ ID NO: 96). The GPD2 portion of
each primer was derived from the 5' region upstream of the GPD2 coding
region and 3' region downstream of the coding region such that integration
of the loxP-URA3-loxP marker resulted in replacement of the GPD2
coding region. The PCR product was transformed into BP913 and
transformants were selected on synthetic complete media lacking uracil
supplemented with 1% ethanol (no glucose). Transformants were
screened to verify correct integration by PCR using primers 0BP582 and
AA270.
The URA3 marker was recycled by transformation with
pRS423::PGAL1-cre and plating on synthetic complete media lacking
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histidine supplemented with 1% ethanol at 30 C. Transformants were
streaked on synthetic complete medium supplemented with 1% ethanol
and containing 5-fluoro-orotic acid (0.1%) and incubated at 3000 to select
for isolates that had lost the URA3 marker. 5-FOA resistant isolates were
grown in YPE (1% ethanol) for removal of the pRS423::PGAL1-cre
plasmid. The deletion and marker removal were confirmed by PCR with
primers 0BP582 (SEQ ID NO: 97) and 0BP591 (SEQ ID NO: 98). The
correct isolate was selected as strain CEN.PK 113-7D Aura3::loxP Ahis3
Apdc6 Apdc1::ilvDSm Apdc5::sadB Agpd2::loxP and designated as
BP1064 (PNY1503).
FRA2 Deletion
The FRA2 deletion was designed to delete 250 nucleotides from
the 3' end of the coding sequence, leaving the first 113 nucleotides of the
FRA2 coding sequence intact. An in-frame stop codon was present 7
nucleotides downstream of the deletion. The four fragments for the PCR
cassette for the scarless FRA2 deletion were amplified using Phusion High
Fidelity PCR Master Mix (New England BioLabs) and CEN.PK 113-7D
genomic DNA as template, prepared with a Gentra Puregene Yeast/Bact
kit (Qiagen). FRA2 Fragment A was amplified with primer 0BP594 (SEQ
ID NO: 99) and primer 0BP595 (SEQ ID NO: 102), containing a 5' tail with
homology to the 5' end of FRA2 Fragment B. FRA2 Fragment B was
amplified with primer 0BP596 (SEQ ID NO: 103), containing a 5' tail with
homology to the 3' end of FRA2 Fragment A, and primer 0BP597 (SEQ ID
NO: 104), containing a 5' tail with homology to the 5' end of FRA2
Fragment U. FRA2 Fragment U was amplified with primer 0BP598 (SEQ
ID NO: 105), containing a 5' tail with homology to the 3' end of FRA2
Fragment B, and primer 0BP599 (SEQ ID NO: 106) containing a 5' tail
with homology to the 5' end of FRA2 Fragment C. FRA2 Fragment C was
amplified with primer 0BP600 (SEQ ID NO: 107), containing a 5' tail with
homology to the 3' end of FRA2 Fragment U, and primer 0BP601 (SEQ ID
NO: 108). PCR products were purified with a PCR Purification kit
(Qiagen). FRA2 Fragment AB was created by overlapping PCR by mixing
FRA2 Fragment A and FRA2 Fragment B and amplifying with primers
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0BP594 (SEQ ID NO: 99) and 0BP597 (SEQ ID NO: 104). FRA2
Fragment UC was created by overlapping PCR by mixing FRA2 Fragment
U and FRA2 Fragment C and amplifying with primers oBP598 (SEQ ID
NO: 105) and 0BP601 (SEQ ID NO: 108). The resulting PCR products
were purified on an agarose gel followed by a Gel Extraction kit (Qiagen).
The FRA2 ABUC cassette was created by overlapping PCR by mixing
FRA2 Fragment AB and FRA2 Fragment UC and amplifying with primers
0BP594 (SEQ ID NO: 99) and 0BP601 (SEQ ID NO: 108). The PCR
product was purified with a PCR Purification kit (Qiagen).
Competent cells of PNY1503 were made and transformed with the
FRA2 ABUC PCR cassette using a Frozen-EZ Yeast Transformation II kit
(Zymo Research). Transformation mixtures were plated on synthetic
complete media lacking uracil supplemented with 1% ethanol at 30 C.
Transformants with a fra2 knockout were screened for by PCR with
primers 0BP602 (SEQ ID NO: 109) and 0BP603 (SEQ ID NO: 110) using
genomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen).
A correct transformant was grown in YPE (yeast extract, peptone, 1 `)/0
ethanol) and plated on synthetic complete medium containing 5-fluoro-
orotic acid (0.1`)/0) at 30 C to select for isolates that lost the URA3
marker.
The deletion and marker removal were confirmed by PCR with primers
0BP602 (SEQ ID NO: 109) and 0BP603 (SEQ ID NO: 110) using genomic
DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). The
absence of the FRA2 gene from the isolate was demonstrated by a
negative PCR result using primers specific for the deleted coding
sequence of FRA2, 0BP605 (SEQ ID NO: 111) and 0BP606 (SEQ ID NO:
112). The correct isolate was selected as strain CEN.PK 113-7D MATa
ura3A::loxP his3A pdc6A pdc1A::P[PDC1]-DHADlilvD_Sm-PDC1t
pdc5A::P[PDC5]-ADHI5adB_Ax-PDC5t gpd2A::loxP fra2A and designated
as PNY1505 (BP1135).
ADH1 Deletion and kivD Ll(y) Integration
The ADH1 gene was deleted and replaced with the kivD coding
region from Lactococcus lactis codon optimized for expression in S.
cerevisiae. The scarless cassette for the ADH1 deletion-kivD Ll(y)
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integration was first cloned into plasmid pUC19-URA3MCS.
The kivD coding region from Lactococcus lactis codon optimized for
expression in S. cerevisiae was amplified using pLH468 (SEQ ID NO: 129)
as template with primer 0BP562 (SEQ ID NO: 113), containing a Pmel
restriction site, and primer 0BP563 (SEQ ID NO: 114), containing a 5' tail
with homology to the 5' end of ADH1 Fragment B. ADH1 Fragment B was
amplified from genomic DNA prepared as above with primer 0BP564
(SEQ ID NO: 115), containing a 5' tail with homology to the 3' end of
kivD Ll(y), and primer 0BP565 (SEQ ID NO: 116), containing a Fsel
restriction site. PCR products were purified with a PCR Purification kit
(Qiagen). kivD_LI(y)-ADH1 Fragment B was created by overlapping PCR
by mixing the kivD_LI(y) and ADH1 Fragment B PCR products and
amplifying with primers 0BP562 (SEQ ID NO: 113) and 0BP565 (SEQ ID
NO: 116). The resulting PCR product was digested with Pmel and Fsel
and ligated with T4 DNA ligase into the corresponding sites of pUC19-
URA3MCS after digestion with the appropriate enzymes. ADH1 Fragment
A was amplified from genomic DNA with primer 0BP505 (SEQ ID NO:
117), containing a Sac restriction site, and primer 0BP506 (SEQ ID NO:
118), containing an Ascl restriction site. The ADH1 Fragment A PCR
product was digested with Sac! and Ascl and ligated with T4 DNA ligase
into the corresponding sites of the plasmid containing kivD_LI(y)-ADH1
Fragment B. ADH1 Fragment C was amplified from genomic DNA with
primer 0BP507 (SEQ ID NO: 119), containing a Pad l restriction site, and
primer 0BP508 (SEQ ID NO: 120), containing a Sall restriction site. The
ADH1 Fragment C PCR product was digested with Pad l and Sall and
ligated with T4 DNA ligase into the corresponding sites of the plasmid
containing ADH1 Fragment A-kivD_LI(y)-ADH1 Fragment B. The hybrid
promoter UAS(PGK/)-PFBAi was amplified from vector pRS316-
UAS(PGK/)-PFBA1-GUS (SEQ ID NO: 130) with primer 0BP674 (SEQ ID
NO: 121), containing an Ascl restriction site, and primer 0BP675 (SEQ ID
NO: 122), containing a Pmel restriction site. The UAS(PGK/)-PFBAi PCR
product was digested with Ascl and Pmel and ligated with T4 DNA ligase
into the corresponding sites of the plasmid containing kivD_LI(y)-ADH1
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Fragments ABC. The entire integration cassette was amplified from the
resulting plasmid with primers 0BP505 (SEQ ID NO: 117) and 0BP508
(SEQ ID NO: 120) and purified with a PCR Purification kit (Qiagen).
Competent cells of PNY1505 were made and transformed with the
ADH1-kivD_LI(y) PCR cassette constructed above using a Frozen-EZ
Yeast Transformation II kit (Zymo Research). Transformation mixtures
were plated on synthetic complete media lacking uracil supplemented with
1% ethanol at 30 C. Transformants were grown in YPE (1% ethanol) and
plated on synthetic complete medium containing 5-fluoro-orotic acid
(0.1%) at 30 C to select for isolates that lost the URA3 marker. The
deletion of ADH1 and integration of kivD Ll(y) were confirmed by PCR
with external primers 0BP495 (SEQ ID NO: 123) and 0BP496 (SEQ ID
NO: 124) and with kivD Ll(y) specific primer 0BP562 (SEQ ID NO: 113)
and external primer 0BP496 (SEQ ID NO: 124) using genomic DNA
prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). The correct
isolate was selected as strain CEN.PK 113-7D MATa ura3A::loxP his3A
pdc6A
pdc1A::P[PDC1]-DHADlilvD_Sm-PDC1tpdc5A::P[PDC5]-
ADHIsadB_Ax-PDC5t gpd2A::loxP fra2A adh1A::UAS(PGK1)P[FBA1]-
kivD_LI(y)-ADH1t and designated as PNY1507 (BP1201).
Construction of integration vector pUC19-kan::pdc1::FBA-alsS::TRX1
The FBA-alsS-CYCt cassette was constructed by moving the 1.7kb
BbvCl/Pacl fragment from pRS426::GPD::alsS::CYC (described in US
Patent No. 7,851,188, which is herein incorporated by reference in its
entirety) to pRS426::FBA::ILV5::CYC (described in US Patent No.
7,851,188, which is herein incorporated by reference in its entirety), which
had been previously digested with BbvCIIPacl to release the ILV5 gene.
Ligation reactions were transformed into E. coli TOP10 cells and
transformants were screened by PCR using primers N98SegF1 (SEQ ID
NO: 125) and N99SeqR2 (SEQ ID NO: 126). The FBA-alsS-CYCt
cassette was isolated from the vector using Bg/II and Notl for cloning into
pUC19-URA3::ilvD-TRX1 at the AM! site (Klenow fragment was used to
make ends compatible for ligation). Transformants containing the alsS
cassette in both orientations in the vector were obtained and confirmed by
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PCR using primers N98SeqF4 (SEQ ID NO: 125) and N1111 (SEQ ID NO:
128) for configuration "A" and N98SeqF4 (SEQ ID NO: 125) and N1110
(SEQ ID NO: 153) for configuration "B". A geneticin-selectable version of
the "A" configuration vector was then made by removing the URA3 gene
(1.2 kb NotlINael fragment) and adding a geneticin cassette. Klenow
fragment was used to make all ends compatible for ligation, and
transformants were screened by PCR to select a clone with the geneticin
resistance gene in the same orientation as the previous URA3 marker
using primers BK468 (SEQ ID NO: 131) and N160SeqF5 (SEQ ID NO:
154). The resulting clone was called pUC19-kan::pdc1::FBA-alsS::TRX1
(clone A) (SEQ ID NO: 155).
Construction of alsS integrant strains
The pUC19-kan::pdc1::FBA-alsS integration vector described
above was linearized with Pmel and transformed into PNY1507. Pmel
cuts the vector within the cloned pdc1-TRX1 intergenic region and thus
leads to targeted integration at that location (Rodney Rothstein, Methods
in Enzymology, 1991, volume 194, pp. 281-301). Transformants were
selected on YPE plus 50 pg/ml G418. Patched transformants were
screened by PCR for the integration event using primers N160SeqF5
(SEQ ID NO: 154) and oBP512 (SEQ ID NO: 70). Two transformants
were tested indirectly for acetolactate synthase function by evaluating the
strains' ability to make isobutanol. To do this, additional isobutanol
pathway genes were supplied on E. coli-yeast shuttle vectors
(pYZ090AalsS and pBP915; SEQ ID NOs: 43 and 44, respectively). One
clone was designated as PNY2205. The plasmid-free parent strain was
designated PNY2204 (MATa ura3A::loxP his3A pdc6A pdc1A::P[PDC1]-
DHADlilvD_Sm-PDC1t-pUC19-loxP-kanMX-IoxP-P[FBA1]-ALSIalsS_Bs-
CYC1t pdc5A::P[PDC5]-ADHI5adB_Ax-PDC5t gpd2A::loxP fra2A
adh1A::UAS(PGK1)P[FBA1 ]-kivD_LI(y)-ADH1t).
Construction of Strain PNY2211
PNY2211 was constructed in several steps from S. cerevisiae strain
PNY1507 as described in the following paragraphs. First the strain was
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modified to contain a phosphoketolase gene. Next, an acetolactate
synthase gene (alsS) was added to the strain, using an integration vector
targeted to sequence adjacent to the phosphoketolase gene. Finally,
homologous recombination was used to remove the phosphoketolase
gene and integration vector sequences, resulting in a scarless insertion of
alsS in the intergenic region between pdc1.6::ilvD and the native TRX1
gene of chromosome XII. The resulting genotype of PNY2211 is MATa
ura3A::loxP his3A pdc6A pdc1A::P[PDC1]-DHADlilvD_Sm-PDC1t-
P[FBA1]-ALSIalsS_Bs-CYC1t pdc5A::P[PDC5]-ADH I sadB_Ax-PDC5t
gpd2A::loxP fra2A adh1A::UAS(PGK1)P[FBA1 ]-kivD_LI(y)-ADH it.
A phosphoketolase gene cassette was introduced into PNY1507 by
homologous recombination. The integration construct was generated as
follows. The plasmid pRS423::CUP1-alsS+FBA-budA (previously
described in US2009/0305363, which is herein incorporated by reference
in its entirety) was digested with Notl and Xmal to remove the 1.8 kb FBA-
budA sequence, and the vector was religated after treatment with Klenow
fragment. Next, the CUP1 promoter was replaced with a TEF1 promoter
variant (M4 variant previously described by Nevoigt et al. Appl. Environ.
Microbiol. 72: 5266-5273 (2006), which is herein incorporated by reference
in its entirety) via DNA synthesis and vector construction service from
DNA2.0 (Menlo Park, CA). The resulting plasmid, pRS423::TEF(M4)-alsS
was cut with Stul and M/ul (removes 1.6 kb portion containing part of the
alsS gene and CYC/ termintor), combined with the 4 kb PCR product
generated from pRS426::GPD-xpk1+ADH-eutD (SEQ ID NO: 175) with
primers N1176 (SEQ ID NO: 164) and N1177 (SEQ ID NO: 165) and an
0.8 kb PCR product DNA generated from yeast genomic DNA (EN01
promoter region) with primers N822 (SEQ ID NO: 160) and N1178 (SEQ
ID NO: 166) and transformed into S. cerevisiae strain BY4741 (ATCC
#201388) using gap repair cloning methodology, see Ma et al. Gene
58:201-216 (1987). Transformants were obtained by plating cells on
synthetic complete medium without histidine. Proper assembly of the
expected plasmid (pRS423::TEF1(M4)-xpk1+EN01-eutD, SEQ ID NO:
156) was confirmed by PCR primers N821 and and N1115 (SEQ ID NOs:
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159 and 163, respectively) and by restriction digest (Bg11). Two clones
were subsequently sequenced. The 3.1 kb TEF(M4)-xpk1 gene was
isolated by digestion with Sac! and Notl and cloned into the pUC19-
URA3::ilvD-TRX1 vector (Clone A, cut with Af111). Cloning fragments were
treated with Klenow fragment to generate blunt ends for ligation. Ligation
reactions were transformed into E. coli Stb13 cells, selecting for ampicillin
resistance. Insertion of TEF1(M4)-xpk1 was confirmed by PCR (primers
N1110 (SEQ ID NO: 153) and N1114 (SEQ ID NO: 162)). The vector was
linearized with Afill and treated with Klenow fragment. The 1.8 kb Kpnl-
Hincll geneticin resistance cassette described in W02011159853A1
(incorporated herein by reference) was cloned by ligation after Klenow
fragment treatment. Ligation reactions were transformed into E. coli Stb13
cells, selecting for ampicillin resistance. Insertion of the geneticin
cassette
was confirmed by PCR (primers N160SeqF5 (SEQ ID NO: 154) and
BK468 (SEQ ID NO: 131)). The plasmid sequence is provided herein
(pUC19-URA3::pdc1::TEF(M4)-xpk1::kan, SEQ ID NO: 157).
The resulting integration cassette
(pdc1::TEF1(M4)-
xpk1::KanMX::TRX1) was isolated (Ascl and Nael digestion generated a
5.3 kb band that was gel purified) and transformed into PNY1507 using
the Zymo Research Frozen-EZ Yeast Transformation Kit (Cat. No. T2001).
Transformants were selected by plating on YPE plus 50 pg/ml G418.
Integration at the expected locus was confirmed by PCR (primers N886
and N1214, SEQ ID NOs: 161 and 167, respectively). Next, plasmid
pRS423::GAL1p-Cre (SEQ ID NO: 169), encoding Cre recombinase, was
used to remove the loxP-flanked KanMX cassette. Proper removal of the
cassette was confirmed by PCR (primers oBP512 and N160SeqF5 (SEQ
ID NOs: 168 and 154, respectively)). Finally, the alsS integration plasmid
described herein (SEQ ID NO: 155; pUC19-kan::pdc1::FBA-alsS::TRX1,
clone A) was transformed into this strain using the included geneticin
selection marker. Two integrants were tested for acetolactate synthase
activity by transformation with plasmids pYZ090AalsS (SEQ ID NO: 43)
and pBP915 (SEQ ID NO: 44) transformed using Protocol #2 in Amberg,
Burke and Strathern "Methods in Yeast Genetics" (2005), and evaluation
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of growth and isobutanol production in glucose-containing media (methods
for growth and isobutanol measurement are as follows: All strains were
grown in synthetic complete medium, minus histidine and uracil containing
0.3 (:)/0 glucose and 0.3 (:)/0 ethanol as carbon sources (10 mL medium in
125 mL vented Erlenmeyer flasks (VWR Cat. No. 89095-260). After
overnight incubation (30 C, 250 rpm in an Innova 40 New Brunswick
Scientific Shaker), cultures were diluted back to 0.2 OD (Eppendorf
BioPhotometer measurement) in synthetic complete medium containing
2% glucose and 0.05% ethanol (20 ml medium in 125 mL tightly-capped
Erlenmeyer flasks (VWR Cat. No. 89095-260)). After 48 hours incubation
(30 C, 250 rpm in an Innova 40 New Brunswick Scientific Shaker),
culture supernatants (collected using Spin-X centrifuge tube filter units,
Costar Cat. No. 8169) were analyzed by HPLC per methods described in
U.S. Appl. Pub. No. 20070092957). One of the two clones was positive
and was named PNY2218.
PNY2218 was treated with Ore recombinase, and the resulting
clones were screened for loss of the xpk1 gene and pUC19 integration
vector sequences by PCR (primers N886 and N160SeqR5; SEQ ID NOs:
161 and 158, respectively). This left only the alsS gene integrated in the
pdc1-TRX1 intergenic region after recombination the DNA upstream of
xpk1 and the homologous DNA introduced during insertion of the
integration vector (a "scarless" insertion since vector, marker gene and
loxP sequences are lost). Although this recombination could have
occurred at any point, the vector integration appeared to be stable even
without geneticin selection, and the recombination event was only
observed after introduction of the Ore recombinase. One clone was
designated PNY2211.
Construction of Saccharomyces cerevisiae strain PNY2242
Strain PNY2242 was constructed in several steps from PNY1507
(described above). First, a chimeric gene comprised of the FBA1
promoter, the alsS coding region and the CYC/ terminator was integrated
into Chromosome XII, upstream of the TRX1 gene. The sequence of the
modified locus is provided as SEQ ID No. 176. Next, two copies of a gene
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encoding horse liver alcohol dehydrogenase were integrated into
Chromsomes VII and XVI. On Chromosome VII, a chimeric gene
comprised of the PDC1 promoter, the hADH coding region and the ADH1
terminator were placed into the fra2A locus (the original deletion of FRA2
is described above). The sequence of the modified locus is provided as
SEQ ID No. 177. On Chromosome XVI, a chimeric gene comprised of the
PDC5 promoter, the hADH coding region and the ADH1 terminator were
integrated in the region formerly occupied by the long term repeat element
YPRCdelta15. The sequence of the modified locus is provided as SEQ ID
No. 178. Then the
native genes YMR226c and ALD6 were deleted.
Elimination of YMR226c was a scarless deletion of only the coding region.
The sequence of the modified locus is provided as SEQ ID No. 179. The
ALD6 coding region plus 700 bp of upstream sequence were deleted
using ORE-lox mediated marker removal (methodology described above),
so the resulting locus contains one loxP site. The sequence of the
modified locus is provided as SEQ ID No. 180. Finally, plasmids were
introduced into the strain for expression of a variant of Anaerostipes
caccae KARI (pLH702, SEQ ID. No. 181) and DHAD
(pYZ067DkivDDhADH, SEQ ID. No. 182), resulting in strain PNY2242.
Example 1
Expression of Candida deformans LIP1 Lipase in Yeast
The DNA sequence of the native LIP1 lipase from C. deformans
was obtained from GenBank (accession number AJ428393), and the open
reading frame (ORF) was optimized for expression in yeast (DNA 2.0).
The resulting DNA sequence had 76% sequence identity with the wild type
sequence, and encoded an identical protein.
The DNA comprising the expression-optimized ORF sequence was
synthesized (DNA 2.0), and the resulting DNA molecule was cloned into a
yeast-E. co/i shuttle vector by gap-repair cloning (Oldenburg KR, Vo KT,
Michaelis S, & Paddon C (1997) Recombination-mediated PCR-directed
plasmid construction in vivo in yeast. Nucleic Acids Res 25:451-452).
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Briefly, the LIP1 lipase ORF was amplified using primers AK10-33_CdL5
and AK10-34 CdL3 (SEQ ID NOs: 10 and 11, respectively), which include
5' regions having homology to regions in plasmid pNAK34 (SEQ ID NO:
x). The resulting PCR product was co-transformed into S. cerevisiae strain
PNY1500 with pNAK34 that had been linearized with the Pad l restriction
endonuclease, by lithium acetate/PEG transformation essentially as
described (Gietz RD & Woods RA (2006) Yeast transformation by the
LiAc/SS Carrier DNA/PEG method. Methods Mol Biol 313:107-120). The
transformation reaction was plated onto synthetic complete agar medium
(Sherman F (2002) Getting started with yeast. Methods in Enzymology
350:3-41) containing 2% glucose and dropout mix minus histidine
(Formedium, UK, catalog number DSCK-042; SCD-His medium). After
incubation at 30 C for 3 d, His colonies were picked for further analysis.
LIP1 lipase-positive isolates were plated onto SC-His medium
containing tributyrin and incubated at 30 C for 3 d. The LIP1 lipase-
positive isolates had a zone of clearing around them, indicating that they
were secreting a functional lipase enzyme capable of hydrolyzing
tributyrin; in contrast, a control yeast strain did not cause clearing of
tributyrin in the agar medium. The plasmids from 3 isolates were
recovered by plasmid rescue (Robzyk K & Kassir Y (1992) A simple and
highly efficient procedure for rescuing autonomous plasmids from yeast.
Nucleic Acids Res. 20:3790) and sequenced using M13-reverse and T7-
promoter primers (SEQ ID NOs: 16 and 17, respectively) on an ABI Prism
3730x1 DNA Analyzer using BigDye Terminator Cycle Sequencing
chemistry. The sequences were a perfect match for the predicted plasmid
product of the gap-repair cloning strategy (data not shown). The resulting
plasmid is pNAK10 (SEQ ID NO: 45; FIGURE 3).
Example 2
Expression of Thermomyces lanuqinosus Lipase in Yeast
The DNA
sequence of the native lipase from The rmomyces
lanuginosus (Tlan lipase) was obtained from GenBank (accession number
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AF054513), and the sequence was optimized for expression in yeast
(DNA 2.0). The resulting DNA sequence had 76% sequence identity with
the wildtype sequence, and encoded an identical protein.
The DNA comprising the expression-optimized ORF sequence was
synthesized (DNA 2.0), and the resulting DNA molecule was cloned into a
yeast-E. coil shuttle vector by gap-repair cloning as in Example 1. Briefly,
the synthesized T. lanuginosus Tlan lipase ORF was amplified using
primers AK10-42_T15-1 and AK10-43_T13 (SEQ ID NOs: 12 and 13,
respectively), which include 5' regions having homology to regions in
plasmid pNAK10 (SEQ ID NO: 45; FIGURE 3). The resulting PCR product
was co-transformed into S. cerevisiae strain PNY1500 with pNAK10 that
had been linearized with the Spel restriction endonuclease, by lithium
acetate/PEG transformation. The transformation reaction was plated onto
SOD-His medium). After incubation at 30 C for 3 d, colonies were
analyzed for plasmid containing the Tlan lipase sequence by colony PCR
using primers AK10-41_T15-1 and AK10-42_T13 (SEQ ID NOs: 12 and 13).
Tlan lipase-positive isolates were plated onto SOD-His medium
containing tributyrin and incubated at 30 C for 3 d. The Tlan lipase-
positive isolates had a zone of clearing around them, indicating that they
were secreting a functional lipase activity; in contrast, a control yeast
strain did not cause clearing of tributyrin in the agar medium. The plasm ids
from three isolates were recovered by plasmid rescue and sequenced
using M13-reverse and T7-promoter primers (SEQ ID NOs: 16 and 17).
The sequences were a perfect match for the predicted plasmid product of
the gap-repair cloning strategy (data not shown). One plasmid was named
pTVAN2 (SEQ ID NO: 100).
Example 3
Scale-up Expression of T. lanuqinosus Lipase in Yeast
One positive isolate from Example 2, PNY1020, was pre-cultured
overnight in SOD-His medium, and this was used to inoculate four 500 mL
cultures of SOD-His medium; two cultures were treated with the
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asparaginyl glycosylation inhibitor tunicamycin (5 pg/mL; Sigma-Aldrich,
St. Louis MO). The flasks were incubated at 30 C and 250 rpm in a
shaking incubator. After 8 h 50 mL of YPD medium (yeast extract, 10 g/L;
peptone, 20 g/L; glucose, 20 g/L) was added to each flask, and the
cultures were incubated overnight. The following morning, after glucose
was exhausted, the cultures were centrifuged at 8000 rpm for 10 min at 4
C. The supernatants were concentrated approximately 500-fold under
pressure through a 10,000 dalton molecular weight cutoff filter. The protein
concentration of the retentates was measured, and 20 pg of protein was
analyzed by SDS-polyacrylamide gel electrophoresis, using a 4-12%
acrylamide Bis-Tris gel (Invitrogen, Carlsbad CA) according to the
manufacturer's instructions. The gel was stained with Coomassie Blue R-
250, and destained. The tunicamycin-treated protein had a lower
molecular weight, as demonstrated by its higher mobility in the gel (not
shown). The identity of the band as Tlan lipase was confirmed by amino-
terminal sequencing.
The concentration of Tlan lipase protein (expressed with or without
tunicamycin treatment) in the retentates was estimated to be 25% of total
soluble protein based on SDS-PAGE analysis, and these two retentates
containing Tlan lipase protein (expressed with or without tunicamycin
treatment) were employed as catalyst for in-vitro esterification of
isobutanol with corn oil fatty acids (Example 5).
Example 4
Production of Corn Oil Fatty Acids
A 5-L round bottom flask was equipped with a mechanical stirrer,
thermocouple, heating mantle, condenser and nitrogen tee and charged
with 750 g of crude corn oil, 2112 g of water and 285 g of 50% sodium
hydroxide solution. Mixture was heated to 90 C and held for two hours,
during which time it became a thick, emulsion-like single phase. At the
end of this time thin-layer chromatography indicated no remaining corn oil
in the mixture. The mixture was then cooled to 74 C and 900 g of 25%
sulfuric acid was added to acidify the mixture, which was then cooled to 50
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C and the aqueous layer was separated. The oil layer was washed twice
with 1500 mL of 40 C water and then once with 1 L of saturated brine,
and then dried over magnesium sulfate and filtered through Celite. Yield
was 610 g of clear red oil. Titration for Free Fatty Acids via AOCS method
Ca 5a-40 shows a fatty acid content of 95 A) expressed as oleic acid. A
sample (104 mg) was silanized by reaction with 100 uL of N-methyl-N-
(trimethylsily1)-trifluoroacetamide in 1 mL of dry pyridine. Gas
chromatography-mass spectrometry (GCMS) analysis of the silanized
product indicated the presence of the TMS derivatives of the 16:0, 18:2,
18:1, 18:0, and 20:0 carboxylic acids.
Example 5
Production of isobutyl-COFA esters by reaction of isobutanol and corn oil
fatty acids catalyzed by secreted lipase
Reaction mixtures containing 3.6 g isobutanol (2-methy1-1-
propanol), 14.7 g corn oil fatty acids (COFA) prepared from corn oil
(Example 4), 45.1 g of aqueous 2-(N-morpholino)ethanesulfonic acid
buffer (0.20 M, pH 5.4), and either 0.487 mg (10 ppm in aqueous phase;
Table 7) or 0.974 mg (20 ppm in aqueous phase; Table 9) of Tlan lipase
protein (expressed with or without tunicamycin treatment; Example 3)
were stirred at 30 C, and samples were withdrawn from each reaction
mixture at predetermined times, immediately centrifuged, and the aqueous
and organic layers separated and analyzed for isobutanol (iBuOH) and
isobutyl-esters of corn oil fatty acids (iBuO-COFA) (Table 10). The
reactions containing Tlan lipase produced considerably more iBuO-COFA
than the control reaction; the lipase samples that were secreted from yeast
in the presence of tunicamycin produced considerably more iBuO-COFA
than that produced without the inhibitor being present..
Table 9. Tlan concentrations in reactions for conversion of isobutanol
(iBuOH) to iso-butyl esters of corn oil fatty acids (iBuO-COFA).
Tlan expressed with
Reaction (PPrn) tunicamycin
1 10 no
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2 20 no
3 10 yes
4 20 yes
5 0 not applicable
Table 10. Weights of isobutanol (iBuOH) and isobutyl esters of corn oil
fatty acids (iBuO-COFA) present in the aqueous fraction (AQ) and organic
fraction (ORG) for reactions described in Table 9.
free iBuOH from
total iBuOH total iBuOH iBuOH iBuO-COFA iBuO-COFA
reaction time (h) (g) (AQ) (g) (ORG) (g) (ORG)
(g) (ORG) (g) (ORG)
1 0.1 1.45 2.15 2.14 0.01 0.03
1 16 1.27 2.33 2.31 0.02 0.09
1 21 1.27 2.33 2.30 0.03 0.14
1 47 1.24 2.36 2.28 0.08 0.36
1 89 1.23 2.37 2.22 0.15 0.67
2 0.1 1.22 2.39 2.38 0.01 0.03
2 16 1.29 2.32 2.30 0.03 0.11
2 21 1.25 2.36 2.32 0.04 0.17
2 47 1.38 2.23 2.14 0.09 0.38
2 89 1.21 2.40 2.18 0.22 0.98
3 0.1 1.22 2.43 2.42 0.01 0.03
3 16 1.28 2.37 2.28 0.09 0.41
3 21 1.24 2.41 2.29 0.12 0.55
3 47 1.22 2.43 2.15 0.28 1.27
3 89 1.17 2.48 1.94 0.54 2.42
4 0.1 1.38 2.22 2.21 0.01 0.03
4 16 1.30 2.30 2.19 0.11 0.49
4 21 1.21 2.39 2.23 0.15 0.69
4 47 1.36 2.24 1.90 0.34 1.51
4 89 1.12 2.48 1.78 0.70 3.16
5 0.1 1.29 2.30 2.30 0.01 0.03
5 16 1.27 2.32 2.30 0.02 0.08
5 21 1.24 2.35 2.33 0.02 0.10
5 47 1.35 2.24 2.20 0.05 0.21
5 89 1.25 2.35 2.27 0.07 0.33
Example 6
Production of fatty acid butyl esters during yeast cultivation
The Tlan lipase isolate PNY1020 and the control strain PNY908
were pre-cultured in SOD-His medium, and used to inoculate flasks (with
non-vented caps) containing 25 mL of SC-His medium. The flasks were
amended with 8.25 g sterile COFA (33% w/w), isobutanol (0.50 g, added
after 8 h of growth), and tunicamycin (Tnm, final concentration 5 pg/ml) as
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follows (Table 11):
Table11.
PNY908 PNY1020
Flask COFA iBuOH Tnm flask COFA iBuOH Tnm
F1 - - - F5 - -
F2 + - - F6 +- -
F3 - + - F7 - + -
F4 + + - F8 + + -
F9 + + +
F10 +- +
The flasks were incubated at 30 C and 250 rpm, and sampled after 24 h
and 96 h of incubation. Samples were analyzed for glucose, ethanol,
isobutanol, and fatty acid alkyl esters in the aqueous phase by HPLC or
GC, and for isobutanol and fatty acid alkyl esters in the organic phase by
GC (Tables 12 and 13). When both isobutanol and COFA were added to
the cultures, the lipase-expressing strain (flasks F8 and F9) produced
more iBuO-COFA than the control strain (flask F4). The cells treated with
tunicamycin produced more ester than the cells without inhibitor treatment.
Table 12. HPLC analysis of aqueous F1 - F10 samples.
sample time glucose glycerol acetate ethanol iBuOH
(h) (mM) (mM) (mM) (mM) (mM)
F1 24 0.1 2.5 8.2 170.4 0.8
F2 24 0.1 3.3 7.3 179.4 1.2
F3 24 50.9 1.6 3.5 106.4 266.6
F4 24 0.0 1.5 5.5 182.0 144.3
F5 24 0.1 3.9 10.5 171.1 0.5
F6 24 0.1 5.0 7.9 184.7 0.1
F7 24 89.9 1.1 2.4 39.0 267.2
F8 24 0.1 1.8 1.7 189.9 144.6
F9 24 65.9 1.7 2.6 80.7 143.5
F10 24 10.8 16.6 5.4 141.6 0.4
F1 96 0.0 0.7 91.7 0.3
F2 96 0.0 3.1 7.0 179.3 0.1
F3 96 50.6 1.2 3.0 108.6 247.9
F4 96 0.0 1.6 7.2 183.8 130.0
F5 96 0.0 3.3 9.2 163.9 0.1
F6 96 0.0 4.9 9.7 182.6 0.1
F7 96 90.3 0.9 1.5 38.6 248.6
F8 96 0.0 1.9 2.0 190.7 128.1
F9 96 0.7 2.1 1.2 184.2 128.5
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F10 96 0.0 17.8 2.2 153.2 0.1
Table 13. Weights of isobutanol (iBuOH) and isobutyl esters of corn oil
fatty acids (iBuO-COFA) present in the aqueous fraction (AQ) and organic
fraction (ORG) for shake flask cultures described in Table 11.
free iBuOH from
total iBuOH total iBuOH iBuOH iBuO-COFA iBuO-COFA
flask time (h) (mg) (AQ) (mg) (ORG) (mg) (ORG) (mg)
(ORG) (mg) (ORG)
F1 24 0
F1 96 0
F2 24 0 0 0 0.0 0.0
F2 96 0 0 0 0.0 0.0
F3 24 439
F3 96 447
F4 24 239 262 257 4.7 21.1
F4 96 247 255 239 15.4 69.5
F5 24 0
F5 96 0
F6 24 0 0 0 0.0 0.0
F6 96 0 0 0 0.0 0.0
F7 24 446
F7 96 443
F8 24 231 271 266 5.0 22.5
F8 96 234 267 251 16.0 72.1
F9 24 234 267 262 4.9 22.0
F9 96 224 278 261 16.6 74.9
F10 24 0 0 0 0.0 0.0
F10 96 0 0 0 0.0 0.0
Example 7
Expression of Candida antarctica Lipase B in Yeast
The DNA sequence for the Candida antarctica lipase B (CalB
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lipase) was obtained from GenBank (accession number Z30645), and the
sequence was optimized for expression in yeast (DNA 2.0, Menlo Park,
CA). The resulting DNA sequence had 72% sequence identity with the
wildtype sequence, and encoded an identical protein.
The DNA comprising the expression-optimized CalB open reading
frame (ORF) sequence was synthesized (DNA 2.0), and the resulting DNA
molecule was cloned into a yeast-E. co/i shuttle vector by gap-repair
cloning. Briefly, the CalB lipase ORF was amplified using primers
CALBL_gap_for and CALBL_gap_rev (SEQ ID NOs: 14 and 15), which
include 5' regions having homology to regions in plasmid pNAK34 (SEQ
ID NO: 232). The resulting PCR product was co-transformed into S.
cerevisiae strain PNY1500 with plasmids pNAK33 (SEQ ID NO: 231),
pNAK34 (SEQ ID NO: 232), or pNAK35 (SEQ ID NO: 233) that had been
linearized with the Hpal restriction endonuclease, by lithium acetate/PEG
transformation. The transformation reaction was plated onto SCD-His
medium. After incubation at 30 C for 3 days, colonies were analyzed for
plasmid containing the CalB lipase sequence by colony PCR using
primers CALBL_gap_for and CALBL_gap_rev (SEQ ID NOs: 14 and 15).
CalB lipase-positive isolates were plated onto SCD-His medium
containing tributyrin and incubated at 30 C for 3 days. The CalB lipase-
positive isolates had a zone of clearing around them, indicating that they
were secreting a functional lipase activity; in contrast, a control yeast
strain did not cause clearing of tributyrin in the agar medium. The plasmids
from 3 isolates were recovered by plasmid rescue and sequenced using
M13-reverse and T7-promoter primers (SEQ ID NOs: 16 and 17). The
sequences were a perfect match for the predicted plasmid product of the
gap-repair cloning strategy. The resulting plasmids were named pTVAN7
(TEF1(M2) promoter), pTVAN3 (TEF1(M4) promoter), and pTVAN8
(TEF1(M6) promoter) (SEQ ID NOs: 278 , 277, and 240, respectively).
Example 8
Surface display of Tlan lipase
A domain that tethers the secreted T. lanuginosus lipase to the
yeast cell surface was introduced as follows. Yeast genomic DNA
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(PNY1500) was used as template in a PCR reaction with primers AK11-46
and AK11-47 (SEQ ID NOs: 215 and 216, respectively), which amplified
the codons for the C-terminal 320 amino acids of the yeast a-agglutinin
protein encoded by SAG1 , and added a sequence at the 5' end containing
a glycine- and serine-rich linker region. Amplification was done with
Phusion DNA polymerase (New England Biolabs) according to the
manufacturer's instructions.
This GS-SAG1 DNA was TOPO cloned into pCR-Blunt1I-TOPO
(InVitrogen) and transformed into DH5a. The pGS-SAG1 plasmid (SEQ ID
NO: 217) was recovered by mini-prep (Qiagen) and the correct sequence
was confirmed by DNA sequencing. The DNA was amplified with primers
Sagtgap1 and Sagtgap2 (SEQ ID NOs: 218 and 219, respectively) which
include regions of homology for gap-repair cloning into lipase expression
vectors pTVAN11, pTVAN12, and pTVAN13 (SEQ ID NOs: 220, 221, and
222, respectively). The purified PCR products were transformed into yeast
strain PNY1500 along with Pad-digested pTVAN11 (TEF1(M2) promoter),
pTVAN12 (TEF1(M4) promoter), or pTVAN13 (TEF1(M6) promoter). The
transformation reactions were plated to SCD-His medium; colonies that
appeared tested positive for expression of lipase activity on tributyrin
plates. Plasmids were rescued from these isolates (Yeast Plasmid
Miniprep Kit, Zymo Research) and transformed into E. coli DH5a and
purified. Sequence analysis showed the expected nucleotide sequence of
the lipase-SAG1 chimera.
The lipase-expressing strains (PNY1052, PNY1053, and PNY1054)
and the control strain (PNY1500) were grown overnight in 50 mL SCD-His
medium, in a 250 mL vented-cap flask incubated at 30 C and 250 rpm.
The following morning, 21.5 mL of the culture was transferred to a 125 mL
flask (unvented cap), with addition of 1.75 mL glucose (500 g/L), 2.5 mL
10X YEP (100 g/L yeast extract, 200 g/L peptone), and 0.313 mL
isobutanol. A sample was taken, then 10.3 mL COFA and a sterile stir bar
were added and the flasks returned to incubation. A sample (5 mL) was
taken after 24 h for HPLC and GC analysis, and 1.75 mL glucose and
0.313 mL isobutanol were added. A second sample was taken after 72 h.
Samples were analyzed as described above (Table 14). The strains
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expressing the SAG1-lipase chimera produced more fatty acid butyl ester
(FABE) than the control strain. Strain PNY1054, which had the strongest
promoter driving transcription of the chimera, produced greater than 6-fold
more FABE than the control, whereas the strains with weaker promoters
produced only ¨30% more FABE than the control.
Table 14. Measured amounts of isobutanol (iBuOH) and fatty acid isobutyl
ester (FABE) in aqueous and organic phases of shake flask cultivations of
the strain indicated.
24 h
iBuOH in rxn, mg iBuOH in rxn, mg FABE
in rxn, mg
Strain
(AQ) (ORG) (ORG)
PNY1052 226 255 29
PNY1053 224 252 30
PNY1054 230 248 167
PNY1500 221 245 25
72 h
PNY1052 201 204 52
PNY1053 197 202 52
PNY1054 206 188 264
PNY1500 195 196 39
Example 9
Cell surface display: cell-association test
This experiment was conducted to determine whether the lipase
activity expressed by the SAG1-lipase chimera was in fact cell-associated
or was secreted into the culture broth.
The lipase-expressing strains (PNY1052, PNY1053, and PNY1054)
and the control strain (PNY1500) were grown for 24 h in 25 mL SCD-His
medium (6.7 g/L yeast nitrogen base without amino acids, 1926 mg
dropout mix minus histidine, 20 g/L glucose), in a 250 mL vented-cap flask
incubated at 30 C and 250 rpm. Then the cells and culture broth were
separated by centrifugation; the cell pellet was washed twice and
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resuspended in 25 mL 50 mM MES buffer pH 5.5. The spent cell-free
culture medium was amended with 1 M MES buffer pH 5.5 to 50 mM.
Isobutanol (final concentration 20 g/L) and COFA (33% wt/wt) were added
to the spent medium and to the cell suspension. The two reactions were
incubated for 72 h at 30 C and 250 rpm. Samples were analyzed by GC
as described above (Table 15). The suspensions of lipase-expressing
cells formed ¨2.5-fold more FABE than the control cell suspension, after
incubation for 72 h with COFA and isobutanol. In contrast, there was no
difference in FABE accumulation in the cell-free medium samples,
demonstrating that the Sag1-lipase chimeric protein is exclusively cell-
associated under these conditions.
Table 15. Measured amounts of isobutanol (iBuOH) and fatty acid isobutyl
ester (FABE) in aqueous and organic phases of shake flask cultivations of
the strain indicated.
Cell-free medium
iBuOH in rxn, mg iBuOH in rxn, mg FABE in rxn, mg
Strain
(AQ) (ORG) (ORG)
PNY1052 215.1 236.3 56.2
PNY1053 211.0 236.8 56.1
PNY1054 221.1 269.1 63.5
PNY1500 208.7 256.6 60.3
Cells suspended in MES buffer
PNY1052 226.3 204.1 143.0
PNY1053 224.2 209.2 151.9
PNY1054 230.3 211.6 137.8
PNY1500 221.1 225.8 55.9
Example 10
Engineering isobutanol-producing yeast to secrete T. lanuqinosus lipase
The Tlan lipase transgene was amplified from plasmid pTVAN6
(SEQ ID NO: 183) with oligonucleotides AK11-24 (SEQ ID NO: 132) and
AK11-25 (SEQ ID NO: 133), which include Ascl sites at their 5' ends. The
PCR products were digested with Ascl and ligated into Ascl-digested
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pBP1236 (SEQ ID NO: 185). This plasmid is used to apply the technique
of Akada et al. (Akada R et al. (2006) PCR-mediated seamless gene
deletion and marker recycling in Saccharomyces cerevisiae. Yeast
23:399-405) for integration of transgenes at the fra2A locus of yeast. The
ligation mixture was transformed into competent E. coli DH5a (Invitrogen,
Carlsbad CA) and plated onto LB-ampicillin agar. Colonies from this plate
were grown overnight in LB-ampicillin, and plasmid DNA was isolated
using the Qiaprep Spin Miniprep kit. Recombinant plasm ids were identified
by digestion with Ascl and agarose gel electrophoresis. DNA sequencing
was used to identify the orientation of the lipase transgenes in the
construct. Plasmid pNAK15 (SEQ ID NO: 186) contains the wildtype lipase
transgene in the reverse direction, and pNAK16 (SEQ ID NO: 187)
contains the wildtype lipase transgene in the forward orientation.
The lipase transgenes were amplified from these plasmids along
with flanking DNA that targets them for integration at fra2A (and which
includes the URA3 gene as a selectable marker) using primers 0BP691
(SEQ ID NO: 136) and 0BP696 (SEQ ID NO: 137). The PCR products
were purified and concentrated using a QIAQuick PCR Purification kit.
Yeast strain PNY2211 (construction described above) was grown
overnight in YPE medium (10 g/L yeast extract, 20 g/L peptone, 20 mL/I
95% ethanol) at 30 C and 250 rpm, and transformed with the PCR
products followed by plating to SCE-Ura agar medium (6.7 g/L yeast
nitrogen base without amino acids (YNB; Difco 291940, BD, Franklin
Lakes NJ), 1926 mg/L dropout mix ¨Ura (DSCK102, Formedium, Norfolk
UK), 20 mL/L 95% ethanol). Ura+ colonies were plated to fresh medium,
and then re-plated to FOA medium (6.7 g/L YNB, 1 g/L 5-fluoroorotic acid,
200 mg/L uracil, 20 mL/I 95% ethanol) to select for isolates that had lost
the URA3 selection marker.
FOA-resistant transformants were checked for correct integration of
the transgene and loss of the selection marker by colony PCR using
primer pairs for each flank of the integration cassette as follows: for the
construct with the transgene in the forward orientation (from pNAK16),
primer pairs AK11-26 (SEQ ID NO: 134) and 0BP730 (SEQ ID NO: 138),
and AK11-27 (SEQ ID NO: 135) and 0BP731 (SEQ ID NO: 139), were
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used; for the construct with the transgene in the reverse orientation (from
pNAK15), primer pairs AK11-27 (SEQ ID NO: 135) and 0BP730 (SEQ ID
NO: 138), and AK11-26 (SEQ ID NO: 134) and 0BP731 (SEQ ID NO:
139), were used. Isolates that produced the correct PCR products were
chosen for further study, and named PNY931 (reverse orientation) and
PNY932 (forward orientation).
The lipase integrant yeast strains, and their parent strain PNY2211,
were transformed with plasmids pBP915 (SEQ ID NO: 44) and
pYZ090AalsS (SEQ ID NO: 43) in order to introduce an isobutanol
metabolic pathway. The strains were cultivated overnight in YPE medium,
then transformed with plasmid DNA as described above, and plated to
SCE ¨His ¨Ura agar medium (6.7 g/L YNB, 1850 mg/L dropout mix ¨His ¨
Ura (DSCK162, Formedium), 20 mL/I 95% ethanol). Colonies were re-
plated to SCE ¨His ¨Ura agar medium, and named PNY934 and PNY935.
Example 11
Production of isobutanol and fatty acid isobutyl esters by heterologous
lipase expression
Strains PNY934 and PNY935 were replated to SC ¨His ¨Ura DE
agar medium (6.7 g/L YNB, 1850 mg/L dropout mix ¨His ¨Ura, 3 g/L
glucose, 3 mL/I 95% ethanol); these cells, along with cells of a control
strain PNY2242 (which produces isobutanol but does not secrete
heterologous lipase) were used to inoculate 3 mL pre-cultures of SC ¨His
¨Ura DE medium. These were grown ¨6 h. Two mL were used to
inoculate 50 mL of the same medium in 250 mL flasks with vented caps;
these were grown overnight to an optical density (0D600) of ¨1. The next
morning, glucose, yeast extract, and peptone were added to
concentrations of 35, 10, and 20 g/L, respectively, with a final volume of
75 mL. This was divided evenly among triplicate 125 mL shake flasks
(non-vented caps, containing a stir bar), and then 10.3 mL of corn oil fatty
acid (COFA) was added and the flasks were incubated at 30 C and 250
rpm. Samples were taken at 0 h (0.6 mL, before COFA addition), and at
24 h and 72 h (5 mL each, after thorough mixing of the aqueous and
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COFA phases). Samples were analyzed by HPLC and GC as previously
described. At 24 h, 1.5 mL of 500 g/L glucose was added.
The isobutanol produced in these fermentations was distributed
among 3 fractions: free isobutanol in the aqueous and COFA phases, and
a fatty acid isobutyl ester (FABE) fraction produced by the esterification of
isobutanol with fatty acids. As shown in Table 16, the lipase-secreting
strains produced considerable amounts of FABE, whereas the control
strain produced only a low amount, -12% of that produced by the lipase-
secreting strains. The lipase-catalyzed esterification of isobutanol into
FABE resulted in a decrease in the aqueous isobutanol concentration as a
percent of the total amount of isobutanol in the system by about 10%, from
53% to 43% at 24 h, and from 45% to -31% at 72 h.
Table 16. Measured amounts of isobutanol (iBuOH) and fatty acid isobutyl
ester (FABE) in aqueous and organic phases of shake flask cultivations of
the strain indicated. Mean standard deviation of triplicate flasks is
shown. Amounts are corrected for volume loss due to sampling.
24 h
iBuOH in rxn, mg iBuOH in rxn, mg FABE in
rxn, mg
(AQ) (ORG) (ORG)
PNY934
42.2 0.9 32 3.4 103.3 4.7
PNY935
49.7 14.9 41.3 15.6 106.5 13.8
PNY2242
93 0.4 78.3 2.5 15.1 0.1
72 h
PNY934
37.9 3.2 32.8 1.1 283 43.3
PNY935
50.4 20.8 32 1.2 284.4 57.6
PNY2242
97.6 57.3 112.5 31.8 34.9 3.5
Example 12
Production of isobutanol and fatty acid isobutyl esters by heterologous
lipase secretion - comparing qlycosylated and non-qlycosylated lipase
The experiment of the previous example was repeated, with the
inclusion of strain PNY936, which secretes the N55A mutant of Tlan
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lipase. In this experiment, glucose was added twice, after 24 h and again
after 48 h (1.5 mL of 500 g/L glucose).
The lipase-secreting strains produced more FABE than the control
strain (in this instance -5-6-fold more). The proportion of the total
isobutanol in the aqueous fraction was decreased as a consequence of
FABE formation, by -10% at 24 h and by -15% at 72 h. The cultures in
which lipase-secreting isobutanologens were grown produced significantly
more FABE fraction than the control. The amount of FABE produced in the
fermentation with the PNY936 strain (secreting glycosylation-mutant
lipase) did not differ significantly from that produced by the strains
secreting the wildtype lipase enzyme.
Table 17. Measured amounts of isobutanol (iBuOH) and fatty acid
isobutyl ester (FABE) in aqueous and organic phases of shake flask
cultivations of the strain indicated. Mean standard deviation of triplicate
flasks is shown. Amounts are corrected for volume loss due to sampling.
24 h
iBuOH in rxn, mg iBuOH in rxn, mg FABE in
rxn, mg
(AQ) (ORG) (ORG)
PNY934
36.3 6.5 31.3 5.8 89.1 6.8
PNY935
33.2 2 27.5 1.4 76.5 5.5
PNY936
34.8 1 28.8 0.8 74.5 1.3
PNY2242
75.1 4.3 63.4 3 14 0.1
72 h
PNY934
48.3 6.2 41 4.6 197.6 20.3
PNY935
45.2 3.6 39.6 5.8 184.2 13.4
PNY936
46.4 1.8 38.7 1.3 203.7 24.4
PNY2242
143.4 20.8 135.9 22.7 34.3 2.1
Example 13
Engineering isobutanol-producing yeast to express C. deformans and C.
antarctica lipases
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The LIP1 and CalB lipase transgenes encoding the wildtype lipases
from C. deformans and C. antarctica, respectively, were amplified from
plasmids pNAK10 (SEQ ID NO: 45), pNAK31 (SEQ ID NO: 238), and
pTVAN8 (SEQ ID NO: 240) with oligonucleotides AK11-24 (SEQ ID NO:
132) and AK11-25 (SEQ ID NO: 133), which include Ascl sites at their 5'
ends. The PCR products were digested with Ascl and ligated into Ascl-
digested pNAK36 (SEQ ID NO: 223). The ligation mixture was
transformed into competent E. coli DH5a (Invitrogen) and plated onto LB-
ampicillin agar. Colonies from this plate were grown overnight in LB-
ampicillin, and plasmid DNA was isolated using the Qiaprep Spin Miniprep
kit. Recombinant plasmids were identified by digestion with Ascl and
agarose gel electrophoresis. Plasmid pNAK38 (SEQ ID NO: 224) contains
the CalB lipase under control of the TEF1(M6) promoter, pNAK37 (SEQ ID
NO: 225) contains the LIP1 lipase under control of the TEF1(M4)
promoter, and pNAK39 (SEQ ID NO: 226) contains the LIP1 lipase under
control of the TEF1(M6) promoter.
The lipase transgenes were amplified from these plasmids along
with flanking DNA that targets them for integration at gpd2.6 (and which
includes the URA3 gene as a selectable marker) using primers 0BP691
(SEQ ID NO: 136) and 0BP696 (SEQ ID NO: 137). The PCR products
were purified and concentrated using a QIAQuick PCR Purification kit.
Yeast strain PNY1556 was grown overnight in YPE medium (10 g/I yeast
extract, 20 g/I peptone, 20 m1/I 95% ethanol) at 30 C and 250 rpm, and
transformed with the PCR products followed by plating to SCE ¨Ura agar
medium. Ura+ colonies were plated to fresh medium, and then re-plated to
FOA medium to select for isolates that had lost the selectable marker.
FOA-resistant transformants were checked for correct integration of
the transgene and loss of the selectable marker by colony PCR using
primer pairs for each flank of the integration cassette as follows: genomic
DNA was purified using the PureGene kit (Qiagen) essentially as
described by the manufacturer. This was used as template for a PCR
reaction with oligos HY48 (SEQ ID NO: 227) and HY49 (SEQ ID NO: 228).
Positive integrants were plated to FOA medium, and FOA-resistant
isolates were recovered. Isolates which had lost the URA3 marker from
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the gpd2A locus were identified by PCR using oligos HY48 and HY49 as
described above.
The lipase integrant yeast strains and the control strain, PNY1556,
were transformed with plasmid pBP2092 (SEQ ID NO: 237) in order to
introduce an isobutanol metabolic pathway, as follows: The strains were
cultivated overnight in YPE medium, then transformed with plasmid DNA
as described above, and plated to SCE ¨His agar medium. Colonies were
re-plated to SCE ¨Ura agar medium, and named PNY1022 (TEF1(M4)-
LIP1), PNY1023 (TEF1(M6)-LIP1), and PNY1024 (TEF1(M4)-CalB).
Example 14
Production of isobutanol and fatty acid isobutyl esters by
heterologous expression of C. deformans and C. antarctica lipases in an
isobutanologen
Strains PNY1022, PNY1023, and PNY1024 were replated to SC ¨
Ura DE agar medium; these cells were used to inoculate 3 ml pre-cultures
of SC ¨His ¨Ura DE medium, which were grown ¨6 h. Two ml were used
to inoculate 50 ml of the same medium in 250 ml flasks with vented caps;
these were grown overnight to an optical density (0D600) of ¨1. The next
morning glucose, yeast extract, and peptone were added to
concentrations of 35, 10, and 20 g/I, respectively, with a final volume of 75
ml. This was divided evenly among triplicate 125 ml shake flasks (non-
vented caps, containing a stir bar), 10.3 ml of corn oil fatty acid (COFA)
was added, and the flasks were incubated at 30 C and 250 rpm. Samples
were taken at 0 h (before COFA addition), and at 24 h, 48 h, and 94 h,
after thorough mixing of the aqueous and COFA phases. Samples were
analyzed by HPLC and GC. At 24 h, 1.5 mL of 500 g/L glucose was added
(1.2 mL to the PNY1556 culture); at 48 h, 1.6 mL of glucose was added to
each flask..
The isobutanol produced in these fermentations was distributed
among 3 phases: free isobutanol in the aqueous and COFA phases, and
fatty acid isobutyl ester (FABE) produced the esterification of isobutanol
with fatty acids. As shown in Table 18, the strains expressing the C.
deformans lipase produced considerable amounts of FABE at both 24 and
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94 h. PNY1023, which has a stronger promoter driving expression of the
C. deformans lipase transgene, makes approx. twice as much FABE as
PNY1022. Interestingly, by 94 h PNY1023 produced significantly more
total isobutanol than the other strains.
The strain expressing the CalB lipase produced much less FABE
than the strains expressing the C. deformans enzyme, although there was
significantly more FABE in its flasks than in the control fermentations. The
control strain (with no lipase transgene) esterified only 10 mg of isobutanol
into FABE by 94 h, presumably due to endogenous lipase activity. The
fermentations carried out by lipase-expressing isobutanologens are all
marked by a significantly lower aqueous isobutanol concentration than the
control fermentations. In the case of the fermentations with C. deformans-
expressing strains (PNY1022 and PNY1023), this corresponds with a
significant accumulation of FABE; the strain expressing the C. antarctica
accumulated much less ester. For the C. deformans-expressing strains,
the total isobutanol production is comparable to the no-lipase control when
a weak promoter is used to express the lipase gene; when a strong
promoter is used, the total isobutanol production is 24% higher than the
control.
Table 18. Measured amounts of isobutanol (iBuOH) and fatty acid
isobutyl ester (FABE) in aqueous and organic phases of shake flask
cultivations of the strain indicated.
24 h
iBuOH in rxn, mg iBuOH in rxn, mg FABE
in rxn, mg
Strain
(AQ) (ORG) (ORG)
PNY1022 126 3 98 2 290.7
16.6
PNY1023 94 5 67 0 534.2
26.1
PNY1024 157 4 133 4 23.7 2
PNY1556 187 5 112 3 7.6
0.5
94 h
PNY1022 211 3 139 3 559.8
8.3
PNY1023 206 19 126 14 1182.7
14.3
PNY1024 204 37 143 31 131.1
11.7
PNY1556 271 4 195 1 42.8
0.6
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Example 15
Expression of Asperaillus tubingensis LIP3 lipase in yeast
The DNA encoding the Aspergillus tubingensis LIP3 lipase was
synthesized (DNA 2.0) with codon usage optimized for expression in S.
cerevisiae. This DNA was amplified using primers Atublip1 and AtubLip2
(SEQ ID NOs: 229 and 230, respectively) with Phusion DNA polymerase
(New England Biolabs). The PCR product was transformed into yeast
strain PNY1500 along with gapped plasmids pNAK33 (TEF1(M2)
promoter), pNAK34 (TEF1(M4) promoter), and pNAK35 (TEF1(M6)
promoter) (SEQ ID NOs: 231, 232, and 233, respectively). The
transformation reactions were plated to SOD-His medium; colonies that
appeared tested positive for expression of lipase activity on tributyrin
plates. Plasmids were rescued from these isolates (Yeast Plasmid
Miniprep Kit, Zymo Research) and transformed into E. coli DH5a and
purified. Sequence analysis showed the expected nucleotide sequence of
the A. tubingensis lipase transgenes. They were named pTVAN9,
pTVAN4, and pTVAN10, respectively, for the TEF1(M2), TEF1(M4), and
TEF1(M6) promoter variants, respectively ((SEQ ID NOs: 234, 235, and
236, respectively)).
The lipase-expressing strains PNY1055 (pTVAN9), PNY1056
(pTVAN4), and PNY1057 (pTVAN10) and the wildtype control strain
(PNY827) were grown overnight in 50 mL SOD-His medium in a 250 mL
vented-cap flask incubated at 30 C and 250 rpm. The following morning,
22 mL of the culture was transferred to a 125 mL flask (unvented cap),
with addition of 1.75 mL glucose (500 g/L), 2.5 mL 10X YEP (100 g/L
yeast extract, 200 g/L peptone), and 0.313 mL isobutanol. A sample was
taken, then 10.3 mL COFA and a sterile stir bar were added and the flasks
returned to incubation. A sample (5 mL) was taken after 24 h for HPLC
and GC analysis, and 1.75 mL glucose and 0.313 mL isobutanol were
added. A second sample was taken after 96 h. Samples were analyzed as
described above. The lipase-expressing strains were able to esterify
isobutanol into FABE; at 96 h, the amount of FABE formed by these
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strains was more than ten-fold the amount formed by the control strain. As
shown, the greatest amount of FABE formation was achieved by the strain
with an intermediate-strength promoter driving lipase transcription.
Table 19. Measured amounts of isobutanol (iBuOH) and fatty acid isobutyl
ester (FABE) in aqueous and organic phases of shake flask cultivations of
the strain indicated.
24 h
iBuOH in rxn, mg iBuOH in rxn, mg FABE in rxn, mg
Strain
(AQ) (ORG) (ORG)
PNY1055 108 101 183
PNY1056 98 83 272
PNY1057 103 91 209
PNY827 130 116 15
96 h
PNY1055 171 159 649
PNY1056 141 122 886
PNY1057 163 144 700
PNY827 237 232 52
Example 16
Genetic abolition of the qlycosylation of lipase expressed in yeast
N-glycosylation sequences matching to the consensus site of
asparaginyl glycosylation, N-X-SIT (Drickamer K & Taylor ME (2006)
Introduction to Glycobiology (2nd ed.). Oxford University Press, USA)
were identified in LIP1 and CalB. LIP1 has two glycosylation sites (NIS at
residue 146 and NNT at residue 167), and CalB has one (NDT at residue
99). These were altered by site-directed mutagenesis to substitute N with
A in all cases (and to create the double mutant in LIP1) as follows.
Mutagenesis was carried out with the QuikChange Site-Directed
Mutagenesis Kit (Strategene, La Jolla CA) according to the manufacturer's
instructions, combining the following plasm ids and primers:
Protein, site Primers Primer Plasmid Plasmid
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SEQ ID SEQ ID
NOs: NO:
CalB, N99 Ca_NA99_for 264 pTVAN8 240
Ca_NA99_rev 265
LIP1, N146 Cd_N146A _for 266 pNAK31 238
Cd N146A_rev 267
LIP1, N167 Cd_NA167_for 268 pNAK31 238
Cd NA167 rev 269
LIP1/N167, Cd_N146A _for 266 pTVAN26 270
N146 Cd N146A_rev 267
After amplification of the plasmid backbone with mutagenic primers
using the thermostable polymerase provided with the kit, the DNA was
digested with Dpnl restriction endonuclease. The treated plasmids were
transformed into E. coli XL1-Blue competent cells, and recovered using
the Qiaprep Spin Miniprep Kit (Qiagen). Mutated clones were identified by
DNA sequence analysis of the mutagenized plasmids. The plasmids were
named pTVAN20, pTVAN25, pTVAN26, and pTVAN27, respectively. The
plasmids (and control plasmids with the wildtype lipase genes) were
transformed into the PNY1500 yeast strain.
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