U.S. patent application number 14/148369 was filed with the patent office on 2014-05-22 for recombinant host cells and media for ethanol production.
This patent application is currently assigned to UNIVERSITY OF FLORIDA RESEARCH FOUNDATION, INC.. The applicant listed for this patent is UNIVERSITY OF FLORIDA RESEARCH FOUNDATION, INC.. Invention is credited to Lonnie O' Neal Ingram, Brent E. Wood, Lorraine P. Yomano, Sean W. York.
Application Number | 20140141493 14/148369 |
Document ID | / |
Family ID | 37605060 |
Filed Date | 2014-05-22 |
United States Patent
Application |
20140141493 |
Kind Code |
A1 |
Ingram; Lonnie O' Neal ; et
al. |
May 22, 2014 |
RECOMBINANT HOST CELLS AND MEDIA FOR ETHANOL PRODUCTION
Abstract
Disclosed are recombinant host cells suitable for degrading an
oligosaccharide that have been optimized for growth and production
of high yields of ethanol, and methods of making and using these
cells. The invention further provides minimal media comprising
urea-like compounds for economical production of ethanol by
recombinant microorganisms. Recombinant host cells in accordance
with the invention are modified by gene mutation to eliminate genes
responsible for the production of unwanted products other than
ethanol, thereby increasing the yield of ethanol produced from the
oligosaccharides, relative to unmutated parent strains. The new and
improved strains of recombinant bacteria are capable of superior
ethanol productivity and yield when grown under conditions suitable
for fermentation in minimal growth media containing inexpensive
reagents. Systems optimized for ethanol production combine a
selected optimized minimal medium with a recombinant host cell
optimized for use in the selected medium. Preferred systems are
suitable for efficient ethanol production by simultaneous
saccharification and fermentation (SSF) using lignocellulose as an
oligosaccharide source. The invention also provides novel isolated
polynucleotide sequences, polypeptide sequences, vectors and
antibodies.
Inventors: |
Ingram; Lonnie O' Neal;
(Gainesville, FL) ; Yomano; Lorraine P.;
(Gainesville, FL) ; York; Sean W.; (Gainesville,
FL) ; Wood; Brent E.; (Gainesville, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF FLORIDA RESEARCH FOUNDATION, INC. |
GAINESVILLE |
FL |
US |
|
|
Assignee: |
UNIVERSITY OF FLORIDA RESEARCH
FOUNDATION, INC.
GAINESVILLE
FL
|
Family ID: |
37605060 |
Appl. No.: |
14/148369 |
Filed: |
January 6, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11922631 |
Apr 1, 2010 |
8652817 |
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PCT/US2006/025655 |
Jun 30, 2006 |
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14148369 |
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60696076 |
Jul 1, 2005 |
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Current U.S.
Class: |
435/252 ;
435/253.6 |
Current CPC
Class: |
C12N 1/22 20130101; Y02E
50/10 20130101; C12P 7/065 20130101; Y02E 50/17 20130101 |
Class at
Publication: |
435/252 ;
435/253.6 |
International
Class: |
C12N 1/22 20060101
C12N001/22 |
Goverment Interests
STATEMENT OF U.S. GOVERNMENT INTEREST
[0002] Funding for the present invention was provided in part by
the Government of the United States under Grant Nos.:
01-35504-10669 from the U.S. Department of Agriculture, and
FG02-96ER20222 from the U.S. Department of Energy. The Government
of the United States has certain rights in and to the invention.
Claims
1-65. (canceled)
66. A minimal medium that supports growth and ethanol production by
a recombinant host cell suitable for degrading a saccharide,
comprising: a defined nitrogen source; a complex nitrogen source; a
source of phosphate; and a source of magnesium.
67. The minimal medium according to claim 66, wherein the defined
nitrogen source is a urea-like compound.
68. The minimal medium according to claim 67, wherein the defined
nitrogen source is urea.
69. The minimal medium according to claim 66, wherein the complex
nitrogen source is selected from the group consisting of corn steep
liquor (CSL), yeast autolysate and/or extract, corn processing
by-product, soy processing by-product, and spent fermentation
broth.
70. The minimal medium according to claim 67, wherein the
concentration of urea nitrogen is from about 0.1 mM to about 100
mM.
71. The minimal medium according to claim 70, wherein the
concentration of urea nitrogen is from about 2 mM to about 20
mM.
72. The minimal medium according to claim 71, wherein the
concentration of urea nitrogen is from about 8 mM to about 12
mM.
73. The minimal medium according to claim 69, wherein the
concentration of CSL is from about 0.1 g L.sup.-1 to about 100 g
L.sup.-1.
74. The minimal medium according to claim 73, wherein the
concentration of CSL is from about 1 g L.sup.-1 to about 20 g
L.sup.-1.
75. The minimal medium according to claim 74, wherein the
concentration of CSL is from about 5 g L.sup.-1 to about 10 g
L.sup.-1.
76. The minimal medium claim 66, wherein the concentration of total
phosphate is from about 1 mM to about 100 mM.
77. The minimal medium according to claim 76, wherein the
concentration of phosphate is from about 10 mM to about 12 mM.
78. The minimal medium of claim 66, wherein the concentration of
magnesium is from about 0.1 mM to about 5.0 mM.
79. The minimal medium according to claim 78, wherein the
concentration of magnesium is from about 0.25 mM to about 1.0
mM.
80. The minimal medium of claim 66, optimized to support growth and
ethanol production by a recombinant host cell suitable for
degrading a saccharide.
81. The minimal medium according to claim 80, optimized to support
growth and ethanol production at acidic pH.
82. The minimal medium of claim 66, optimized to support growth and
ethanol production by a recombinant host cell suitable for
degrading a saccharide comprising: (a) a heterologous
polynucleotide sequence that codes for an enzyme that converts
sugars to ethanol, wherein said host cell expresses said
heterologous polynucleotide sequence at a sufficient functional
level so as to facilitate production of ethanol as a primary
fermentation product by said cell; and (b) a mutation in at least
one polynucleotide sequence that codes for a protein in a metabolic
pathway in said cell that produces a product other than ethanol
from sugars, wherein said mutation results in increased ethanol
production by said cell, as compared to ethanol production by the
cell in the absence of said mutation.
83. The minimal medium according to claim 80, optimized to support
growth and ethanol production by strains of Klebsiella oxytoca.
84. The minimal medium of claim 66, further comprising a source of
saccharide.
85. The minimal medium according to claim 84 wherein the source of
saccharide comprises lignocellulose.
86-111. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 11/922,631, filed Apr. 1, 2010, granted, which is the U.S.
national phase application, pursuant to 35 U.S.C. .sctn.371, of PCT
international application Ser. No. PCT/US2006/025655, filed Jun.
30, 2006, designating the United States and published in English on
Jan. 11, 2007 as publication WO 2007/005646 claims the benefit of
U.S. provisional application Ser. No. 60/696,076, filed Jul. 1,
2005, the entire content of which is expressly incorporated herein
by reference.
BACKGROUND
[0003] The use of ethanol as an automotive fuel provides a cleaner
burning and renewable alternative to petroleum-based fuels [1].
Technology currently in use for ethanol production is based on
edible crops such as sugar cane juice (molasses) and corn starch
[2] that have alternative markets. The cost of these feedstocks has
been estimated to represent 40% of total production costs [3]. In
contrast, inedible lignocellulosic biomass is available at a cost
competitive with petroleum [4]. The continued development of
improved microorganisms for the conversion of lignocellulosic
sugars into ethanol offers the potential to decrease dependence on
petroleum and create new manufacturing opportunities from existing
plant materials.
[0004] Ethanologenic strains of Klebsiella oxytoca have been
developed [5,6]. These strains have been shown to metabolize a
variety of sugar monomers (such as glucose, xylose, and arabinose)
derived from lignocellulosic biomass [5-7]. Such strains can
function well in simultaneous saccharification and fermentation
(SSF) processes with cellulose [8-10]. An ethanologenic strain of
K. oxytoca known as K. oxytoca P2 has been described that contains
genes from Zymomonas mobilis encoding pyruvate decarboxylase (pdc)
and alcohol dehydrogenase (adhB), enzymes involved in converting
oligosaccharides to ethanol. These genes are chromosomally
integrated into the genome of strain P2.
[0005] In contrast to analogous strains of Escherichia coli
[11-13], K. oxytoca has the native ability to metabolize many
soluble products from lignocellulosic biomass, including
cellobiose, cellotriose, xylobiose, xylotriose, and arabinosides
[6, 14, 15]. The ability of K. oxytoca P2 to efficiently metabolize
incompletely hydrolyzed products from lignocelluose at pH 5.2 (near
optimal for fungal enzymes) provides an added advantage during
simultaneous saccharification and fermentation (SSF) processes [8].
Under these conditions, K. oxytoca P2 required less than half of
the fungal cellulase required by Saccharomyces cerevisiae to
achieve equivalent fermentation rates and yields [9].
[0006] The availability of inexpensive industrial media for growth
of ethanologenic bacteria that support high ethanol productivity
and yield is essential for ethanol production from biomass
feedstocks [17,18]. However, unlike grain, hydrolysates of
cellulosic biomass are inherently nutrient poor and must be
supplemented [16]. Accordingly, previous use of K. oxytoca P2 for
ethanol production has involved culture of the cells in complex
growth media containing laboratory nutrients such as yeast extract
and Difco Tryptone.TM.. Unfortunately, it is impractical to use
such nutrients for commercial production of commodity chemicals
such as ethanol from lignocellulose.
[0007] To fully realize the potential of recombinant ethanologenic
bacterial strains to serve as a source of ethanol, there is a clear
need for new and improved strains of such bacteria that can
efficiently produce ethanol while growing in inexpensive minimal
media, and new media that can support these cells.
SUMMARY OF THE INVENTION
[0008] The invention relates to recombinant host cells that have
been optimized for growth and production of high yields of ethanol,
and methods of making and using these cells. The invention further
relates to novel optimized media for economical production of
ethanol by recombinant microorganisms.
[0009] During fermentation by microorganisms, sugar-containing
substrates in the media are converted into ethanol and a variety of
unwanted co-products. Recombinant cells in accordance with the
invention are modified by genetic manipulation to control (e.g.,
down regulate) genes responsible for the production of one or more
products other than ethanol, thereby increasing the yield of
ethanol produced by these cells from the sugars, relative to
unmutated parent strains. The new and improved strains of
recombinant bacteria are capable of superior ethanol productivity
and yield when grown under conditions suitable for fermentation in
minimal growth media containing inexpensive reagents. Certain
strains are optimized for superior ethanol production in particular
embodiments of the optimized media. Systems optimized for ethanol
production combine a selected optimized minimal medium and a
recombinant host cell optimized for use in the selected medium.
Preferred systems are suitable for efficient ethanol production by
SSF using lignocellulose as a saccharide source.
[0010] Accordingly, one aspect of the invention is a recombinant
host cell suitable for degrading a saccharide comprising:
[0011] (a) a heterologous polynucleotide sequence that codes for an
enzyme that converts sugars to ethanol, wherein the host cell
expresses the heterologous polynucleotide sequence at a sufficient
functional level so as to facilitate production of ethanol as a
primary fermentation product by the bacterium; and
[0012] (b) a mutation in at least one polynucleotide sequence that
codes for a protein in a metabolic pathway in the cell that
produces a product other than ethanol from sugars, wherein the
mutation results in increased ethanol production by the cell, as
compared to ethanol production by the cell in the absence of the
mutation. In some embodiments of the recombinant host cell, the
mutation is a deletion, insertion, or base change mutation.
[0013] Recombinant host cells in accordance with the invention can
be produced from any suitable host organism, including
single-celled or multicellular microorganisms such as bacteria,
fungi or yeast, and higher eukaryotic organisms including
nematodes, insects, reptiles, birds, amphibians and mammals. Yeast
host cells are derived, e.g., from Saccharomyces,
Schizosacharomyces, Hansenula, Pachyosolen, Kluyveromyces,
Debaryomyces, Yarrowia, and Pichia. Bacterial host cells are
selected from Gram-positive and Gram-negative bacteria. Preferred
Gram-negative bacteria are enteric bacteria such as strains of
Erwinia and Klebsiella. Gram-positive bacterial host cells include
Bacillus, Geobacillus, Clostridium, Streptococcus, and
Cellulomonas.
[0014] In some embodiments of bacterial host cells in accordance
with the invention, the heterologous polynucleotide sequence codes
for alcohol dehydrogenase and/or pyruvate decarboxylase, enzymes
involved in the conversion of sugars to ethanol.
[0015] Some embodiments of the recombinant cells are derived from
Klebsiella oxytoca, which has the native ability to use urea as a
nitrogen source. These strains are especially suitable host
organisms for use in minimal media comprising urea-like compounds
as a defined nitrogen source. One such suitable host strain is
Klebsiella oxytoca strain P2 (ATCC 55307).
[0016] As discussed above, recombinant host cells in accordance
with the invention comprise a mutation in at least one
polynucleotide sequence that codes for a protein in a metabolic
pathway in the cell that produces a product other than ethanol from
sugars. In various embodiments of the cells, the product other than
ethanol is selected from formate, lactate, succinate, acetate,
acetoin, butanediol, 2,3-butanediol, xylitol, butyrate, pyruvate,
proprionate, isopropyl alcohol, 1-propanol, 2-propanol,
propanediol, citrate, glutamate, and acetone.
[0017] In one embodiment of the recombinant cells, the metabolic
pathway is the butanediol pathway. Co-products of sugar metabolism
resulting from this pathway are acetoin and 2,3-butanediol, which
are produced by the enzymes .alpha.-acetolactate decarboxylase and
.alpha.-acetolactate synthase, respectively. Accordingly, in some
embodiments, the cells include a mutation in at least one
polynucleotide sequence that codes for an enzyme involved in the
butanediol metabolic pathway. The mutated polynucleotide sequence
can comprise a nucleotide sequence from a budA, budB, budR, or budC
gene, or a homolog or functional variant thereof. Some embodiments
comprise a deletion mutation in one or both of the budA and budB
genes. The deletion mutation decreases or eliminates expression of
at least one and preferably both of the enzymes
.alpha.-acetolactate decarboxylase and .alpha.-acetolactate
synthase in the cell, thereby increasing ethanol production by the
cell, as compared to ethanol production by the cell in absence of
the mutation.
[0018] Any of the above-described bacterial strains can be used to
obtain the genes for genetic manipulation and in some embodiments
to serve as hosts for reinsertion of DNA fragments comprising the
altered gene sequences.
[0019] Some preferred embodiments of recombinant bacterial host
cells in accordance with the invention are represented by
Klebsiella oxytoca strains, including strains BW15 (NRRLB-30857),
BW19 (NRRLB-30858), and BW21 (NRLLB-30859), which were deposited on
Jun. 28, 2005 with the Agricultural Research Service Culture
Collection (ARSCC) of the National Center for Agricultural
Utilization Research (Peoria, Ill., USA).
[0020] In another aspect, the invention provides a method for
producing ethanol from a source of saccharide. The saccharide
source is contacted with a recombinant host cell according to the
invention, as described above, to thereby produce ethanol from the
source of saccharide.
[0021] The invention further provides a method for producing a
recombinant host cell optimized for producing ethanol from a
saccharide source. The method comprises:
[0022] (a) contacting a parent ethanologenic host cell with a
selected medium and an oligosaccharide source, under conditions
suitable for ethanol production by the parent cell;
[0023] (b) determining the level of ethanol produced from the
saccharide source in the medium under the selected conditions;
[0024] (c) determining the level of at least one product other than
ethanol produced from the saccharide source, to identify an
undesirable co-product having increased expression in the medium
under the selected conditions; and
[0025] (d) mutating a polynucleotide sequence of a gene encoding a
protein in a metabolic pathway that produces the undesirable
co-product, wherein the mutation decreases or eliminates expression
of at least one protein in the metabolic pathway, and increases
ethanol production by the cell as compared to ethanol production by
the parent cell in the absence of the mutation, thereby producing a
recombinant host cell optimized for ethanol production from a
saccharide source.
[0026] Some embodiments of the method further comprise producing an
isolated polynucleotide fragment comprising a mutation of the gene;
and introducing the mutated polynucleotide fragment into the parent
cell. In some embodiments of the method, the mutation is a
deletion, insertion, or base change mutation.
[0027] In some embodiments of the method, the cell is optimized for
ethanol production in a minimal medium. Any suitable host cell as
described above can be used as the parent host cell strain to be
optimized by the method.
[0028] In yet another aspect, the invention provides novel minimal
media that support growth and ethanol production by a recombinant
host cell suitable for degrading a saccharide. A medium in
accordance with the invention includes a defined nitrogen source; a
complex nitrogen source such as corn steep liquor (CSL), yeast
autolysate and/or extract, corn processing by-product, soy
processing byproduct, or spent fermentation broth; a source of
phosphate; and source of magnesium.
[0029] Some embodiments of the minimal media include a urea-like
compound as a defined source of nitrogen. In various embodiments of
urea-based minimal media in accordance with the invention, the
concentration of urea nitrogen is from about 0.1 to 100 mM,
preferably from about 2.0-20 mM, and more preferably from about
8-12 mM. Also contributing to low production cost, minimal media in
accordance with the invention contain low levels of complex
nitrogen sources, for example, corn steep liquor (CSL). In several
embodiments, the concentration of CSL is from about 0.1-100 g
L.sup.-1, preferably from about 1-20 g L.sup.-1, and more
preferably from about 5-10 g L.sup.-1.
[0030] Some embodiments of the minimal media are optimized to
support growth and ethanol production by a recombinant host cell
suitable for degrading a saccharide. For example, some media are
optimized to support growth and ethanol production at acidic pH,
making them suitable for use in simultaneous saccharification and
fermentation (SSF).
[0031] Some media in accordance with the invention are optimized to
support growth and ethanol production by recombinant host cells
described herein, made according to the methods described above.
One preferred embodiment is a medium optimized to support growth
and ethanol production by recombinant strains of Klebsiella
oxytoca.
[0032] Media in accordance with the invention can further comprise
a source of saccharide. In some embodiments, the source of
saccharide comprises lignocellulose. The use of urea-like compounds
in the novel media of the invention is particularly advantageous
due to its relatively low cost as a source of nitrogen, relative to
proteinaceous sources such as peptone and also to small molecule
sources of nitrogen, such as ammonia, commonly used in growth media
for bacterial culture.
[0033] Also encompassed by the invention are isolated
polynucleotide sequences, vectors comprising these sequences, and
isolated polypeptide sequences. The sequences are useful for many
purposes, including construction of recombinant host cells
expressing isolated polynucleotide sequences in accordance with the
invention, and construction of recombinant host cells comprising
mutations in these sequences.
[0034] In various embodiments, the invention further provides
isolated polypeptides. Thus, in one embodiment, the invention
provides a fragment of a polypeptide comprising the amino acid
sequence of SEQ ID NO:6 or 7, wherein the fragment comprises at
least 15 contiguous amino acids of SEQ ID NO: 6 or 7.
[0035] Another embodiment is a naturally occurring allelic variant
of a polypeptide comprising the amino acid sequence of SEQ ID NO:6
or 7, wherein the polypeptide is encoded by a nucleic acid molecule
which hybridizes to a nucleic acid molecule consisting of SEQ ID
NO:3 or 4 under stringent conditions.
[0036] Other embodiments include polypeptides encoded by a nucleic
acid molecule comprising a nucleotide sequence which is at least
60% identical to a nucleic acid comprising the nucleotide sequence
of SEQ ID NO:3 or 4; and polypeptides comprising an amino acid
sequence which is at least 50% identical to the amino acid sequence
of SEQ ID NO:6 or 7.
[0037] Another aspect of the invention is a vector comprising a
deletion mutation in a polynucleotide sequence of a bacterial gene
coding for at least one of an .alpha.-acetolactate decarboxylase
and an .alpha.-acetolactate synthase protein. The vector is capable
of decreasing or eliminating expression of the proteins when
integrated into a bacterial host cell.
[0038] The invention further features antibodies, such as
monoclonal or polyclonal antibodies, that specifically bind
proteins/polypeptides of the invention.
[0039] In some embodiments, the mutated polynucleotide sequence is
derived from genes selected from budA and budB of Klebsiella
species. The polynucleotide sequence can comprise deletion
mutations in both budA and budB.
[0040] A preferred vector of this type comprises deletion mutations
in polynucleotide sequences of bacterial genes coding for
.alpha.-acetolactate decarboxylase and .alpha.-acetolactate
synthase proteins, wherein the vector is capable of decreasing or
eliminating expression of these gene products when integrated into
a bacterial host cell.
[0041] One embodiment of such a vector comprises a mutated budAB
polynucleotide sequence that is at least 80% identical to SEQ ID
NO:5. Another embodiment comprises a mutated budAB polynucleotide
sequence that is at least 80% identical to SEQ ID NO:8.
[0042] Particular embodiments of vectors in accordance with the
invention are plasmids, in which the polynucleotide sequences are
derived from genes selected from budA and budB of Klebsiella
species. Exemplary plasmids are designated herein as pLOI3310 or
pLOI3313.
[0043] Yet a further aspect of the invention is a system optimized
for ethanol production from a saccharide source by a recombinant
host cell suitable for degrading a saccharide. The system
comprises:
[0044] (a) a selected medium that supports optimal growth and
ethanol production by the host cell under selected conditions;
[0045] (b) a saccharide source; and
[0046] (c) a recombinant host cell optimized for ethanol production
in the selected medium and conditions, the cell comprising: [0047]
a heterologous polynucleotide sequence that codes for an enzyme
that converts sugars to ethanol, wherein said cell expresses said
heterologous polynucleotide sequence at a sufficient functional
level so as to facilitate production of ethanol as a primary
fermentation product by said host cell; and [0048] a mutation in at
least one polynucleotide sequence that codes for a protein in a
metabolic pathway in the cell that produces a product other than
ethanol from the saccharide source in the medium under the selected
conditions, wherein the mutation decreases or eliminates expression
of the protein, thereby increasing ethanol production by the host
cell, as compared to ethanol production by the cell lacking the
mutation, thereby optimizing ethanol production.
[0049] In one embodiment of the system, a fermentation reaction is
conducted in a minimal medium. In some embodiments, the minimal
medium is a urea-based medium as described above.
[0050] Any suitable recombinant host cell that is optimized for
ethanol production in the selected medium in accordance with the
invention can be used in the system.
[0051] In some embodiments of the system, the source of saccharide
comprises lignocellulose.
[0052] In one system in accordance with the invention suitable for
use in SSF using lignocellulose as a saccharide source, the
selected medium is a minimal urea-based medium as described above,
and the recombinant host cell is a strain of Klebsiella oxytoca
comprising a deletion mutation in a gene coding for a protein in
the butanediol pathway.
[0053] In yet another aspect, the invention provides a kit
comprising a recombinant host cell as described above, packaged
with instructions for using the recombinant host cell according to
the methods or systems of the invention.
[0054] Other aspects and advantages of the invention are discussed
below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0055] FIGS. 1A and 1B are diagrams showing vector constructs and
steps used in the construction of a recombinant ethanologenic
bacterium lacking budAB genes of the butanediol pathway, according
to an embodiment of the invention. FIG. 1A is a schematic
illustration showing operon and transcriptional regulation. Circled
"+" signs denote that expression of the budAB operon is increased
by low pH, and positively regulated by BudR and Fnr. FIG. 1B (upper
left) shows PCR primers used to clone budAB'. In the lower portion
of the drawing, plasmids (e.g., pLOI3301) used in the construction
of a recombinant ethanologenic microorganism optimized for ethanol
production in a minimal medium, according to an embodiment of the
invention, are shown diagrammatically and indicated by numbers.
[0056] FIGS. 2A-2D are four graphs showing fermentation by
recombinant strains of ethanologenic microorganism K. oxytoca P2 in
various minimal media and Luria broth containing 90 g L.sup.-1
glucose, according to an embodiment of the invention. FIGS. 2A and
2B show results in LB media and media containing ammonia nitrogen.
Symbols: .DELTA.-LB; .largecircle.-M9+Fe; .box-solid.-0.5% CSL+M.
FIGS. 2C and 2D show results in media with urea nitrogen. Symbols:
.largecircle.-U-M9+Fe; .box-solid.-U-0.5%; CSL+M; -OUM1. Standard
errors are included for data with n.gtoreq.3.
[0057] FIGS. 3A and 3B are two graphs showing aspects of
fermentation of glucose to ethanol by K. oxytoca P2 in a urea-based
minimal medium (OUM1) optimized for K. oxytoca P2 strains,
according to an embodiment of the invention. Data depict growth at
48 hr in flask cultures (hatched bars, OD.sub.550) and ethanol
production (solid bars) in media comprising varying concentrations
of corn steep liquor (FIG. 3A) and urea (FIG. 3B). Standard errors
are included for data with n.gtoreq.3.
[0058] FIGS. 4A and 4B are two graphs showing effects of deletion
of budAB gene products on growth and ethanol production by
ethanologenic strains of K. oxytoca (90 g L.sup.-1 glucose). FIG.
4A shows growth; FIG. 4B shows ethanol production. Symbols:
.largecircle.-strain BW21 (.DELTA.budAB); -strain P2 (parent).
Standard errors are included for data with n.gtoreq.3. Improved
ethanol production is achieved by the mutated strain lacking budAB
genes, which are involved in competing metabolic pathways that
produce products other than ethanol from sugar substrates.
[0059] FIG. 5 is a graph showing comparison of ethanol yield and
productivity by recombinant bacteria (parent strain P2, and mutant
strain BW21 with budAB deletion), according to an embodiment of the
invention. Ethanol production by the deletion strain exceeds that
of the parent strain.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0060] As used herein the terms "recombinant host cell,"
"recombinant microorganism," and the like, are intended to include
cells suitable for, or subjected to, genetic manipulation, e.g.,
which can incorporate heterologous polynucleotide sequences, e.g.,
which can be transfected, or has been so manipulated. The cell can
be a microorganism or a higher eukaryotic cell. The term is
intended to include progeny of the cell originally transfected. In
some embodiments, the cell is a bacterial cell, e.g., a
Gram-positive bacterial cell or a Gram-negative bacterial cell. The
latter term is intended to include all facultatively anaerobic
Gram-negative cells of the family Enterobacteriaceae such as
Escherichia, Shigella, Citrobacter, Salmonella, Klebsiella,
Enterobacter, Erwinia, Kluyvera, Serratia, Cedecea, Morganella,
Hafnia, Edwardsiella, Providencia, Proteus, and Yersinia. Preferred
recombinant hosts are Escherichia coli or Klebsiella oxytoca
cells.
[0061] The term "heterologous polynucleotide segment" or
"heterologous polynucleotide sequence" is intended to include a
polynucleotide segment that encodes one or more polypeptides or
portions or fragments of polypeptides. A heterologous
polynucleotide segment may be derived from any source, e.g.,
eukaryotes, prokaryotes, virii, or synthetic polynucleotide
fragments. The term "heterologous polynucleotide sequence" may also
refer to a polynucleotide sequence that is not naturally occurring
in an organism, e.g., a sequence that is introduced into the
organism. In one embodiment, the gene of a polynucleotide sequence
is involved in at least one step in the bioconversion of a
carbohydrate to ethanol. Accordingly, the term is intended to
include any gene encoding a polypeptide such as an alcohol
dehydrogenase, a pyruvate decarboxylase, a secretory protein/s, or
a polysaccharase, e.g., a glucanase, such as an endoglucanase or
exoglucanase, a cellobiohydrolase, .beta.-glucosidase,
endo-1,4-.beta.-xylanase, .beta.-xylosidase, .alpha.-glucuronidase,
.alpha.-L-arabinofuranosidase, acetylesterase, acetylxylanesterase,
.alpha.-amylase, .beta.-amylase, glucoamylase, pullulanase,
.beta.-glucanase, hemicellulase, arabinosidase, mannanase, pectin
hydrolase, or pectate lyase.
[0062] The terms "polysaccharase," "cellulase," or "glucanase" are
used interchangeably herein and are intended to include a
polypeptide capable of catalyzing the degradation or
depolymerization of any linked sugar moiety, e.g., disaccharides,
trisaccharides, oligosaccharides, including complex carbohydrates,
also referred to herein as complex sugars, e.g.,
cellooligosaccharide and lignocellulose, which comprise cellulose,
hemicellulose, and pectin. The terms are intended to include
cellulases such as glucanases, including, preferably,
endoglucanases but also including, e.g., exoglucanase,
.beta.-glucosidase, cellobiohydrolase, endo-1,4-.beta.-xylanase,
.beta.-xylosidase, .alpha.-glucuronidase,
.alpha.-L-arabinofuranosidase, acetylesterase, acetylxylanesterase,
.alpha.-amylase, .beta.-amylase, glucoamylase, pullulanase,
.beta.-glucanase, hemicellulase, arabinosidase, mannanase, pectin
hydrolase, pectate lyase, or a combination of any of these
cellulases.
[0063] The term "endoglucanase" is intended to include a cellulase
which typically hydrolyses internal .beta.1-4 glucosyl linkages in
polymeric substrates and does not preferentially hydrolyze linkages
located at the ends of the chain.
[0064] The terms "saccharide," "saccharide source,"
"oligosaccharide source," "oligosaccharide," "complex cellulose,"
"complex carbohydrate," "complex sugar," "polysaccharide," "sugar
source," "source of a fermentable sugar" and the like are intended
to include any carbohydrate source comprising more than one sugar
molecule.
[0065] The term "saccharide," as used herein, includes, e.g.,
disaccharides, trisaccharides, oligosaccharides, and
polysaccharides. These carbohydrates may be derived from any
unprocessed plant material or any processed plant material.
Examples are wood, paper, pulp, plant derived fiber, or synthetic
fiber comprising more than one linked carbohydrate moiety, i.e.,
one sugar residue.
[0066] One particular saccharide source is "lignocellulose," which
represents approximately 90% of the dry weight of most plant
material and contains carbohydrates, e.g., cellulose,
hemicellulose, pectin, and aromatic polymers, e.g., lignin.
Cellulose makes up 30%-50% of the dry weight of lignocellulose and
is a homopolymer of cellobiose (a dimer of glucose). Similarly,
hemicellulose makes up 20%-50% of the dry weight of lignocellulose
and is a complex polymer containing a mixture of pentose (xylose,
arabinose) and hexose (glucose, mannose, galactose) sugars which
contain acetyl and glucuronyl side chains. Pectin makes up 1%-20%
of the dry weight of lignocellulose and is a methylated homopolymer
of glucuronic acid.
[0067] Other saccharide sources include carboxymethyl cellulose
(CMC), amorphous cellulose (e.g., acid-swollen cellulose), and the
cellooligosaccharides cellobiose, cellotriose, cellotetraose, and
cellopentaose. Cellulose, e.g., amorphous cellulose may be derived
from a paper or pulp source (including, e.g., fluid wastes thereof)
or, e.g., agricultural byproducts such as corn stalks, soybean
solubles, or beet pulp. Any one or a combination of the above
carbohydrate polymers is a potential source of sugars for
depolymerization and subsequent bioconversion to ethanol by
fermentation according to the products and methods of the present
invention.
[0068] The term "nucleic acid" is intended to include nucleic acid
molecules, e.g., polynucleotide sequences which include an open
reading frame encoding a polypeptide, and can further include
non-coding regulatory sequences, and introns. Nucleic acid
molecules in accordance with the invention include DNA molecules
(e.g., linear, circular, cDNA or chromosomal DNA) and RNA molecules
(e.g., tRNA, rRNA, mRNA) and analogs of the DNA or RNA generated
using nucleotide analogs. The nucleic acid molecule can be
single-stranded or double-stranded, but advantageously is
double-stranded DNA.
[0069] An "isolated" nucleic acid molecule of the invention
includes a nucleic acid molecule which is free of sequences which
naturally flank the nucleic acid molecule (i.e., sequences located
at the 5' and 3' ends of the nucleic acid molecule) in the
chromosomal DNA of the organism from which the nucleic acid is
derived. In various embodiments, an isolated nucleic acid molecule
can contain less than about 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,
0.5 kb, 0.1 kb, 50 bp, 25 bp or 10 bp of nucleotide sequences which
naturally flank the nucleic acid molecule in chromosomal DNA of the
microorganism from which the nucleic acid molecule is derived.
Moreover, an isolated nucleic acid molecule, such as a cDNA
molecule, can be substantially free of other cellular materials
when produced by recombinant techniques, or substantially free of
chemical precursors or other chemicals when chemically
synthesized.
[0070] A "gene," as used herein, is a nucleic acid that can direct
synthesis of an enzyme or other polypeptide molecule, e.g., can
comprise coding sequences, for example, a contiguous open reading
frame (ORF) which encodes a polypeptide, or can itself be
functional in the organism. A gene in an organism can be clustered
in an operon, as defined herein, wherein the operon is separated
from other genes and/or operons by intergenic DNA. Individual genes
contained within an operon can overlap without intergenic DNA
between the individual genes. One embodiment of a gene is one or
more genes that map to a functional locus or operon such as the
budA and budB genes of Klebsiella, that encode the proteins
.alpha.-acetolactate decarboxylase and .alpha.-acetolactate
synthase, respectively, which are involved in the butanediol
metabolic pathway. In addition, the term "gene" is intended to
include a specific gene for a selected purpose. A gene can be
endogenous to the host cell or can be recombinantly introduced into
the host cell, e.g., as a plasmid maintained episomally or a
plasmid (or fragment thereof) that is stably integrated into the
genome.
[0071] An "isolated gene," as described herein, includes a gene
which is essentially free of sequences which naturally flank the
gene in the chromosomal DNA of the organism from which the gene is
derived (i.e., is free of adjacent coding sequences which encode a
second or distinct polypeptide or RNA molecule, adjacent structural
sequences or the like), and optionally includes 5' and 3'
regulatory sequences, for example promoter sequences and/or
terminator sequences. In one embodiment, an isolated gene includes
predominantly coding sequences for a polypeptide (e.g., sequences
which encode polypeptides).
[0072] The term "homolog," as used herein, includes a polypeptide
or polypeptide sharing at least about 30-35%, advantageously at
least about 35-40%, more advantageously at least about 40-50%, and
even more advantageously at least about 60%, 70%, 80%, 90% or more
identity with the amino acid sequence of a wild-type polypeptide or
polypeptide described herein and having a substantially equivalent
functional or biological activity as the wild-type polypeptide or
polypeptide. Thus, the term "homolog" in intended to encompass
"functional variants" as well as "orthologs" (equivalent genes from
different species).
[0073] For example, a budA or budB homolog shares at least about
30-35%, advantageously at least about 35-40%, more advantageously
at least about 40-50%, and even more advantageously at least about
60%, 70%, 80%, 90% or more identity with the polypeptide having the
amino acid sequences set forth respectively as SEQ ID NO:6 and SEQ
ID NO:7, and has a substantially equivalent functional or
biological activity (i.e., is a functional equivalent) of the
polypeptide having the amino acid sequence set forth as SEQ ID NO:6
or SEQ ID NO:7 (e.g., has a substantially equivalent
.alpha.-acetolactate decarboxylase or .alpha.-acetolactate synthase
activity). Methods for measuring functional activity of the gene
product of a nucleic acid, or a homolog thereof in accordance with
the invention are known, and are described in Examples below.
[0074] In one embodiment, the gene is involved in at least one step
in the bioconversion of a carbohydrate to a product other than
ethanol. Such products are also referred to herein as "co-products"
or "co-products of fermentation." Co-products are generally
undesirable in ethanol fermentation reactions, reducing yields as a
result of diversion of the carbohydrate (saccharide) substrates
into competing metabolic pathways other than those used for ethanol
production. Accordingly, in one aspect, genes encoding proteins
such as enzymes involved in the latter pathways (for instance, the
butanediol pathway), are of interest in accordance with the
invention as desired targets for elimination from the cells.
Examples of genes involved in the bioconversion of a carbohydrate
to a product other than ethanol in a microorganism are the budA and
budB genes of bacteria, which respectively encode the enzymes
.alpha.-acetolactate decarboxylase and .alpha.-acetolactate
synthase involved in the synthesis of butanediol and acetoin from
carbohydrate sources. Other genes of interest are those involved in
metabolic pathways that produce other undesired co-products of
sugar fermentation including but not limited to formate, lactate,
succinate, acetate, acetoin, xylitol, butyrate, pyruvate,
proprionate, isopropyl alcohol, 1-propanol, 2-propanol,
propanediol, citrate, glutamate, and acetone.
[0075] "Allelic variant(s)," as used herein include both functional
and non-functional proteins. Functional allelic variants will
typically contain only conservative substitution of one or more
amino acids of SEQ ID NO:6 or 7, or substitution, deletion or
insertion of non-critical residues in non-critical regions of the
protein. Non-functional allelic variants will typically contain a
non-conservative substitution, a deletion, or insertion or
premature truncation of the amino acid sequence of SEQ ID NO:6 or
7, or a substitution, insertion or deletion in critical residues or
critical regions.
[0076] A polynucleotide or amino acid sequence of the present
invention can further be used as a "query sequence" to perform a
search against public databases, for example, to identify other
family members or related sequences, e.g., genes related to budA
and budB in organisms in which these genes have not been cloned.
Such searches can be performed using the NBLAST and XBLAST programs
(version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
BLAST nucleotide searches can be performed with the NBLAST program,
score=100, wordlength=12 to obtain nucleotide sequences homologous
to polynucleotide molecules of the invention. BLAST protein
searches can be performed with the XBLAST program, score=50,
wordlength=3 to obtain amino acid sequences homologous to
polypeptide molecules of the invention. To obtain gapped alignments
for comparison purposes, Gapped BLAST can be utilized as described
in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402.
When utilizing BLAST and Gapped BLAST programs, the default
parameters of the respective programs (e.g., XBLAST and NBLAST) can
be used. See http://www.ncbi.nlm.nih.gov.
[0077] The term "mutation," as used herein, is intended to refer to
a relatively permanent change in the hereditary material of an
organism involving either an aberration in one or more chromosomes,
or a change in the DNA sequence that makes up genes. A mutation, as
used herein, includes a change in a DNA sequence created either by
deletion or insertion of a DNA sequence, by a change in one or more
bases (e.g., a point mutation), by duplication, by missense, by
frameshift, by repeat or by nonsense mutation. Methods of creating
insertion, deletion, and base change mutations are known in the art
and are described, for example, in treatises such as Sambrook et
al. [26].
[0078] The terms "fermentation" and "fermenting" are intended to
include the degradation or depolymerization of a complex sugar and
bioconversion of that sugar residue into ethanol, acetate and
succinate. The terms are intended to include the enzymatic process
(e.g. cellular or acellular, e.g. a lysate or purified polypeptide
mixture) by which ethanol is produced from a carbohydrate, in
particular, as a primary product of fermentation.
[0079] The term "simultaneous saccharification and fermentation" or
"SSF" is intended to include the use of one or more recombinant
hosts (or extracts thereof, including purified or unpurified
extracts) for the contemporaneous degradation or depolymerization
of a complex sugar and bioconversion of that sugar residue into
ethanol by fermentation. SSF is a well-known process that can be
used for breakdown of biomass to polysaccharides that are
ultimately convertible to ethanol by bacteria. Reflecting the
breakdown of biomass as it occurs in nature, SFF combines the
activities of fungi (or enzymes such as cellulases extracted from
fungi) with the activities of ethanologenic bacteria (or enzymes
derived therefrom) to break down sugar sources such as
lignocellulose to simple sugars capable of ultimate conversion to
ethanol. SSF reactions are typically carried out at acid pH to
optimize the use of the expensive fungal enzymes.
[0080] The term "transcriptional control" is intended to include
the ability to modulate gene expression at the level of
transcription. In a preferred embodiment, transcription, and thus
gene expression, is modulated by replacing or adding a surrogate
promoter near the 5' end of the coding region of a gene of interest
thereby resulting in altered gene expression. In a preferred
embodiment, the transcriptional control of one or more genes is
engineered to result in the optimal expression of such genes, e.g.,
in a desired ratio. The term also includes inducible
transcriptional control as recognized in the art.
[0081] The term "expression" is intended to include the expression
of a gene at least at the level of mRNA production, and optionally
at the polypeptide level.
[0082] The term "expression product" is intended to include the
resultant product of an expressed gene, e.g., a polypeptide or
protein.
[0083] The terms "increased expression" and "decreased expression"
are intended to include an alteration in gene expression
(up-regulation, and down-regulation, respectively) at least at the
level of mRNA production, and preferably, at the level of
polypeptide or protein expression.
[0084] The terms "increased production" and "decreased or
eliminated production" in reference to a polypeptide are intended
to include an increase or decrease in the amount of a polypeptide
expressed, in the level of the enzymatic activity of the
polypeptide, or a combination thereof.
[0085] The terms "activity" and "enzymatic activity" are used
interchangeably and are intended to include any functional activity
normally attributed to a selected polypeptide when produced under
favorable conditions. The activity of an .alpha.-acetolactate
decarboxylase enzyme (encoded by BudA) is, for example, to produce
an acetoin product from a carbohydrate source. Techniques for
determining activity such as that of .alpha.-acetolactate
decarboxylase are known in the art (see for example Blomqvist et
al. [35]), and are described in Examples herein.
[0086] The term "derived from" is intended to include the isolation
(in whole or in part) of a polynucleotide segment from an indicated
source, or the purification of a polypeptide from an indicated
source. The term is intended to include, for example, direct
cloning, PCR amplification, or artificial synthesis from, or based
on, a sequence associated with the indicated polynucleotide source.
In studies described herein, for example, nucleotide sequences
encoding gene products involved in the butanediol pathway are
derived from budA and budB genes amplified from the genomic DNA of
the bacterium Klebsiella oxytoca.
[0087] The term "ethanologenic" is intended to include the ability
of a microorganism to produce ethanol from a carbohydrate as a
primary fermentation product. The term includes but is not limited
to naturally occurring ethanologenic organisms, organisms with
naturally occurring or induced mutations, and organisms that have
been genetically modified.
[0088] The term "non-ethanologenic" is intended to include cells
that are unable to produce ethanol from a carbohydrate as a primary
non-gaseous fermentation product. The term is intended to include
microorganisms that produce ethanol as the minor fermentation
product comprising less than 40%, for example 20%, 30%, 40%, of
total non-gaseous fermentation products.
[0089] The term "primary fermentation product" is intended to
include non-gaseous products of fermentation that comprise greater
than about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95% of total non-gaseous product. The primary fermentation product
is the most abundant non-gaseous product. In certain embodiments of
the invention, the primary fermentation product is ethanol. In
further embodiments, the primary fermentation products are produced
by the host grown in minimal salts medium.
[0090] The term "minor fermentation product" as used herein is
intended to include non-gaseous products of fermentation that
comprise less than 40%, for example 20%, 30%, 40%, of total
non-gaseous product.
[0091] The terms "Gram-negative bacteria" and "Gram-positive
bacteria" are intended to include the art-recognized definitions of
these terms. Typically, Gram-negative bacteria include, for
example, the family Enterobacteriaceae which comprises Escherichia,
Shigella, Citrobacter, Salmonella, Klebsiella, Enterobacter,
Erwinia, Kluyvera, Serratia, Cedecea, Morganella, Hafnia,
Edwardsiella, Providencia, Proteus, and Yersinia. Other
Gram-negative bacteria include, but are not limited to,
Acinetobacter, Gluconobacter, Geobacter and Shewanella.
Gram-positive bacteria include, but are not limited to, Bacillus,
Geobacillus, Clostridium, Streptococcus, Cellulomonas,
Corynebacterium, Lactobacillis, Lactococcus, Oenococcus and
Eubacterium.
[0092] The term "defined nitrogen source" is intended to mean a
discrete nitrogen source, i.e., a single chemical entity that is
capable providing a source of nitrogen that is suitable for use in
accordance with the invention. Exemplary defined nitrogen sources
include, for example, urea and ammonia. In certain embodiments, two
or more defined nitrogen sources may be used.
[0093] The term "complex nitrogen source", as distinguished from
"defined nitrogen source" is intended to include a mixture of
chemical entities that collectively provide sources of nitrogen
that is suitable for use in accordance with the invention.
[0094] The term "urea" refers to an organic chemical compound
having the formula (NH.sub.2).sub.2 CO. The term "urea-like
compound," as used herein, includes various analogs/derivatives of
urea having the general formula R.sub.1N--(C.dbd.O)--.sub.NR.sub.2.
Urea-like compounds, and methods of making these compounds, are
described, for example, in U.S. Pat. No. 6,875,764 to Muzi et al.,
(2005), the disclosure of which is hereby incorporated by reference
in its entirety.
II. Recombinant Host Cells Comprising Mutations in Genes Encoding
Proteins in Metabolic Pathways Leading to Byproducts of
Fermentation
[0095] As discussed, the invention relates to new and improved
recombinant host cells suitable for degrading saccharides. The
cells comprise mutations in one or more genes associated with a
metabolic pathway for production of unwanted co-products of
fermentation. Perturbation of these pathways results in a greater
percentage of the saccharide starting materials being converted
into ethanol, rather than other, undesired products of
fermentation.
[0096] Accordingly and in one aspect, the invention provides a
recombinant host cell suitable for degrading a saccharide. The cell
comprises a heterologous polynucleotide sequence that codes for an
enzyme that converts sugars to ethanol. The host cell expresses the
heterologous polynucleotide sequence at a sufficient functional
level so as to facilitate production of ethanol as a primary
fermentation product. The recombinant host cell further comprises a
mutation in at least one polynucleotide sequence that codes for a
protein in a metabolic pathway in the cell that produces a product
other than ethanol from the sugar source. The presence of the
mutation decreases or eliminates expression of at least one protein
in the metabolic pathway, thereby increasing ethanol production by
the cell, as compared to ethanol production by the recombinant cell
in absence of the mutation.
[0097] The recombinant host cell suitable for degrading saccharides
can be a cell of a higher eukaryotic organism such as a nematode,
an insect, a reptile, a bird, an amphibian, or a mammal. The cell
can also be a cell of a single-celled or multi-cellular
microorganism, such as a bacterium, yeast or fungus. Recombinant
yeast cells in accordance with the invention are derived, e.g.,
from Saccharomyces, Schizosacharomyces, Hansenula, Pachyosolen,
Kluyveromyces, Debaryomyces, Yarrowia, and Pichia.
[0098] Some bacterial host cells in accordance with the invention
are derived from Gram-positive bacteria. Certain embodiments of the
cells are derived, e.g., from Bacillus, Geobacillus, Clostridium,
Streptococcus, and Cellulomonas.
[0099] Other bacterial host cells are derived from Gram-negative
bacteria. In some embodiments, the bacteria are facultatively
anaerobic. Preferred facultative anaerobes are selected from the
family Enterobacteriaceae, and include but are not limited to
Escherichia, Shigella, Citrobacter, Salmonella, Klebsiella,
Enterobacter, Erwinia, Kluyvera, Serratia, Cedecea, Morganella,
Hafnia, Edwardsiella, Providencia, Proteus, and Yersinia.
[0100] As discussed, recombinant host cells in accordance with the
invention comprise one or more heterologous polynucleotide
sequences that code for an enzyme that converts sugars to ethanol.
Accordingly, these cells are ethanologenic. Suitable polynucleotide
sequences for use in constructing recombinant ethanologenic host
cells may encode, e.g., genes from naturally occurring
ethanologenic strains of bacteria, such as Zymomonas mobilis. Two
preferred heterologous genes that convert sugars to ethanol include
alcohol dehydrogenase (adh) and pyruvate decarboxylase (pdc). The
recombinant cells of the invention include one or both of these
genes and may further include other heterologous nucleotide
sequences that code for enzymes such as polysaccharases that assist
in converting sugar to ethanol. Preferably the heterologous genes
are integrated into the bacterial chromosome.
[0101] Methods of making recombinant ethanologenic microorganisms
are known in the art of molecular biology. Suitable materials and
methods and recombinant host organisms are described, for example,
in U.S. Pat. Nos. 6,849,434, 6,333,181, 5,821,093; 5,482,846;
5,424,202; 5,028,539; 5,000,000; 5,487,989, 5,554,520, and
5,162,516, hereby incorporated by reference, and may be employed in
carrying out the present invention.
[0102] In some embodiments, the recombinant host cell is an
ethanologenic Gram-negative bacterium from the family
Enterobacteriaceae. The ethanologenic hosts of U.S. Pat. No.
5,821,093, hereby incorporated by reference, for example, are
suitable hosts and include, in particular, E. coli strains KO4
(ATCC 55123), KO11 (ATCC 55124), and KO12 (ATCC 55125), and
Klebsiella oxytoca strain P2 (ATCC 55307), discussed infra.
Alternatively, a non-ethanologenic host of the present invention
may be converted to an ethanologenic host by addition of
heterologous polynucleotide sequences that code for one or more
suitable enzymes that convert sugars to ethanol.
[0103] In some embodiments of the invention, a recombinant
ethanologenic bacterial host cell is derived from Erwinia or
Klebsiella. Recombinant hosts derived from Klebsiella oxytoca are
particularly suitable for SSF of lignocellulose, having several
advantages including efficiency of pentose and hexose
co-fermentation, resistance to toxins, production of enzymes for
complex saccharide depolymerization (avoiding or reducing the need
for depolymerization by added fungal cellulases) and environmental
hardiness.
[0104] One suitable ethanologenic Klebsiella host cell is K.
oxytoca P2, a derivative of K. oxytoca M5A1 (See Wood, et al.
(1992) Appl. Environ. Microbiol. 58:2103-2110, and U.S. Pat. No.
5,821,093). Advantageously, K. oxytoca strains possess the native
ability to use urea as a nitrogen source. In one embodiment, the
recombinant ethanologenic bacterium contains at least one
heterologous polynucleotide segment (e.g., celY or celZ derived
from Erwinia) encoding at least one endoglucanase (e.g., EGY or
EGZ). More preferably, the recombinant ethanologenic host cell
contains more than one heterologous polynucleotide segment which
encodes endoglucanases. For example, as described in published U.S.
Patent Application No. 2004/015990, celY and celZ can be
functionally integrated, expressed, and secreted from the
ethanologenic strain K. oxytoca P2 concurrently to produce ethanol
from a saccharide substrate (e.g., crystalline cellulose).
[0105] As discussed above, it is known that the process of ethanol
production by SSF is accompanied by the production of unwanted
co-products of fermentation other than ethanol by recombinant host
cells. Diversion of the substrate sugars into alternative metabolic
pathways that produce products other than ethanol results in lower
productivity and yield of ethanol than theoretically possible in
the absence of the alternative pathways. Recombinant host cells in
accordance with the invention are engineered to reduce this problem
by virtue of their reduced capacity or inability to produce the
unwanted co-products. Various embodiments of the cells are
engineered such that the cells have reduced or absent capacity to
produce selected co-products of fermentation. Some embodiments of
the recombinant host cells are unable to produce co-products
including but not limited to formate, lactate, succinate, acetate,
acetoin, butanediol, 2,3-butanediol, xylitol, butyrate, pyruvate,
proprionate, isopropyl alcohol, 1-propanol, 2-propanol, and
acetone.
[0106] To effect the above-described inability to produce unwanted
co-products, some embodiments of recombinant host cells in
accordance with the invention comprise a deletion mutation in at
least one polynucleotide sequence that codes for a protein in a
metabolic pathway in the cell that produces a product other than
ethanol from a sugar source. Some embodiments of the cells have
deletion mutations in polynucleotide sequences that code for a
protein involved at least one metabolic pathway that produces
formate, lactate, succinate, acetate, acetoin, butanediol,
2,3-butanediol, xylitol, butyrate, pyruvate, proprionate, isopropyl
alcohol, 1-propanol, 2-propanol, or acetone from sugar in the
cell.
[0107] One aspect of the invention involving deletion mutations
relates to "knocking out" genes known to be associated with
selected metabolic pathways in cells. Understanding is well
advanced of the biochemical pathways that exist in cells, such as
bacterial cells, to produce selected products of metabolism.
Metabolic pathways that exist in cells such as bacteria for the
production of products other than ethanol from sugars (e.g.,
products such as formate, lactate, succinate, acetate, acetoin, and
2,3-butanediol) have been described and can be readily ascertained,
for example, by a search of the scientific literature.
[0108] Knockout of genes encoding proteins or functional fragments
thereof involved in the metabolic pathways for production of
formate, lactate, succinate, acetate, acetoin, butanediol,
2,3-butanediol, xylitol, butyrate, pyruvate, proprionate, isopropyl
alcohol, 1-propanol, 2-propanol, or acetone results in recombinant
hosts with inability to produce such unwanted co-products of sugar
fermentation.
[0109] In some embodiments of the recombinant host cells, a
deletion mutation is in at least one polynucleotide sequence that
codes for an enzyme involved in the butanediol metabolic pathway.
This pathway results in the production of acetoin and
2,3-butanediol from sugar sources by bacterial cells. In some
embodiments, the deletion mutation is in one or both of the budA
and budB genes, which respectively encode the enzymes
.alpha.-acetolactate decarboxylase and .alpha.-acetolactate
synthase. Production of these co-products by the butanediol pathway
is brought about by the actions of these two enzymes. The deletion
mutation decreases or eliminates expression of these enzymes,
resulting in a recombinant ethanologenic host cell that is unable
to produce acetoin and 2,3-butanediol from a sugar source, and
which thereby exhibits increased ethanol production from the sugar
source, relative to the recombinant cell in absence of the
mutation.
[0110] One embodiment of a host cell provided by the invention is a
recombinant ethanologenic bacterium comprising a deletion mutation
in both the budA and budB genes. One preferred host cell carrying
deletions in these genes is a variant of K. oxytoca P2 designated
K. oxytoca BW21. As shown below, this strain can be advantageously
used for ethanol production in minimal media containing urea-like
compounds as a nitrogen source, and is particularly suitable for
use in fermentation reactions conducted in media in the acidic pH
range.
III. Isolated Nucleic Acid Molecules and Polypeptides
[0111] In another aspect, the present invention features isolated
nucleic acid molecules comprising budA and budB gene sequences,
which respectively encode the enzymes .alpha.-acetolactate
decarboxylase and .alpha.-acetolactate synthase. The nucleic acids
are derived from Gram-negative and Gram-positive bacteria, for
example, the Gram-negative bacterium Klebsiella oxytoca.
[0112] Also featured are isolated genomic nucleic acids comprising
the above-mentioned genes of the butanediol pathway (i.e., budA,
budB) but also other flanking regions which comprise regulatory
regions (e.g., promoter(s) and ribosome binding sites(s)) as well
as other associated genes involved in ethanologenesis, e.g.,
alcohol dehydrogenase (adh) and pyruvate decarboxylase (pdc).
[0113] As discussed, the invention provides novel nucleic acids
encoding, inter alia, full-length or partial coding sequences,
respectively, of budA and budB genes from Klebsiella strains. These
genes were isolated in response to the discovery of increased
production of unwanted co-products of fermentation that result from
the activity of enzymes encoded by these genes. More specifically,
it was demonstrated that production of these co-products was
increased in ethanologenic bacteria during fermentation reactions
carried out in desirable, inexpensive growth media such as OUM
described above.
[0114] To eliminate production of these products, a genetic
strategy was devised to eliminate the ability of an ethanologenic
producer cell to make the co-products. In one example, the genes
encoding budA and a portion of budB were first cloned from
Klebsiella oxytoca. The isolated sequences were subsequently
subjected to genetic manipulation to delete a large fragment of the
budAB gene, rendering it inoperative. The budA and budB genes had
not been previously isolated from Klebsiella oxytoca. Accordingly,
the invention provides in one aspect novel DNA sequences, and
predicted amino acid sequences based on these DNA sequences.
[0115] One embodiment of a novel nucleic acid in accordance with
the invention is an isolated nucleic acid molecule comprising the
nucleotide sequence set forth in SEQ ID NO:3 or 4. The nucleic acid
designated herein as SEQ ID NO:3 corresponds to a putative
full-length coding sequence from the budA gene of Klebsiella
oxytoca, which encodes the protein product .alpha.-acetolactate
decarboxylase. The nucleic acid designated herein as SEQ ID NO:4
corresponds to a partial coding sequence from the budB gene of the
same species, which encodes the protein .alpha.-acetolactate
synthase.
[0116] Other novel sequences of the invention are predicted amino
acid sequences corresponding to SEQ ID NOS.:3 and 4, (described
infra), which are designated herein as SEQ ID NOS:6 and 7,
respectively.
[0117] One nucleic acid embodiment in accordance with the invention
is an isolated nucleic acid molecule which encodes a polypeptide
comprising the amino acid sequence set forth in SEQ ID NO:6 or
7.
[0118] The invention further provides an isolated nucleic acid
molecule which encodes a naturally occurring allelic variant of a
polypeptide comprising the amino acid sequence set forth in SEQ ID
NO: 6 or 7.
[0119] In another embodiment, an isolated nucleic acid molecule
encodes a homolog of a polypeptide comprising the amino acid
sequence set forth in SEQ ID NO: 6 or 7. Additional budA and budB
nucleic acid sequences are those that encode a homolog of the
polypeptide having the amino acid sequence set forth in SEQ ID NO:6
or SEQ ID NO:7 (e.g., encoding a polypeptide having at least 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95% or more identity to the
polypeptide having the amino acid sequence as set forth in SEQ ID
NO:6 or SEQ ID NO:7, and having a substantially identical activity
as the polypeptide).
[0120] Yet other embodiments include the following isolated nucleic
acid molecules: One embodiment is a nucleic acid molecule
comprising a nucleotide sequence which is at least 60% identical to
the nucleotide sequence of SEQ ID NO:3 or 4, or a complement
thereof. In various embodiments, an isolated nucleic acid molecule
of the present invention comprises a nucleotide sequence which is
at least about 60-65%, advantageously at least about 70-75%, more
preferably at least about 80-85%, and even more advantageously at
least about 90-95% or more identical to a nucleotide sequence set
forth herein as SEQ ID NO:3 or SEQ ID NO:4.
[0121] Yet another embodiment is a nucleic acid molecule comprising
a fragment of at least 100 nucleotides of a nucleic acid comprising
the nucleotide sequence of SEQ ID NO:3 or 4, or a complement
thereof.
[0122] A nucleic acid molecule in accordance with the invention can
also be one that encodes a polypeptide comprising an amino acid
sequence at least about 50% identical to the amino acid sequence of
SEQ ID NO:6 or 7.
[0123] Another embodiment is a nucleic acid molecule which encodes
a fragment of a polypeptide comprising the amino acid sequence of
SEQ ID NO: 6 or 7, wherein the fragment comprises at least 15
contiguous amino acid residues of the amino acid sequence of SEQ ID
NO: 6 or 7.
[0124] In another embodiment, an isolated nucleic acid molecule
hybridizes under stringent conditions to a nucleic acid molecule
having a nucleotide sequence as set forth as SEQ ID NO:3 or SEQ ID
NO:4. In another embodiment, an isolated budA or budB nucleic acid
molecule hybridizes to all or a portion of a nucleic acid molecule
having a nucleotide sequence that encodes a polypeptide having the
amino acid sequence of SEQ ID NO:6, or SEQ ID NO:7.
[0125] Suitable hybridization conditions are known to those skilled
in the art and can be found, e.g., in Current Protocols in
Molecular Biology, Ausubel et al., eds., John Wiley & Sons,
Inc. (1995), sections 2, 4 and 6. Additional stringent conditions
can be found in Molecular Cloning: A Laboratory Manual, Sambrook et
al., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989),
chapters 7, 9 and 11.
[0126] A particular, non-limiting example of stringent
hybridization conditions includes hybridization in 4.times. sodium
chloride/sodium citrate (SSC), at about 65-70.degree. C. (or
hybridization in 4.times.SSC plus 50% formamide at about
42-50.degree. C.) followed by one or more washes in 1.times.SSC, at
about 65-70.degree. C. A particular, non-limiting example of highly
stringent hybridization conditions includes hybridization in
1.times.SSC, at about 65-70.degree. C. (or hybridization in
1.times.SSC plus 50% formamide at about 42-50.degree. C.) followed
by one or more washes in 0.3.times.SSC, at about 65-70.degree. C. A
particular, non-limiting example of reduced stringency
hybridization conditions includes hybridization in 4.times.SSC, at
about 50-60.degree. C. (or alternatively hybridization in
6.times.SSC plus 50% formamide at about 40-45.degree. C.) followed
by one or more washes in 2.times.SSC, at about 50-60.degree. C.
Ranges intermediate to the above-recited values, e.g., at
65-70.degree. C. or at 42-50.degree. C. are also intended to be
encompassed by the present invention. SSPE (1.times.SSPE is 0.15 M
NaCl, 10 mM NaH.sub.2PO.sub.4, and 1.25 mM EDTA, pH 7.4) can be
substituted for SSC (1.times.SSC is 0.15 M NaCl and 15 mM sodium
citrate) in the hybridization and wash buffers; washes are
performed for 15 minutes each after hybridization is complete.
[0127] The hybridization temperature for hybrids anticipated to be
less than 50 base pairs in length should be 5-10.degree. C. less
than the melting temperature (T.sub.m) of the hybrid, where T.sub.m
is determined according to the following equations. For hybrids
less than 18 base pairs in length, T.sub.m(.degree. C.)=2(# of A+T
bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs
in length, T.sub.m(.degree.
C.)=81.5+16.6(log.sub.10[Na.sup.+])+0.41(% G+C)-(600/N), where N is
the number of bases in the hybrid, and [Na.sup.+] is the
concentration of sodium ions in the hybridization buffer
([Na.sup.+] for 1.times.SSC=0.165 M).
[0128] It will also be recognized by the skilled practitioner that
additional reagents can be added to hybridization and/or wash
buffers to decrease non-specific hybridization of nucleic acid
molecules to membranes, for example, nitrocellulose or nylon
membranes, including but not limited to blocking agents (e.g., BSA
or salmon or herring sperm carrier DNA), detergents (e.g., SDS),
chelating agents (e.g., EDTA), Ficoll, PVP and the like. When using
nylon membranes, in particular, an additional, non-limiting example
of stringent hybridization conditions is hybridization in 0.25-0.5M
NaH.sub.2PO.sub.4, 7% SDS at about 65.degree. C., followed by one
or more washes at 0.02M NaH.sub.2PO.sub.4, 1% SDS at 65.degree. C.,
see e.g., Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA
81:1991-1995, (or, alternatively, 0.2.times.SSC, 1% SDS).
[0129] In another embodiment, an isolated nucleic acid molecule
comprises a nucleotide sequence that is complementary to a budA or
budB nucleotide sequence as set forth herein (e.g., is the
complement of the nucleotide sequence set forth as SEQ ID NO:3 or
SEQ ID NO:4).
[0130] Yet another embodiment of the present invention features
mutant budA or budB nucleic acid molecules or genes. Typically, a
mutant nucleic acid molecule or mutant gene, as described herein,
includes a nucleic acid molecule or gene having a nucleotide
sequence which includes at least one alteration (e.g., base change,
insertion, deletion) such that the polypeptide or peptide that can
be encoded by said mutant exhibits an activity that differs from
the polypeptide or peptide encoded by the wild-type nucleic acid
molecule or gene.
[0131] One embodiment of an isolated mutant nucleic acid molecule
in accordance with the invention comprises the mutated budAB
nucleotide sequence from Klebsiella oxytoca set forth in SEQ ID
NO:5. In another version of a nucleic acid comprising a mutated
Klebsiella budAB nucleotide sequence, mutated (truncated) budA and
budB coding sequences are separated by a tetracycline gene flanked
by FRT sites, inserted between the mutated budA and budB genes. A
DNA construct comprising the latter configuration is designated
herein as SEQ ID NO:8. See also Examples 4 and 5, infra.
[0132] A nucleic acid molecule of the present invention (e.g., a
nucleic acid molecule having the nucleotide sequence of SEQ ID NO:3
or SEQ ID NO:4) can be isolated using standard molecular biology
techniques and the sequence information provided herein. For
example, nucleic acid molecules can be isolated using standard
hybridization and cloning techniques (e.g., as described in
Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) or
can be isolated by the polymerase chain reaction using synthetic
oligonucleotide primers designed based upon the sequence of SEQ ID
NO:3 or SEQ ID NO:4. Primers suitable for the amplification of a
DNA fragment from Klebsiella oxytoca and related strains comprising
the putative full-length coding sequence of budA and a partial
coding sequence of budB can have the nucleotide sequences set forth
herein as SEQ ID NOS:1 and 2. A nucleic acid of the invention can
be amplified using cDNA, mRNA or alternatively, genomic DNA, as a
template and appropriate oligonucleotide primers according to
standard PCR amplification techniques. Suitable PCR primers can be
designed, for example, having the sequences of SEQ ID NOS: 1 and
2.
[0133] A mutant nucleic acid molecule or mutant gene can encode a
polypeptide having improved .alpha.-acetolactate decarboxylase or
.alpha.-acetolactate synthase activity, e.g., substrate affinity;
thermostability; activity at a different pH; or codon usage (e.g.,
for improved expression in the recipient host cell).
[0134] Alternatively, a mutant nucleic acid molecule or mutant gene
in accordance with the invention can encode a polypeptide having
reduced or absent .alpha.-acetolactate decarboxylase or
.alpha.-acetolactate synthase activity. A mutant nucleic acid
encoding a mutated budAB polypeptide derived from Klebsiella
oxytoca that lacks expression or activity of .alpha.-acetolactate
decarboxylase and .alpha.-acetolactate synthase can have the
sequence of SEQ ID NO:5 or 8. Methods for detecting reduced or
absent .alpha.-acetolactate decarboxylase or .alpha.-acetolactate
synthase activity are known, and described, for instance, in
Examples, infra.
[0135] The invention further includes an isolated nucleic acid
molecule comprising any of the above-described nucleic acid
molecules and a nucleotide sequence encoding a heterologous
polypeptide. In some embodiments, heterologous polynucleotide
sequences of the present invention feature nucleic acids comprising
isolated pyruvate decarboxylase (pdc) nucleic acid sequences or
genes, and/or isolated alcohol dehydrogenase (adh) nucleic acid
sequences or genes, derived from a Gram-positive or Gram-negative
bacterium. Advantageously, the pdc nucleic acid or gene is derived
from a Gram-negative microorganism selected from the group
consisting of Gluconobacter, Rhizobium, Bradyrhizobium,
Alcaligenes, Rhodobacter, Rhodococcus. Azospirillum,
Rhodospirillum, Sphingomonas, Burkholderia, Desulfomonas,
Geospirillum, Succinomonas, Aeromonas, Shewanella, Halochromatium,
Citrobacter, Escherichia, Klebsiella, Zymomonas (e.g., Zymomonas
mobilis), Zymobacter (e.g., Zymobacter palmae), and Acetobacter
(e.g., Acetobacter pasteurianus).
[0136] In another embodiment, the pdc nucleic acid or gene is
derived from a Gram-positive microorganism selected from the group
consisting of Fibrobacter, Acidobacter, Bacteroides,
Sphingobacterium, Actinomyces, Corynebacterium, Nocardia,
Rhodococcus, Propionibacterium, Bifidobacterium, Bacillus,
Geobacillus, Paenibacillus, Sulfobacillus, Clostridium,
Anaerobacter, Eubacterium, Streptococcus, Lactobacillus,
Leuconostoc, Enterococcus, Lactococcus, Thermobifida, Cellulomonas,
and Sarcina (e.g., Sarcina ventriculi).
[0137] As discussed above, another aspect of the present invention
features novel isolated polypeptides (e.g., isolated enzymes active
in the butanediol metabolic pathway, for example,
.alpha.-acetolactate decarboxylase or .alpha.-acetolactate synthase
derived from Klebsiella oxytoca. In one embodiment, polypeptides
are produced by recombinant DNA techniques and can be isolated from
microorganisms of the present invention by an appropriate
purification scheme using standard polypeptide purification
techniques. In another embodiment, polypeptides are synthesized
chemically using standard peptide synthesis techniques.
[0138] An isolated or purified polypeptide (e.g., an isolated or
purified .alpha.-acetolactate decarboxylase or .alpha.-acetolactate
synthase) is substantially free of cellular material or other
contaminating polypeptides from the microorganism from which the
polypeptide is derived, or substantially free from chemical
precursors or other chemicals when chemically synthesized. In one
embodiment, an isolated or purified polypeptide has less than about
30% (by dry weight) of contaminating polypeptide or chemicals, more
advantageously less than about 20% of contaminating polypeptide or
chemicals, still more advantageously less than about 10% of
contaminating polypeptide or chemicals, and most advantageously
less than about 5% contaminating polypeptide or chemicals.
[0139] In one embodiment, an isolated polypeptide of the present
invention (e.g., an isolated .alpha.-acetolactate decarboxylase or
an isolated .alpha.-acetolactate synthase enzyme), comprises an
amino acid sequence as shown in SEQ ID NO:6 or SEQ ID NO:7,
respectively.
[0140] In other embodiments, an isolated polypeptide of the present
invention is a homolog of at least one of the polypeptides set
forth as SEQ ID NO:6 or SEQ ID NO:7 (e.g., comprises an amino acid
sequence at least about 30-40% identical, advantageously about
40-50% identical, more advantageously about 50-60% identical, and
even more advantageously about 60-70%, 70-80%, 80-90%, 90-95% or
more identical to the amino acid sequence of SEQ ID NO:6 or SEQ ID
NO:7, and has an activity that is substantially similar to that of
the polypeptide encoded by the amino acid sequence of SEQ ID NO:6
or SEQ ID NO:7, respectively.
[0141] To determine the percent identity of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in the
sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a second amino or nucleic acid sequence). When a
position in the first sequence is occupied by the same amino acid
residue or nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position. The
percent identity between the two sequences is a function of the
number of identical positions shared by the sequences (i.e., %
identity=# of identical positions/total # of positions.times.100),
advantageously taking into account the number of gaps and size of
said gaps necessary to produce an optimal alignment.
[0142] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. A particular, non-limiting example of a
mathematical algorithm utilized for the comparison of sequences is
the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci.
USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc.
Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated
into the NBLAST and XBLAST programs (version 2.0) of Altschul et
al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can
be performed with the NBLAST program, score=100, wordlength=12 to
obtain nucleotide sequences homologous to nucleic acid molecules of
the invention. BLAST polypeptide searches can be performed with the
XBLAST program, score=50, wordlength=3 to obtain amino acid
sequences homologous to polypeptide molecules of the invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al. (1997) Nucleic Acids
Research 25(17):3389-3402. When utilizing BLAST and Gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
Another particular, non-limiting example of a mathematical
algorithm utilized for the comparison of sequences is the algorithm
of Myers and Miller (1988) Comput Appl Biosci. 4:11-17. Such an
algorithm is incorporated into the ALIGN program available, for
example, at the GENESTREAM network server, IGH Montpellier, FRANCE
or at the ISREC server. When utilizing the ALIGN program for
comparing amino acid sequences, a PAM120 weight residue table, a
gap length penalty of 12, and a gap penalty of 4 can be used.
[0143] In another embodiment, the percent identity between two
amino acid sequences can be determined using the GAP program in the
GCG software package, using either a Blossom 62 matrix or a PAM250
matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight
of 2, 3, or 4. In yet another embodiment, the percent homology
between two nucleic acid sequences can be accomplished using the
GAP program in the GCG software package (available at
http://www.gcg.com), using a gap weight of 50 and a length weight
of 3.
IV. Vectors
[0144] The present invention further features vectors (e.g.,
recombinant vectors), including plasmid vectors. Vectors in
accordance with the invention include nucleic acid molecules (e.g.,
isolated or recombinant nucleic acid molecules and/or genes)
described herein. In particular, recombinant vectors are featured
that include nucleic acid sequences that encode bacterial gene
products as described herein, advantageously budA and budB gene
products (e.g., SEQ ID NO:3 or 4) or mutated budA and budB
sequences (e.g., SEQ ID NO:5 or 8) that reduce or eliminate
expression of these gene products in host cells. The sequences are
more advantageously budA and budB gene products of a Gram-negative
or a Gram-positive bacterium, and even more advantageously budA and
budB gene products derived from Klebsiella or Erwinia.
[0145] One embodiment of a vector in accordance with the invention
includes a polynucleotide sequence comprising a mutation in a
bacterial gene coding for at least one of an .alpha.-acetolactate
decarboxylase and an .alpha.-acetolactate synthase protein, wherein
the vector is capable of decreasing or eliminating expression of
said protein when integrated into a bacterial host cell.
[0146] In one embodiment of the vector, the polynucleotide sequence
comprises a mutation in an isolated nucleic acid molecule
comprising the nucleotide sequence set forth in SEQ ID NO:3 or 4,
or a functional fragment thereof, as discussed supra. The mutation
can be a deletion, insertion or base change mutation.
[0147] In one preferred embodiment, the mutation in the
polynucleotide sequence is a deletion mutation. Some versions of a
vector of this type comprise a mutated budAB polynucleotide
sequence from Klebsiella oxytoca as set forth in SEQ ID NO:5 or 8.
Other vectors comprise a mutation in a polynucleotide sequence that
is at least 80% identical to SEQ ID NO:5 or 8.
[0148] Another preferred embodiment of a vector in accordance with
the invention is a plasmid vector comprising the mutated budAB
polynucleotide sequence set forth in SEQ ID NO:5 or 8. Examples of
plasmid vectors of this type, which have been designated pLOI3310
or pLOI13313, are described herein. See, for instance Examples 4
and 5, infra.
[0149] Another embodiment of a vector in accordance with the
invention comprises the polynucleotide sequence set forth in SEQ ID
NO:3, that encodes the protein .alpha.-acetolactase decarboxylase
derived from Klebsiella oxytoca.
[0150] The recombinant vector (e.g., plasmid, phage, phasmid,
virus, cosmid or other purified nucleic acid vector) can been
altered, modified or engineered such that it contains greater,
fewer or different nucleic acid sequences than those included in
the native or natural nucleic acid molecule from which the
recombinant vector was derived. Advantageously, the recombinant
vector includes a budA and budB gene or recombinant nucleic acid
molecule including a budA and budB gene or mutant thereof, operably
linked to regulatory sequences, for example, promoter sequences,
terminator sequences and/or artificial ribosome binding sites
(RBSs), as defined herein.
[0151] Typically, the budA and budB gene or mutant is operably
linked to a regulatory sequence(s) in a manner which allows for
expression (e.g., enhanced, increased, constitutive, basal,
attenuated, decreased or repressed expression) of the nucleotide
sequence, advantageously expression of a gene product encoded by
the nucleotide sequence (e.g., when the recombinant nucleic acid
molecule is included in a recombinant vector, as defined herein,
and is introduced into a microorganism).
[0152] The regulatory sequence includes nucleic acid sequences that
affect (e.g., modulate or regulate) expression of other nucleic
acid sequences. In one embodiment, a regulatory sequence is
included in a recombinant nucleic acid molecule or recombinant
vector in a similar or identical position and/or orientation
relative to a particular gene of interest as is observed for the
regulatory sequence and gene of interest as it appears in nature,
e.g., in a native position and/or orientation. For example, a gene
of interest can be included in a recombinant nucleic acid molecule
or recombinant vector operably linked to a regulatory sequence that
accompanies or is adjacent to the gene of interest in the natural
organism (e.g., operably linked to "native" regulatory sequences,
for example, to the "native" promoter). Alternatively, a gene of
interest can be included in a recombinant nucleic acid molecule or
recombinant vector operably linked to a regulatory sequence that
accompanies or is adjacent to another (e.g., a different) gene in
the natural organism.
[0153] Alternatively, a gene of interest can be included in a
recombinant nucleic acid molecule or recombinant vector operably
linked to a regulatory sequence from another organism. For example,
regulatory sequences from other microbes (e.g., other bacterial
regulatory sequences, bacteriophage regulatory sequences, and the
like) can be operably linked to a particular gene of interest.
[0154] In one embodiment, a regulatory sequence is a non-native or
non-naturally-occurring sequence (e.g., a sequence which has been
modified, mutated, substituted, derivatized, or deleted, including
sequences which are chemically synthesized). Advantageous
regulatory sequences include promoters, enhancers, termination
signals, anti-termination signals and other expression control
elements (e.g., sequences to which repressors or inducers bind
and/or binding sites for transcriptional and/or translational
regulatory polypeptides, for example, in the transcribed mRNA).
Such regulatory sequences are described, for example, in Sambrook,
J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory
Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989.
[0155] Regulatory sequences include those which direct constitutive
expression of a nucleotide sequence in a microorganism (e.g.,
constitutive promoters and strong constitutive promoters); those
which direct inducible expression of a nucleotide sequence in a
microorganism (e.g., inducible promoters, for example, xylose
inducible promoters); and those which attenuate or repress
expression of a nucleotide sequence in a microorganism (e.g.,
attenuation signals or repressor sequences). It is also within the
scope of the present invention to regulate expression of a gene of
interest by removing or deleting regulatory sequences. For example,
sequences involved in the negative regulation of transcription can
be removed such that expression of a gene of interest is
enhanced.
[0156] In one embodiment, a recombinant nucleic acid molecule or
recombinant vector of the present invention includes a nucleic acid
sequence or gene that encodes at least one bacterial budA or budB
gene product operably linked to a promoter or promoter sequence.
Advantageous promoters of the present invention include native
promoters, surrogate promoters and/or bacteriophage promoters. In
one embodiment, a promoter is a promoter associated with a
biochemical housekeeping gene or a promoter associated with a
ethanologenic pathway. In another embodiment, a promoter is a
bacteriophage promoter. Other promoters include tef (the
translational elongation factor (TEF) promoter) and pyc (the
pyruvate carboxylase (PYC) promoter), which promote high level
expression in Bacillus (e.g., Bacillus subtilis). Additional
advantageous promoters, for example, for use in Gram positive
microorganisms include, but are not limited to, the amyE promoter
or phage SP02 promoters. Additional advantageous promoters, for
example, for use in Gram negative microorganisms include, but are
not limited to tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacIq,
T7, T5, T3, gal, trc, ara, SP6, .lamda.-P.sub.R or
.lamda.-P.sub.L.
[0157] In another embodiment, a recombinant nucleic acid molecule
or recombinant vector of the present invention includes a
terminator sequence or terminator sequences (e.g., transcription
terminator sequences). Typically, terminator sequences refer to the
regulatory sequences that serve to terminate transcription of a
gene. Terminator sequences (or tandem transcription terminators)
can further serve to stabilize mRNA (e.g., by adding structure to
mRNA), for example, against nucleases.
[0158] In yet another embodiment, a recombinant nucleic acid
molecule or recombinant vector of the present invention includes
sequences which allow for detection of the vector containing said
sequences (i.e., detectable and/or selectable markers), for
example, sequences that overcome auxotrophic mutations, for
example, ura3 or ilvE, fluorescent markers, and/or colorimetric
markers (e.g., lacZ/.beta.-galactosidase), and/or antibiotic
resistance genes (e.g., bla or tet).
[0159] It is understood that any one of the budA and budB genes of
the invention can be introduced into a vector also comprising one
or more ethanologenic genes (e.g., alcohol dehydrogenase (i.e.,
adh) and pyruvate decarboxylase (pdc) and/or a gene encoding a gene
product suitable for fermenting a sugar or degrading a sugar for
subsequent fermentation as described for example, in U.S. Pat. Nos.
5,821,093; 5,482,846; 5,424,202; 5,028,539; 5,000,000; 5,487,989,
5,554,520, and 5,162,516. Such two or more genes can be expressed
independently using separate regulatory elements (e.g., promoters),
common regulatory element(s), native regulatory element(s), or a
combination thereof.
V. Methods of Making Recombinant Host Cells Comprising Gene
Deletions
[0160] As discussed, recombinant host cells in accordance with the
invention comprise a mutation in at least one polynucleotide
sequence that encodes a protein from a metabolic pathway that leads
to the production of products other than ethanol during
fermentation of saccharides. Genetic methods for making
microorganisms comprising mutations such as deletion mutations are
known in the art. Genetic techniques for isolating and manipulating
genetic constructs such as PCR-based gene cloning, plasmid
constructions and genetic analyses are well established and routine
in the art of molecular biology. See, for example, methodology
treatises such as Ausubel et al., 1987; Miller, 1992; and Sambrook
and Russell, 2001 [24-26]. Methods for producing chromosomal
deletions [22] and integrations, including the use of removable
antibiotic resistance genes, have also been described [27-29].
[0161] Use of such methods to produce an exemplary recombinant host
cell having improved ethanologenic properties is further
illustrated in Examples 4 and 5, infra, which provide detailed
description of PCR-based cloning of genes in the butanediol pathway
(budAB) from K. oxytoca, creation of a deletion mutation (knockout)
in these genes, and integration of a DNA construct comprising this
mutation into the chromosome of K. oxytoca P2, to provide improved
strains, designated K. oxytoca B15, B19 and BW21. The B15 and B19
strains comprise a mutated budAB sequence flanking a FRT-flanked
tetracycline gene, i.e., the polynucleotide sequence designated
herein as SEQ ID NO:8 (see also Example 5, infra). The BW21 strain
comprises the same mutated budAB sequence as the BW15 and BW 19
strains (i.e., a mutated budAB sequence designated herein as SEQ ID
NO:5), but lacks the FRT-flanked tetracycline gene, which was
removed using FLP recombinase (see also Example 5). The BW21 strain
was tested and shown to exhibit superior ethanologenic properties
relative to the parent strain, P2.
[0162] In yet another aspect, the invention provides a method for
producing a recombinant host cell that is optimized for producing
ethanol from asaccharide source. The method includes the following
steps:
[0163] (a) contacting a parent ethanologenic host cell with a
selected growth medium and a saccharide source under conditions
suitable for ethanol production by the parent cell;
[0164] (b) determining the level of ethanol produced from the
oligosaccharide source under the selected conditions;
[0165] (c) determining the level of at least one and preferably all
products other than ethanol produced from the saccharide source, to
identify undesirable co-products having increased expression under
said conditions;
[0166] (d) mutating a polynucleotide sequence of a gene encoding a
protein in a metabolic pathway that produces the undesirable
co-product, wherein the mutation decreases or eliminates expression
of at least one gene product in the metabolic pathway, and
increases ethanol production by the mutant cell in the selected
medium, as compared to ethanol production by the parent cell in the
absence of the mutation, thereby producing a recombinant host cell
optimized for ethanol production in the selected medium.
[0167] Some embodiments of the method further comprise producing an
isolated polynucleotide fragment comprising a mutation of the gene;
and introducing the mutated polynucleotide fragment into the parent
cell.
[0168] Advantageously, the method provides for the construction of
recombinant host cells that are custom tailored for optimal ethanol
production in particular selected media, and under particular
culture conditions. Step (c) of the method, involving
identification of co-products that are upregulated in the parent
ethanologenic host cell as a result of growth in the particular
medium, is used to identify the corresponding metabolic pathways
that can be targeted and eliminated by further genetic engineering
of the parent cell.
[0169] In some embodiments of the method, the cell is optimized for
ethanol production in a minimal medium. As discussed, the cost of
growth media used in the production of ethanol by recombinant host
cells such as bacteria is a very significant factor in the overall
production cost. To contain production costs, recent efforts have
resulted in the development of "minimal media" comprising minimal
amounts of required nutrients from inexpensive sources. (See, for
example, Section VI, and Table 2, infra, showing formulations of
several minimal media suitable for SSF, including a novel
urea-based minimal medium disclosed herein, designated "optimized
urea medium," (OUM). One embodiment of the method produces a cell
optimized for ethanol production in OUM, e.g., K. oxytoca BW21,
comprising deletions in budAB genes that encode butanediol products
that are upregulated in this medium at acidic pH, a preferred pH
for SSF.
VI. Minimal Media
[0170] In another aspect, the invention provides minimal media that
support growth and ethanol production by recombinant host cell
suitable for degrading a saccharide. A minimal medium in accordance
with the invention comprises the following base components:
[0171] a defined nitrogen source;
[0172] a complex nitrogen source;
[0173] a source of phosphate; and
[0174] a source of magnesium.
Optionally, sources of metal ions such as FeCL.sub.2 and NiCl.sub.2
are added in some embodiments of the media, as cofactors that are
beneficial for enzymatic activity in certain ethanologenic
bacterial strains such as derivatives of Klebsiella oxytoca.
[0175] In some embodiments of the minimal media in accordance with
the invention, the defined nitrogen sources is a urea-like
compound. The concentration ranges for urea nitrogen can be from
about 0.1 to 100 mM, preferably from about 2.0-20 mM, and more
preferably from about 8-12 mM.
[0176] Complex nitrogen sources of use in the minimal media can be
selected from a variety of sources, including but not limited to
corn steep liquor (CSL), yeast autolysate and/or extract, corn
processing by-product, soy processing byproduct, and spent
fermentation broth, for example from fermentations using
microorganisms such as yeast, streptomycetes, bacilli, etc.
[0177] Some embodiments of the minimal media comprise corn steep
liquor (CSL) as a complex nitrogen source. Preferred concentration
ranges for CSL in these media are from about 0.1-100 g L.sup.-1,
preferably from about 1-20 g L.sup.-1, and more preferably from
about 5-10 g L.sup.-1.
[0178] It has been determined from studies described herein that
concentration ranges of other components, such as total phosphate
and magnesium may be less critical to ethanol production. The
lowest effective concentration of these components can be
determined empirically as described above for urea and CSL.
Generally, advantageous concentrations of total phosphate are from
about 10-100 mM, and preferably about 10-12 mM. Preferred
concentrations of magnesium are in the range of about 0.1-5.0 mM,
and more preferably in the range of about 0.25 to 1.0 mM.
[0179] In one aspect, the invention provides minimal media that are
optimized to support growth and ethanol production by a recombinant
host cell suitable for degrading asaccharide. Some embodiments of
the media are optimized for selected recombinant host cells and
conditions of fermentation. Depending upon the ethanologenic strain
to be used, the concentrations of the base components of the
minimal media are optimized for cost effectiveness by the
determining the minimum concentrations of components consistent
with acceptable cell growth and high levels of ethanol production
by ethanologenic strains during fermentation of the sugar
substrates. (See, for example, studies described in Examples 1-3
and Tables 2 and 3, infra, relating to optimization of minimal
urea-based media particularly suitable for use with ethanologenic
K. oxytoca strains).
[0180] Some embodiments of optimized media in accordance with the
invention are suitable for methods and conditions used in SFF of
lignocellulosic biomass. As discussed, lignocellulose, being
inedible by animals, is attractive as an abundant and inexpensive
starting material for ethanol production. Abundant sources of
lignocellulose are found in waste products including plant residues
such as stems, leaves, hulls, husks, cobs and the like, as well as
wood, wood chips, wood pulp and waste paper. SSF can be conducted
using lignocellulosic biomass as a saccharide source. In the SSF
process, soluble products are produced by fungal enzymes (typically
cellulases and xylanases) that hydrolyze the lignocellulose.
Examples of soluble products released from lignocellulosic products
include cellobiose, cellotriose, xylobiose, xylotriose and
arabinosides. These soluble sugar products are concurrently
converted to ethanol by ethanologenic microorganisms such as
recombinant bacteria. Importantly, the fungal enzymes exhibit
optimal performance at acidic pH (around pH 5.0), necessitating SSF
reactions to be carried out at acidic pH.
[0181] As discussed, countering the advantage of lignocellulose as
an inexpensive source of biomass for ethanol production is its
disadvantage of being poor in nutrients needed to support the
ethanologenic bacteria used in the SSF process. The invention
addresses this problem by providing in one aspect improved minimal
media suitable, for example, for use in SSF with ethanologenic
bacteria and lignocellulosic starting materials.
[0182] The media are optimized to support maximal growth and
ethanol production by ethanologenic strains of bacteria, and to
minimize cost by substituting previously used sources of nitrogen,
including simple chemical agents such as ammonia and
(NH.sub.4).sub.2SO.sub.4 with low levels of urea or urea-like
compounds as the sole source of nitrogen. The substitution of these
compounds provides a significant cost advantage over use of other
nitrogen sources.
[0183] A particularly preferred embodiment of a minimal medium in
accordance with the invention, designated "optimized urea medium 1"
(OUM1) comprises the following components at the indicated
concentrations:
TABLE-US-00001 NH.sub.2CONH.sub.2 (urea) 10.0 mM CSL 10.0 g
L.sup.-1 KH.sub.2PO.sub.4 10.7 mM Na.sub.2HPO.sub.4 1.3 mM
CaCl.sub.2 1 mM MgSO.sub.4 1 mM FeCl.sub.2 0.074 mM NiCl.sub.2
0.0068 mM
For use in fermentation reactions, a source of saccharide is added,
for example glucose (e.g., at 90 g L.sup.-1) or a lignocellulosic
source of sugar as described above.
VII. Methods and Systems for Optimized Ethanol Production During
Fermentation of a Saccharide Source by Recombinant Host Cells
[0184] The above-described optimized minimal media of the invention
and the recombinant organisms optimized by genetic engineering for
maximal ethanol productivity and minimized production of
co-products can be advantageously combined in a system for ethanol
production
[0185] Accordingly, in another aspect the invention provides a
system for optimized ethanol production from a saccharide source by
a recombinant host cell suitable for degrading a saccharide. The
system includes the following components:
[0186] (a) a selected medium that supports optimal growth and
ethanol production by a host cell under the selected
conditions;
[0187] (b) a source of a saccharide; and
[0188] (c) a recombinant host cell optimized for ethanol production
in the selected medium and conditions, the cell comprising: [0189]
a heterologous polynucleotide sequence that codes for an enzyme
that converts sugars to ethanol, wherein the cell expresses the
heterologous polynucleotide sequence at a sufficient functional
level so as to facilitate production of ethanol as a primary
fermentation product by the host cell; and [0190] a mutation in at
least one polynucleotide sequence that codes for a protein in a
metabolic pathway in the cell that produces a product other than
ethanol from the oligosaccharide source in the selected medium
under the selected conditions, wherein the mutation decreases or
eliminates expression of the protein, thereby increasing ethanol
production the host cell, as compared to ethanol production by the
cell when lacking the mutation, thereby optimizing ethanol
production.
[0191] In embodiments of the system preferred for commercial use,
the selected medium is a minimal medium, such as a urea-based
minimal medium in accordance with the present invention, as
described above.
[0192] Any suitable recombinant host cell optimized for ethanol
production can be used in the system, as can any suitable
saccharide or oligosaccharide source. A preferred inexpensive
saccharide source is lignocellulosic material. As discussed, SSF
reactions are optimally carried out in the acidic pH range, to
optimize efficiency of the fungal enzymes. However, the acidic
conditions can have detrimental effects on fermentation by the
bacteria, such as increased production of unwanted co-products and
concomitant decreased production of ethanol at the preferred
pH.
[0193] In an optimized system in accordance with the present
invention, the detrimental effects of a condition of growth (such
as pH), or of the minimal media itself, can be minimized by pairing
a particular growth medium with a recombinant bacterium
specifically optimized for growth and ethanol production in that
medium. For example, if it is known that in a particular medium an
undesired metabolic pathway in the host is altered (e.g.,
upregulated) under the particular conditions of culture, then a
system can be designed that pairs that medium with a selected
recombinant host cell comprising genetic alterations (e.g.,
deletion mutations) that target the altered pathway, thereby
reducing or eliminating the unwanted product under those
conditions. Accordingly, the system is optimized for ethanol
production during fermentation reactions in the particular minimal
medium.
[0194] A preferred embodiment of the optimized system of the
invention combines a novel OUM medium as described herein, a
recombinant optimized Klebsiella oxytoca strain, such as strain
BW21, and a lignocellulosic source of sugar. This optimized system
for the production of ethanol provides an attractive and
inexpensive means of producing ethanol from biomass containing
lignocellulose.
[0195] A suitable recombinant ethanologenic bacterial strain for
fermentation of biomass by SSF is Klebsiella oxytoca P2. An
exemplary recombinant host cell of the present invention optimized
for fermentation of biomass by SSF is an improved ethanologenic
strain derived from Klebsiella oxytoca P2, designated BW21, that
differs from the parent strain in having a deletion mutation that
eliminates the co-products of the butanediol pathway (acetoin and
2,3-butanediol). As shown in studies herein, this pathway is
specifically upregulated when the P2 cells are grown and used in
fermentation studies in a preferred minimal medium (OUM1)
containing urea as the defined nitrogen source. In the optimized
BW21 cells, expression of these unwanted gene products was
eliminated by gene deletion. Assays of ethanol production by BW21
cells demonstrated that these cells possessed superior ability to
produce ethanol at acidic pH in the minimal medium than the parent
P2 strain, even when grown in rich medium such as Luria broth.
Thus, by combining an optimized minimal medium comprising a
urea-like compound with a recombinant Klebsiella strain optimized
for ethanol production in this particular medium, ethanol
production was optimized in the system at acidic pH, under
conditions suitable for SFF, to levels achievable using more
expensive media.
[0196] In yet a further aspect, the invention provides kits
comprising a recombinant host cell as described above, packaged
with instructions for using the recombinant host cell according to
the methods or systems of the invention.
[0197] Materials and methods generally useful in the practice of
the above method are further described below, and in the following
Examples.
EXAMPLES
[0198] The invention is further illustrated by reference to the
following Examples, which should not be construed as limiting.
Materials and Methods:
[0199] The following materials and methods were used throughout the
Examples below.
[0200] 1a. Strains and Plasmids:
[0201] Table 1 lists the organisms and plasmids used to construct
the recombinant microorganisms of the invention.
TABLE-US-00002 TABLE 1 Strains and Plasmids Strain or Traits
Source/Reference DH5a lacZ.DELTA.M15 recA Bethesda Res Lab K.
oxytoca M5A1 prototroph [5] P2 pflB::(Zm pdc, adhB) cat [6] BW15
M5A1 .DELTA.budAB::FKT-tet-FKT See text BW19 P2 transductant from
BW15, .DELTA.budAB::FRT-tet-FRT See text BW21 BW19 Tc.sup.s,
.DELTA.budAB::FRT See text pCR2.1-TOPO TOPO T/A PCR cloning vector
bla kan Invitrogen pLOI2065 FRT-tet-FRT [20] pFT-K FLP Recombinase
kan [21] pKD46 Red recombinase, bla [22] pHP45W aac [23] pLOI2745
temperature conditional vector, pSC101.sup.ts, kan See text
pLOI3301 pCR2.1 budAB' See text pLOI3310 pLOI3301 budA' FRT-tet-FRT
`budB` See text pLOI3313 pLOI2745 budA' FRT-tet-FRT `budB` See text
pLOI3421 1.8 kbp SmaI frag.containing aac from pHP45W XmnI See
text
[0202] 1b. Growth Media and Conditions
[0203] Ethanologenic strains were maintained on Luria agar [24]
containing 2% glucose and chloramphenicol (40 or 600 mg L.sup.-1 on
alternate days) under argon. Other strains were maintained on Luria
agar plates lacking added sugar with appropriate antibiotics.
Unless otherwise noted, ampicillin (50 mg L.sup.-1), kanamycin (50
mg L.sup.-1), apramycin (50 mg L.sup.-1), and tetracycline (12.5 mg
L.sup.-1) were used for selection. Strains harboring plasmids with
temperature conditional replicons were grown at 30.degree. C. All
other strains were maintained at 37.degree. C., except where noted.
Plasmid preparations were stored at -20.degree. C. Stock cultures
were stored in glycerol at -75.degree. C.
[0204] 1c. Genetic Methods:
[0205] Standard methods were used for PCR-based gene cloning,
plasmid constructions, and genetic analyses [24-26]. Methods for
integration, chromosomal deletions, integration, and the use of
removable antibiotic resistance genes were used as previously
described [22, 27-29]. E. coli DH5.alpha. was used for
constructions.
[0206] 1d. Deletion of budA and budB Genes.
[0207] A DNA fragment containing .alpha.-acetolactate decarboxylase
(budA) and the 5' end of .alpha.-acetolactate synthase (budB) was
amplified by PCR using genomic DNA from K. oxytoca as a template
and Taq PCR Master Mix (Qiagen). After an initial denaturation at
94.degree. C. for 3 min, DNA was amplified for 30 cycles
(denaturation at 94.degree. for 30 s, annealing at 55.degree. C.
for 30 s and extension at 72.degree. C. for 70 s). A final
elongation step at 72.degree. C. for 10 min was also included.
[0208] The budAB' DNA fragment was amplified using the following
primers:
TABLE-US-00003 forward 5'GCTGAATCGGGTCAACATTT-3' (SEQ ID NO:
primer: 1) reverse 5'-TTTCGGTTTGTCCAGGTAGT-3' (SEQ ID NO: primer
2)
and cloned into pCR2.1-TOPO. This fragment, designated pLOI3301,
was sequenced and has been deposited in GenBank (Accession No.
AY722056).
[0209] For preparation of a DNA fragment containing deletions in
budAB, a cloning/deletion strategy was used in which the central
region of the budA and budB genes was deleted and replaced with a
tetracycline gene flanked by two FRT sites. The resulting
construct, carrying budA and budB deletions (budA' FRT-tet-FRT
`budB) (SEQ ID NO: 8) was integrated into a temperature conditional
vector, pSC100.sup.ts and used to stably transform ethanologic
bacteria.
[0210] FIG. 1 is a schematic diagram showing steps in the
preparation of exemplary constructs (pLOI3301, pLOI 3310 and pLOI
3313) comprising deletion mutations in budAB that result in
elimination of the butanediol products of fermentation when
introduced into ethanologenic bacteria. Further detailed
description of FIG. 1 and of the methods used to produce these
plasmids is found infra, in particular in Example 4.
[0211] 1e. Production of Strain BW21 from Klebsiella oxytoca
P2.
[0212] DNA constructs comprising deletion mutations in budA and
budB, prepared as described in 1d above, were used to produce
recombinant bacteria transformed with these constructs. See, for
instance, Example 5 describing transformation of ethanologenic
strain K. oxytoca M5A1. Bacteriophage P1 (an E. coli phage) was
then used to transduce the budAB chromosomal deletion constructed
in K. oxytoca M5A1 into strain P2 to produce strain BW21.
[0213] 1f. Assessment of Butanediol Pathway by Screening for
Absence of Acetoin Product of BudA:
[0214] Strains were screened for acetoin production using a
modification of the Vogues-Proskaur (VP) agar method described by
Blomqvist, et al. [35] that used microtiter plates instead of petri
plates, increasing the sensitivity by limiting diffusion of the
colored product. Each well was filled with 1 ml of the medium (per
liter: 2.5 g Difco Bactopeptone, 1.0 g Difco yeast extract, 10 g
glucose, 1.0 g sodium pyruvate, and 25 g agar), and inoculated.
After 24 hours, 200 .mu.L of a 5% .alpha.-napthol solution in 2.5 N
NaOH was added to each well. Color development was monitored for 1
h at room temperature. The absence of red color confirmed the lack
of acetoin (product of BudA activity). Additional confirmation was
provided by HPLC analysis of fermentation products.
[0215] 2a. Minimal Media for Ethanologenic Bacteria:
[0216] Components of media, both previously known and as developed
herein (optimized urea medium 1, OUM1), are summarized in Table
2.
TABLE-US-00004 TABLE 2 Composition of Media (excluding fermentable
sugar) Media Composition (mM) Component.sup.a LB.sup.b
M9(+Fe).sup.c U-M9(+Fe).sup.d 0.5% CSL + M.sup.e U-0.5% CSL +
M.sup.f OUM1.sup.g KH.sub.2PO.sub.4 22 22 7.4 7.4 10.7
K.sub.2HPO.sub.4 2.9 2.9 Na.sub.2HPO.sub.4 42 42 1.3 Total PO.sub.4
64 64 10.3 10.3 12 NaCl 85.6 9 9 CaCl.sub.2 0.1 0.1 1 1 1
MgSO.sub.4 1 1 2 2 1 FeCl.sub.3 0.074 0.074 0.074 0.074 0.074
NiCl.sub.2 0.0068 0.0068 0.0068 NH.sub.4Cl 19
(NH.sub.4).sub.2SO.sub.4 23.5 NH.sub.2CONH.sub.2 10 23.5 10 Total
Nitrogen.sup.b 19 20 47 47 20 Tryptone (gL.sup.-1) 10 Yeast extract
5 CSL (gL.sup.-1) 10 10 10 .sup.aDegree of hydration is omitted for
simplicity. .sup.bLuria Broth .sup.cM9 medium (30). .sup.dM9 with
NH.sub.4Cl replaced with equivalent urea nitrogen. .sup.e0.5% CSL +
M (32). .sup.f0.5% CSL + M media except that
(NH.sub.4).sub.2SO.sub.4 was replaced with equivalent urea
nitrogen. .sup.gOUM1, optimized urea media number 1. .sup.hTotal
mmoles of available nitrogen.
[0217] Each medium tested in fermentation reactions with either 50
g L.sup.-1 (278 mM) glucose or 90 g L.sup.-1 (500 mM) glucose.
Components were purchased from either the Fisher Scientific Company
or the Sigma Chemical Company. Inorganic salts were reagent grade.
Urea was technical grade. M9 medium was prepared as previously
described [30] and further supplemented with 0.07 mM FeCl.sub.3 to
ensure adequate levels for iron-requiring Z. mobilis alcohol
dehydrogenase [31]. Corn steep liquor (CSL) medium for
ethanologenic E. coli, (0.5% CSL+M), has been previously described
[32]. Both M9 and 0.5% CSL+M media were used as starting points to
optimize a medium for ethanologenic derivatives of K. oxytoca M5A1.
When urea was used as the nitrogen source, 0.007 mM NiCl.sub.2 was
added for urease activity. CSL levels are expressed on a dry weight
basis. Stock solutions of CSL were prepared and sterilized as
previously described [33].
[0218] K. oxytoca P2 was used in all media optimization studies.
Isolated colonies from freshly grown plates (24 h) were resuspended
in 1 ml of deionized H.sub.20 and used to inoculate 125 ml flasks
(.about.50 .mu.L inoculum) containing 75 ml of medium (50 g
L.sup.-1 glucose). Growth and ethanol production were monitored
after 24 and 48 hours.
[0219] 2b. Fermentation Conditions:
[0220] Seed cultures (150 ml in 250 ml flasks) were grown for 16 h
at 35.degree. C. (120 rpm) in media containing 50 g L.sup.-1
glucose. Cells were harvested by centrifugation (5000.times.g, 5
min) and used as inocula to provide an initial concentration of 33
mgL.sup.-1 dry cell weight (OD.sub.550nm=0.1). Respective media
used for fermentations were also used for seed growth but with a
lower concentration of glucose (50 gL.sup.-1). Fermentation vessels
were previously described [34] and contained an initial volume of
350 ml (90 gL.sup.-1 glucose). Cultures were incubated at
35.degree. C. (150 rpm). Broth was maintained at pH 5.2 (except
where noted) by the automatic addition of 2N KOH.
[0221] 2c. Analytical Methods:
[0222] Cell mass was estimated by determining OD.sub.550nm with a
Bausch & Lomb Spectronic 70 spectrophotometer. With this
instrument, 1 OD.sub.550nm corresponds to a cell density of 0.33 mg
(dry wt.) L.sup.-1. Measurements of cell density for K. oxytoca
have a large error due to the clumping nature of the cells. Ethanol
was measured by gas chromatography using a Varian model 3400.times.
as previously described [11]. Other fermentation products were
determined by high-performance liquid chromatography (HPLC) using a
Hewlett-Packard model 1090 series II chromatograph and a Bio-Rad
Aminex 87H ion partition column (45.degree. C.; 4 mM
H.sub.2SO.sub.4; 0.4 ml min.sup.-1; 10 .mu.L, injection volume)
with dual detectors (refractive index and UV.sub.210nm) [33].
Carbon balances were calculated as previously described [29, 36].
When LB was used as the fermentation medium, cell mass was assumed
to be produced exclusively from the complex media components and
was not included in calculations of carbon balance.
Example 1
Minimal Media for Growth and Ethanol Production by K. oxytoca
P2
[0223] This example describes the development of a novel minimal
medium (OUM1) comprising urea as the sole source of nitrogen,
optimized for growth and ethanol production by recombinant
ethanologenic bacteria such as Klebsiella strains. The new medium
was developed and optimized using strain K. oxytoca P2, by
comparing its growth and ethanol production capacity with that of
several known media.
[0224] Previously described media used for comparative purposes
were the following:
Luria broth (LB) (Ausubel et al., 1989); M9 medium (+Fe) (Neidhardt
et al., 1974); U-M9 (+Fe), in which NH.sub.4Cl is replaced with
equivalent nitrogen from urea; 0.5% corn steep liquor, CSL+M
(Martinez et al., 1999); U-0.5% CSL+M, in which
(NH.sub.4).sub.2SO.sub.4 is replaced with equivalent nitrogen from
urea. The new media is referred to herein as "optimized urea medium
number 1" (OUM1). The formulations of each of the above media are
listed in Table 2, supra.
[0225] In initial studies to develop the optimized media, cell
growth and ethanol production capacity were compared in K. oxytoca
P2 tested at pH 5.2 in M9 (+Fe) and 0.5% CSL+M media, the latter
having ammonia as the sole nitrogen source. LB medium at pH 5.2 was
included as a control to provide a benchmark for performance.
Referring to FIGS. 2A and 2B, as expected [32], LB medium supported
the highest cell yield (FIG. 2A) and the most rapid ethanol
production (FIG. 2B). Equivalent levels of ethanol were produced in
0.5% CSL+M and LB (FIG. 2B).
[0226] Table 3 shows, inter alia, the effect of replacing ammonia
with urea on ethanol production and yield in both M9 and CSL+M
media. As can be seen, this change resulted in a small decrease in
ethanol production (71% vs. 76% yield for M9; 78% vs. 83% for
CSL+M). As is also shown in Table 3, in general, ethanol
productivity and yields increased with the richness of the media
(LB>0.5% CSL+M>M9+Fe), regardless of nitrogen source.
TABLE-US-00005 TABLE 3 Production of Ethanol and Co-products in
Various Media (90 gL.sup.-1 glucose, 72 h)..sup.a Acetoin + Carbon
Ethanol Ethanol Formate Lactate Succinate Acetate 2,3 Butanediol
Recovery.sup.c Strain Medium pH n (mM) Yield.sup.b(%) (mM) (mM)
(mM) (mM) (mM) (% total) P2 LB 5.2 2 848 85 <1 25 8 <1 31 110
P2 M9 + NH.sub.4 5.2 2 761 76 <1 22 18 <1 24 96 P2 0.5% CSL +
NH.sub.4 5.2 2 831 83 <1 13 8 <1 23 101 P2 U-M9 5.2 2 708 71
<1 33 12 <1 18 89 P2 U-0.5% CSL + M 5.2 2 776 78 <1 15 20
<1 19 95 P2 OUM1 5.2 10 825 (65) 83 <1 10 (5) 13 (5) 9 (3) 72
(20) 101 (6) BW21 OUM1 5.2 4 926 (17) 93 <1 4 (1) 13 (3) 5 (1) 2
(1) 100 (2) P2 LB 6.0 2 998 100 11 37 12 25 39 111 P2 LB 6.8 2 979
98 66 28 11 34 38 112 P2 OUM1 6.8 2 806 81 44 30 17 47 16 96
.sup.aValues are corrected for dilution by added base. Standard
deviations are shown in parentheses for n values of 3 or more
values. .sup.bPercentage of theoretical yield based on total
glucose (90 gL.sup.-1). .sup.cIncludes unmetabolized glucose
remaining after 72 hours.
Example 2
Optimized Minimal Media Comprising Urea as the Defined Nitrogen
Source
[0227] This example describes a procedure for developing optimized
minimal media comprising urea for use with ethanologenic bacteria,
and provides the formulation of an embodiment of such a medium
designated "OUM1."
[0228] A urea-based medium, designated OUM1 (having urea as the
only defined nitrogen source), was tested in pH-controlled
fermentations at pH 5.2 with 90 gL.sup.-1 glucose, using methods as
described above. Ethanol production in this medium was slightly
superior to urea-containing formulations of M9 (+Fe) and CSL+M
media, confirming that higher levels of nitrogen, phosphate, and
CSL are not necessary (FIGS. 2C and 2D).
[0229] Results of cell growth studies showed that maximum cell
densities were quite similar in M9 (+Fe) and 0.5% CSL+M media (FIG.
2A), and in OUM1 medium (FIG. 2C), suggesting that the lower levels
of nitrogen (19.0 mM in M9) and phosphate (10.3 mM in 0.5% CSL+M)
in these respective media are adequate.
[0230] Based on the compositions of M9 (+Fe) and 0.5% CSL+M, flask
experiments were designed to evaluate different levels of nutrients
in OUM1 medium: phosphate (12-72 mM), magnesium (0.25-1.0 mM), CSL
(0-15 g L.sup.-1), and urea nitrogen (2.5-15 mM). Results of
experiments varying the concentrations of corn steep liquor and
urea are shown in FIGS. 3A and 3B, respectively. The data showed
that over the stated ranges, only CSL and urea had clear optima,
i.e., 10 g L.sup.-1 and 10 mM, respectively. Similar concentrations
of ethanol (13.9.+-.1.3 gL.sup.-1) were produced after 48 h with
all levels of other components. Although it is possible that lower
concentrations may be adequate, 12 mM PO.sub.4.sup.-3, 1 mM
MgSO.sub.4, and 1 mM CaCl.sub.2 were selected for the optimized
urea medium (OUM1). (See Table 2 for complete formulation of OUM1
medium).
Example 3
Effect of Acidic pH on Ethanol Titers and Production of Co-Products
of Fermentation
[0231] As discussed above, for certain types of fermentation
reactions, for example those in which fermentable sugars are
derived from lignocellulosic feedstocks, it is desirable to conduct
the reactions at acidic pH, as this is the range in which fungal
hydrolases and cellulases exhibit optimal performance. This example
describes the effect of pH on production of ethanol and byproducts
of fermentation by ethanologenic bacterial strain P2 grown in
various media, including newly developed medium OUM1.
[0232] Referring again to Table 3, results of ethanol production
from glucose by strain P2 grown in OUM1 medium is compared with
results for these cells grown in previously described media. As can
be seen in Table 3, ethanol titers with OUM1 media were generally
equivalent to those obtained with LB media at pH 5.2.
[0233] It was noted in these studies, however, that ethanol titers
with all media were lower at pH 5.2 than previously reported in
rich media, e.g., Luria broth (LB) at more neutral pH [5,6]. The
detrimental effect of low pH on ethanol production was confirmed
for fermentations with both LB and OUM1 media. As shown in Table 3,
strain P2 in LB media produced ethanol yields of 100% and 98%,
respectively, at pH 6.0 and 6.8, whereas yields were reduced to 85%
at pH 5.2.
[0234] In the fermentation experiments shown in Table 3, the levels
were also determined for several undesired co-products (in addition
to ethanol) made from the glucose substrate, i.e., formate,
lactate, succinate, acetate, acetoin and 2,3-butanediol. Although
co-products were made by strain P2 in all media at pH 5.2, it was
seen that an unexpectedly high level (72 mM) of products from the
2,3-butanediol pathway was produced with OUM1 medium at this pH
(Table 3). In both LB medium and OUM1 medium, fermentations at pH
5.2 contained a higher proportion of neutral co-products (acetoin
and 2,3-butanediol) than at pH 6.8. More particularly, in OUM1
medium, the levels of neutral co-products were 4.5 fold higher at
pH 5.2 than at pH 6.8. In contrast, in OUM1 medium at pH 6.8,
butanediol+acetoin levels were reduced (16 mM vs. 72 mM), and
acetate and formate levels were increased at pH 6.8 (47 mM vs. 9
mM, and 44 mM vs. <1 mM, respectively). Unlike the results in
OUM1 medium, in LB medium, the levels of neutral co-products
remained relatively constant at pH 5.2 and 6.8 (31 mM vs. 38 mM)
while acidic co-products declined (Table 3).
[0235] Without intending to be bound by theory, it is believed that
the increased formation by strain P2 of neutral co-products from
the 2,3-butanediol pathway may be related both to the composition
of the OUM1 medium and to the low pH. The findings appear to be
consistent with reported activities of the corresponding enzymes in
native strains of K. oxytoca. For example, enzyme activities
concerned with the production of co-products of the 2,3-butanediol
pathway are known to increase in response to low pH [35, 37, 38].
At more neutral pH, acidogenic activities such as pyruvate
formate-lyase, acetate kinase, and lactate dehydrogenase produce
more acidic products [39].
Example 4
Constructs for Deletion of budAB Operon in Ethanologenic
Bacteria
[0236] This example describes the isolation of a putative
full-length cDNA sequence for the budA gene, and a partial cDNA
sequence for the budB gene, derived from bacteria (Klebsiella
oxytoca), and construction of DNA fragments in which these genes in
the butanediol pathway have been disrupted. Following introduction
into bacterial cells, these constructs are useful for eliminating
the production of unwanted 2,3-butanediol and acetoin co-products
during fermentation of saccharides by the cells.
[0237] The budA and budB genes are contiguous in the K. oxytoca
genome and are designated together as budAB. The two genes encode,
respectively, two enzymes involved in the production of
2,3-butanediol and acetoin, i.e., .alpha.-acetolactate
decarboxylase and .alpha.-acetolactate synthase. In this Example,
deletions in the budAB genes of K. oxytoca M5A1 were constructed
and transduced into K. oxytoca strain P2, to produce strain BW21.
In general, the methods used to construct the deletion mutation
strains such as BW21 are described in Materials and Methods,
sections 1a-c, supra. The plasmids and strains used to construct
the new strain with budAB deletions are listed in Table 1,
supra.
[0238] The budAB genes have not been previously described in K.
oxytoca. Homologous genes are known, however, from two related
organisms, i.e., Enterobacter aerogenes and Roultella terrigena
(formerly Klebsiella terrigena; [35, 37]). Based on these sequences
and the partial (unannotated) genome of K. pneumoniae [40], primers
as described above (SEQ ID NOS: 1 and 2) were designed for PCR
amplification of a DNA fragment containing budAB'.
[0239] FIG. 1 is a schematic diagram showing the Bud operon and the
steps in the construction of plasmids used to delete the
2,3-butanediol fermentation pathway involving this operon in K.
oxytoca. The inset (FIG. 1A) is a diagrammatic representation of
the operon. As indicated in FIG. 1A, expression of this operon is
increased by low pH, and positively regulated by BudR and Fnr.
[0240] As described in the Methods above, a DNA fragment containing
the putative full length coding sequence of .alpha.-acetolactate
decarboxylase (budA) (SEQ ID NO. 3) and a partial coding sequence
comprising the 5' end of .alpha.-acetolactate synthase (budB) (SEQ
ID NO:4) was amplified by PCR using genomic DNA from K. oxytoca as
a template. (The predicted amino acid sequences of the polypetides
encoded by SEQ ID NOS: 3 and 4 are set forth in SEQ ID NOS: 6 and
7, respectively.) The diagram in the upper left of FIG. 1B shows
the portion of the budA and budB genes of K. oxytoca amplified by
the indicated PCR primers (SEQ ID NOS: 1 and 2).
[0241] Referring to the lower portion of FIG. 1B, the amplified DNA
fragment comprising budAB was cloned into PCR cloning vector
pCR2.1-TOPO (described in Table 1), to produce pLOI301 (pCR2.1
budAB'; indicated by (1) in FIG. 1B).
[0242] To eliminate the budAB gene product, a large central region
of the budAB' fragment was deleted and replaced with a tet gene
flanked by two FRT (Flp recombinase Recognition Target) sites, to
produce pLOI3310 (budA'-FRT-tet-FRT-`budB`; FIG. 1B, (3)). A
nucleic acid construct comprising the truncated budA and budB
sequences and the intervening FRT-flanked tetracycline gene
(budA'-FRT-tet-FRT-`budB)` is designated herein as SEQ ID NO:8.
Deletion of the central portion of the budAB sequence, and
replacement with the tet-containing construct was accomplished by
standard techniques known in the art using pLOI2065 (FIG. 1, (2))
which contains the tet gene flanked by two FRT sequences (Underwood
et al., 2002; see also Table 1). Flanking FRT sites were included
to facilitate marker removal after chromosomal integration [20, 21,
27].
[0243] To minimize background during subsequent integration, the
2.1 kbp
[0244] HindIII-ApaI fragment comprising budA'-FRT-tet-FRT-`budB`
from pLOI3310 was ligated into corresponding sites of pLOI2745
(FIG. 1, (4)). This vector contains a temperature-conditional
pSC101 replicon (see Table 1 for further description). The
resulting 5502 bp plasmid was designated (pLOI3313) (pLOI2745
(budA'-FRT-tet-FRT-`budB`) (FIG. 1B, (5)).
Example 5
Production of Recombinant Ethanologenic Klebsiella Bacterial
Strains with budAB Deletions
[0245] As demonstrated in Example 3 and Table 3 above, during
fermentation reactions, recombinant ethanologenic strain K. oxytoca
P2 exhibits reduced ethanol productivity and increased production
of co-products of the butanediol pathway when grown at acidic pH in
OUM1 fermentation medium. As discussed, the budAB genes encode two
enzymes involved in the production of 2,3-butanediol and acetoin,
i.e., .alpha.-acetolactate decarboxylase and .alpha.-acetolactate
synthase, respectively. This Example describes the production of a
new ethanologenic strain of K. oxytoca, strain BW21, derived from
strain P2, that comprises deletions in the budAB genes that result
in elimination of expression of the budAB gene products in the
mutant cells.
[0246] For integration of the budA'-FRT-tet-FRT-`budB` fragment
into Klebsiella strains useful for ethanol production, the pLOI3313
plasmid, described in Example 4 above, was linearized and used as a
template for PCR amplification. The PCR product containing the
budAB deletion was integrated into strain K. oxytoca strain M5A1 by
electroporation in the presence of Red recombinase (pLOI3421; see
Table 1).
[0247] To verify functional deletion of the budAB gene products,
ten clones were grown in optimized urea medium (OUM1; see Table 2
supra) containing 5% glucose, and screened for the presence or
absence of acetoin and 2,3-butanediol as described in Methods, 1f.
Absence of detectable levels of these products confirmed deletion
of the budAB pathway in the successfully transformed cells, also
termed "deletion clones." Deletion of budAB in the clones was also
confirmed by PCR analysis.
[0248] For construction of ethanologenic strains of K. oxytoca
(such as P2) having deletions in the butanediol pathway, one
deletion clone of K. oxytoca strain M5A1 (designated BW15) was
selected and used as a donor for transduction into K. oxytoca P2
using bacteriophage P1, as described above. Ten resulting
transductants were screened for acetoin and butanediol production
as described. One deletion clone was selected for further study,
and designated strain BW19. The FRT-flanked tet gene was
subsequently removed by standard procedures using FLP recombinase
(pFT-K). The resultant strain, having the budAB deletion but
lacking FRT-flanked tet was designated K. oxytoca strain BW21. An
isolated nucleic acid fragment comprising the truncated budA and
budB sequences that remain in the cells after removal of the
FRT-flanked tet gene is designated herein as SEQ ID NO:5.
Example 6
Deletion of budAB Increases Ethanol Yields by Ethanologenic
Bacteria
[0249] This example describes the improved ethanologenic properties
and the decreased production of co-products of fermentation of
glucose by the newly developed ethanologenic strain K. oxytoca BW21
(described in Example 5), in comparison with the parent strain K.
oxytoca P2.
[0250] Referring again to Table 3, a comparison is now made of the
effect of budAB deletion (strain BW21 vs. parent strain P2) on
production of ethanol and co-products of fermentation by cells
grown in OUM1 (with 90 g L.sup.-1 glucose).
[0251] As shown in the table and previously discussed, in
fermentations by parent strain P2 carried out at pH 5.2, unwanted
co-products (acetoin+2,3-butanediol) from the butanediol pathway (2
mol pyruvate per mol product) were produced from approximately 14%
of the glucose available for ethanol production. In striking
contrast, deletion of the genes encoding acetolactate synthase and
acetolactate decarboxylase in strain BW21 essentially eliminated
both of these co-products (Table 3). Lactate and acetate levels
were also lower in strain BW21 than in parent strain P2.
[0252] In OUM1 medium at pH 5.2, the decrease in co-products by
strain BW21 was accompanied by a 12% increase in ethanol titer and
yield in comparison to strain P2 (See Table 3 and FIG. 4.) The
graphs in FIG. 4 show a comparison of cell growth (4A) and ethanol
production (4B) by K. oxytoca strains BW21 and P2 in OUM1 medium.
Although the growth rates of BW21 and P2 were essentially the same,
ethanol production was consistently higher in strain BW21.
[0253] FIG. 5 shows a comparison of the ethanol yield (expressed in
grams, per gram of glucose) and ethanol productivity (expressed as
maximum and average volumetric rates of ethanol production, in mM
ethanol per hour) for parent P2 strain grown in three
urea-containing media, i.e., U-M9, U-0.5% CSL+M and OUM1 (refer to
Table 2 for media formulations), and for strain BW21 with budAB
deletions, grown in OUM1 medium. Average productivities are
calculated for the initial 72 h. Ethanol yields are calculated
after 72 h. Maximal volumetric productivity occurs early in
fermentation, between 8 h and 24 h.
[0254] Importantly, as can be seen in FIG. 5, both ethanol yield
and productivity were consistently higher for strain BW21 grown in
OUM1 medium than for the parent P2 strain grown in any of the media
tested.
Example 7
Economical Production of Ethanol by Recombinant Bacteria Lacking
budAB Genes Grown in OUM1 Medium
[0255] As discussed above, the product yields and costs associated
with production materials such as bacterial culture media are
important factors in the economics of commodity chemicals such as
ethanol. K. oxytoca is an advantageous choice as an ethanologenic
microorganism because this bacterium has the native ability to use
urea as a nitrogen source. On an equivalent nitrogen basis, urea is
typically sold for about half the cost of ammonium salts. The use
of urea as a nitrogen source has further additional benefits.
Unlike the metabolism of ammonium salts, the metabolism of urea
does not contribute to the acidification of the media [41] and thus
reduces the amount of base required for pH control.
[0256] The new media described herein, designated OUM1, offers
further potential savings from the low concentrations of other
salts and corn steep liquor. On a weight basis, OUM1 medium
consists of 0.5% CSL, 0.06% urea, and 0.2% inorganic salts, plus
fermentable sugar. The low pH used in these fermentations is
particularly appropriate for lignocellulosic feedstocks because
fungal cellulases and xylanases typically exhibit optima around pH
5 [42].
[0257] As recognized herein, despite these advantages of low pH for
SSF, a disadvantage to conducting fermentation reactions at acidic
pH is that the pathway for butanediol (and acetoin) production in
the ethanologenic bacteria is activated by low pH [43], leading to
an increase in co-products and decline in ethanol yield. However,
as demonstrated herein, this problem can be successfully overcome
by constructing improved recombinant ethanologenic bacterial
strains having deletions in the two genes uniquely involved in this
pathway (budAB). As shown above, elimination of the butanediol
reaction products by deletion of these genes resulted in an
improved ethanologenic Klebsiella strain (exemplified by strain
BW21) that attained 12% higher ethanol yields than the parent
strain P2. Most significantly, ethanol production from glucose by
BW21 at pH 5.2 in OUM1 was essentially complete after 48 h and
exceeded that of the parent (strain P2) in LB medium. Thus this new
strain, together with the novel minimal medium optimized for
production of ethanol by this organism from inexpensive reagents,
provide a significant advance in the goal of generating affordable,
renewable energy sources from biomass.
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INCORPORATION BY REFERENCE
[0302] All patents, published patent applications and other
references disclosed herein are hereby expressly incorporated
herein in their entireties by reference.
EQUIVALENTS
[0303] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
8120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1gctgaatcgg gtcaacattt 20220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2tttcggtttg tccaggtagt 203780DNAKlebsiella oxytoca 3atgaaccatt
ctgttgaatg ctcttgtgaa gagagcctgt gtgaaactct acgaggattt 60tccgcgcaac
atcccgatag cgtcatctac cagacctctc tgatgagcgc gctgcttagc
120ggcgtttatg aaggtaatac gaccatcgcc gatttgctca cccacggtga
tttcggcctc 180gggaccttta atgaactgga cggcgagctg atcgcgttta
gcagtgaagt ttaccagctg 240cgcgccgacg gcagcgcccg caaagcccgt
atggaacagc gtacgccgtt cgcggtgatg 300acctggtttc agccgcagta
tcgcaaaacg ttcgataaac cggtcagccg cgaacagctg 360cacaacatca
tcgaccagca aatcccgtcg gacaatctgt tctgcgccct gcgtattaac
420ggccattttc gccacgccca tacccgcacg gtaccgcgcc agacgccgcc
ctaccgggcg 480atgaccgacg tactcgacga ccagccggtt tttcgcttca
accagcgcga gggggtcctg 540gttgggttca gaacgccgca gcatatgcag
ggcattaacg tggccggcta ccacgaacac 600ttcatcaccg atgaccgcca
gggcggcggc catctgctcg attatcagct cgaccacggc 660gtgctgacct
ttggcgagat ccacaaattg atgattgacc ttcctgccga tagcgccttc
720ctgcaggcgg atctgcatcc agacaatctt gatgccgcca ttcgctcagt
cgaaaactaa 7804359DNAKlebsiella oxytoca 4gtggataatc aacatcaacc
gcgccagtgg gcgcacggcg ccgacctcat cgtcagccag 60cttgaggccc agggagtacg
ccaggtgttc ggcattccgg gggccaaaat cgataaagtc 120ttcgattcgc
tgctcgactc ctccattcgc attatcccgg tgcgccacga agccaacgcc
180gcctttatgg ccgccgcggt tggccgcatc accggcaaag cgggcgtcgc
gctggtcacg 240tccggaccgg gctgctctaa cctgattacc gggatggcaa
cggccaatag cgagggcgac 300ccggtggtgg cgctgggcgg cgcggtaaaa
cgcgccgata aagccaaaca ggtccatca 3595433DNAKlebsiella oxytoca
5gctgaatcgg gtcaacattt atttaacctt tctgatattc gttgaacgag gaagtgggca
60atgaaccatt ctgttgaatg ctcttgtgaa gagagcctgt gtgaaactct acgaggattt
120tccgcgcaac atcccgatag cgtcatctac cagacctctc tgatgagcgc
gctgcttagg 180gtaccgagct cgaattcccg cgcccgatga attgatccga
agttcctatt ctctagaaag 240tataggaact tcgaattgtc gacaagctcc
ccggttggcc gcatcaccgg caaagcgggc 300gtcgcgctgg tcacgtccgg
accgggctgc tctaacctga ttaccgggat ggcaacggcc 360aatagcgagg
gcgacccggt ggtggcgctg ggcgcgcggt aaaacgcgcc gataaagcca
420aacaggtcca tca 4336259PRTKlebsiella oxytoca 6Met Asn His Ser Val
Glu Cys Ser Cys Glu Glu Ser Leu Cys Glu Thr 1 5 10 15 Leu Arg Gly
Phe Ser Ala Gln His Pro Asp Ser Val Ile Tyr Gln Thr 20 25 30 Ser
Leu Met Ser Ala Leu Leu Ser Gly Val Tyr Glu Gly Asn Thr Thr 35 40
45 Ile Ala Asp Leu Leu Thr His Gly Asp Phe Gly Leu Gly Thr Phe Asn
50 55 60 Glu Leu Asp Gly Glu Leu Ile Ala Phe Ser Ser Glu Val Tyr
Gln Leu 65 70 75 80Arg Ala Asp Gly Ser Ala Arg Lys Ala Arg Met Glu
Gln Arg Thr Pro 85 90 95 Phe Ala Val Met Thr Trp Phe Gln Pro Gln
Tyr Arg Lys Thr Phe Asp 100 105 110 Lys Pro Val Ser Arg Glu Gln Leu
His Asn Ile Ile Asp Gln Gln Ile 115 120 125 Pro Ser Asp Asn Leu Phe
Cys Ala Leu Arg Ile Asn Gly His Phe Arg 130 135 140 His Ala His Thr
Arg Thr Val Pro Arg Gln Thr Pro Pro Tyr Arg Ala 145 150 155 160Met
Thr Asp Val Leu Asp Asp Gln Pro Val Phe Arg Phe Asn Gln Arg 165 170
175 Glu Gly Val Leu Val Gly Phe Arg Thr Pro Gln His Met Gln Gly Ile
180 185 190 Asn Val Ala Gly Tyr His Glu His Phe Ile Thr Asp Asp Arg
Gln Gly 195 200 205 Gly Gly His Leu Leu Asp Tyr Gln Leu Asp His Gly
Val Leu Thr Phe 210 215 220 Gly Glu Ile His Lys Leu Met Ile Asp Leu
Pro Ala Asp Ser Ala Phe 225 230 235 240Leu Gln Ala Asp Leu His Pro
Asp Asn Leu Asp Ala Ala Ile Arg Ser 245 250 255 Val Glu Asn
7119PRTKlebsiella oxytoca 7Met Asp Asn Gln His Gln Pro Arg Gln Trp
Ala His Gly Ala Asp Leu 1 5 10 15 Ile Val Ser Gln Leu Glu Ala Gln
Gly Val Arg Gln Val Phe Gly Ile 20 25 30 Pro Gly Ala Lys Ile Asp
Lys Val Phe Asp Ser Leu Leu Asp Ser Ser 35 40 45 Ile Arg Ile Ile
Pro Val Arg His Glu Ala Asn Ala Ala Phe Met Ala 50 55 60 Ala Ala
Val Gly Arg Ile Thr Gly Lys Ala Gly Val Ala Leu Val Thr 65 70 75
80Ser Gly Pro Gly Cys Ser Asn Leu Ile Thr Gly Met Ala Thr Ala Asn
85 90 95 Ser Glu Gly Asp Pro Val Val Ala Leu Gly Gly Ala Val Lys
Arg Ala 100 105 110 Asp Lys Ala Lys Gln Val His 115
85844DNAKlebsiella oxytoca 8gctgaatcgg gtcaacattt atttaacctt
tctgatattc gttgaacgag gaagtgggca 60atgaaccatt ctgttgaatg ctcttgtgaa
gagagcctgt gtgaaactct acgaggattt 120tccgcgcaac atcccgatag
cgtcatctac cagacctctc tgatgagcgc gctgcttagg 180gtaccgagct
cgaattcccg cgcccgatga attgatccga agttcctatt ctctagaaag
240tataggaact tcgaattgtc gacaagctag cttgcatgcc tgcaggtcga
ctctagagga 300tccccgtact atcaacaggt tgaactgcgg atcttgcggc
ccgcgtcagc ttgatcaagg 360gttggtttgc gcattcacag ttctccgcaa
gaattgattg gctccaattc ttggagtggt 420gaatccgtta gcgaggtgcc
gccggcttcc attcaggtcg aggtggcccg gctccatgca 480ccgcgacgca
acgcggggag gcagacaagg tatagggcgg cgcctacaat ccatgccaac
540ccgttccatg tgctcgccga ggcggcataa atcgccgtga cgatcagcgg
tccagtgatc 600gaagttaggc tggtaagagc cgcgagcgat ccttgaagct
gtccctgatg gtcgtcatct 660acctgcctgg acagcatggc ctgcaacgcg
ggcatcccga tgccgccgga agcgagaaga 720atcataatgg ggaaggccat
ccagcctcgc gtcgcgaacg ccagcaagac gtagcccagc 780gcgtcggccg
ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
840ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
cgacaggccg 900atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
tgacccagag cgctgccggc 960acctgtccta cgagttgcat gataaacaag
acagtcataa gtgcggcgac gatagtcatg 1020ccccgcgccc accggaagga
gctgactggg ttgaaggctc tcaagggcat cggtcggcgc 1080tctcccttat
gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
1140accgccgccg caaggaatgg tgcatgtaag gagatggcgc ccaacagtcc
cccggccacg 1200gggcctgcca ccatacccac gccgaaacaa gcgctcatga
gcccgaagtg gcgagcccga 1260tcttccccat cggtgatgtc ggcgatatag
gcgccagcaa ccgcacctgt ggcgccggtg 1320atgccggcca cgatgcgtcc
ggcgtagaga atccacagga cgggtgtggt cgccatgatc 1380gcgtagtcga
tagtggctcc aagtagcgaa gcgagcagga ctgggcggcg gccaaagcgg
1440tcggacagtg ctccgagaac gggtgcgcat agaaattgca tcaacgcata
tagcgctagc 1500agcacgccat agtgactggc gatgctgtcg gaatggacga
tatcccgcaa gaggcccggc 1560agtaccggca taaccaagcc tatgcctaca
gcatccaggg tgacggtgcc gaggatgacg 1620atgagcgcat tgttagattt
catacacggt gcctgactgc gttagcaatt taactgtgat 1680aaactaccgc
attaaagcta gcttatcgat gataagctgt caaacatgag aattgacgcg
1740cgatgaattg atccgaagtt cctattctct agaaagtata ggaacttcga
attgtcgaca 1800agctccccgg ttggccgcat caccggcaaa gcgggcgtcg
cgctggtcac gtccggaccg 1860ggctgctcta acctgattac cgggatggca
acggccaata gcgagggcga cccggtggtg 1920gcgctgggcg cgcggtaaaa
cgcgccgata aagccaaaca ggtccatcaa agggcgaatt 1980ctgcagatat
ccatcacact ggcggccgct cgagcatgca tctagagggc ccaattcgcc
2040ctatagtgag tcgtattaca attcactggc cgtcgtttta caacgtcgtg
actgggaaaa 2100ccctggcgtt acccaactta atcgccttgc agcacatccc
cctttcgcca gctggcgtaa 2160tagcgaagag gcccgcaccg atcgcccttc
ccaacagttg cgcagcctga atggcgaatg 2220ggacgcgccc tgtagcggcg
cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 2280cgctacactt
gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
2340cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
ggttccgatt 2400tagagcttta cggcacctcg accgcaaaaa acttgatttg
ggtgatggtt cacgtagtgg 2460gccatcgccc tgatagacgg tttttcgccc
tttgacgttg gagtccacgt tctttaatag 2520tggactcttg ttccaaactg
gaacaacact caaccctatc gcggtctatt cttttgattt 2580ataagggatt
ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaattca
2640gggcgcaagg gctgctaaag gaaccggaac acgtagaaag ccagtccgca
gaaacggtgc 2700tgaccccgga tgaatgtcag ctactgggct atctggacaa
gggaaaacgc aagcgcaaag 2760agaaagcagg tagcttgcag tgggcttaca
tggcgatagc tagactgggc ggttttatgg 2820acagcaagcg aaccggaatt
gccagctggg gcgccctctg gtaaggttgg gaagccctgc 2880aaagtaaact
ggatggcttt cttgccgcca aggatctgat ggcgcagggg atcaagatct
2940gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
gcacgcaggt 3000tctccggccg cttgggtgga gaggctattc ggctatgact
gggcacaaca gacaatcggc 3060tgctctgatg ccgccgtgtt ccggctgtca
gcgcaggggc gcccggttct ttttgtcaag 3120accgacctgt ccggtgccct
gaatgaactg caggacgagg cagcgcggct atcgtggctg 3180gccacgacgg
gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
3240tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcgcct
tgctcctgcc 3300gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
atacgcttga tccggctacc 3360tgcccattcg accaccaagc gaaacatcgc
atcgagcgag cacgtactcg gatggaagcc 3420ggtcttgtcg atcaggatga
tctggacgaa gagcatcagg ggctcgcgcc agccgaactg 3480ttcgccaggc
tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgat ccatggcgat
3540gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcaa
cgactgtggc 3600cggctgggtg tggcggaccg ctatcaggac atagcgttgg
atacccgtga tattgctgaa 3660gagcttggcg gcgaatgggc tgaccgcttc
ctcgtgcttt acggtatcgc cgctcccgat 3720tcgcagcgca tcgccttcta
tcgccttctt gacgagttct tctgaattga aaaaggaaga 3780gtatgagtat
tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
3840ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
cagttgggtg 3900cacgagtggg ttacatcgaa ctggatctca acagcggtaa
gatccttgag agttttcgcc 3960ccgaagaacg ttttccaatg atgagcactt
ttaaagttct gctatgtcat acactattat 4020cccgtattga cgccgggcaa
gagcaactcg gtcgccgggc gcggtattct cagaatgact 4080tggttgagta
ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
4140tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
ctgacaacga 4200tcggaggacc gaaggagcta accgcttttt tgcacaacat
gggggatcat gtaactcgcc 4260ttgatcgttg ggaaccggag ctgaatgaag
ccataccaaa cgacgagagt gacaccacga 4320tgcctgtagc aatgccaaca
acgttgcgca aactattaac tggcgaacta cttactctag 4380cttcccggca
acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
4440gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
gagcgtgggt 4500ctcgcggtat cattgcagca ctggggccag atggtaagcc
ctcccgtatc gtagttatct 4560acacgacggg gagtcaggca actatggatg
aacgaaatag acagatcgct gagataggtg 4620cctcactgat taagcattgg
taactgtcag accaagttta ctcatatata ctttagattg 4680atttaaaact
tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
4740tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
gtagaaaaga 4800tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
ctgctgcttg caaacaaaaa 4860aaccaccgct accagcggtg gtttgtttgc
cggatcaaga gctaccaact ctttttccga 4920aggtaactgg cttcagcaga
gcgcagatac caaatactgt ccttctagtg tagccgtagt 4980taggccacca
cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
5040taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
tcaagacgat 5100agttaccgga taaggcgcag cggtcgggct gaacgggggg
ttcgtgcaca cagcccagct 5160tggagcgaac gacctacacc gaactgagat
acctacagcg tgagcattga gaaagcgcca 5220cgcttcccga agggagaaag
gcggacaggt atccggtaag cggcagggtc ggaacaggag 5280agcgcacgag
ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
5340gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
agcctatgga 5400aaaacgccag caacgcggcc tttttacggt tcctggcctt
ttgctggcct tttgctcaca 5460tgttctttcc tgcgttatcc cctgattctg
tggataaccg tattaccgcc tttgagtgag 5520ctgataccgc tcgccgcagc
cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 5580aagagcgccc
aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
5640ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
aatgtgagtt 5700agctcactca ttaggcaccc caggctttac actttatgct
tccggctcgt atgttgtgtg 5760gaattgtgag cggataacaa tttcacacag
gaaacagcta tgaccatgat tacgccaagc 5820ttggtaccga gctcggatcc acta
5844
* * * * *
References