U.S. patent application number 12/371745 was filed with the patent office on 2009-10-08 for process for the biological production of 1,3-propanediol with high titer.
This patent application is currently assigned to E. I. DU PONT DE NEMOURS AND COMPANY. Invention is credited to Mark Emptage, Sharon L. Haynie, Lisa A. Laffend.
Application Number | 20090253192 12/371745 |
Document ID | / |
Family ID | 22530731 |
Filed Date | 2009-10-08 |
United States Patent
Application |
20090253192 |
Kind Code |
A1 |
Emptage; Mark ; et
al. |
October 8, 2009 |
PROCESS FOR THE BIOLOGICAL PRODUCTION OF 1,3-PROPANEDIOL WITH HIGH
TITER
Abstract
The present invention provides an improved method for the
biological production of 1,3-propanediol from a fermentable carbon
source in a single microorganism. In one aspect of the present
invention, an improved process for the conversion of glucose to
1,3-propanediol is achieved by the use of an E. coli transformed
with the Klebsiella pneumoniae dha regulon genes dhaR, orfY, dhaT,
orfX, orfW, dhaB1, dhaB2, dhaB3, and orfZ, all these genes arranged
in the same genetic organization as found in wild type Klebsiella
pneumoniae. In another aspect of the present invention, an improved
process for the production of 1,3-propanediol from glucose using a
recombinant E. coli containing genes encoding a G3PDH, a G3P
phosphatase, a dehydratase, and a dehydratase reactivation factor
compared to an identical process using a recombinant E. coli
containing genes encoding a G3PDH, a G3P phosphatase, a
dehydratase, a dehydratase reactivation factor and a
1,3-propanediol oxidoreductase (dhaT). The dramatically improved
process relies on the presence in E. coli of a gene encoding a
non-specific catalytic activity sufficient to convert
3-hydroxypropionaldehyde to 1,3-propanediol.
Inventors: |
Emptage; Mark; (Wilmington,
DE) ; Haynie; Sharon L.; (Philadelphia, PA) ;
Laffend; Lisa A.; (Claymont, DE) |
Correspondence
Address: |
E I DU PONT DE NEMOURS AND COMPANY;LEGAL PATENT RECORDS CENTER
BARLEY MILL PLAZA 25/1122B, 4417 LANCASTER PIKE
WILMINGTON
DE
19805
US
|
Assignee: |
E. I. DU PONT DE NEMOURS AND
COMPANY
Wilmington
DE
|
Family ID: |
22530731 |
Appl. No.: |
12/371745 |
Filed: |
February 16, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11282497 |
Jan 16, 2006 |
7504250 |
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12371745 |
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10277249 |
Oct 21, 2002 |
7067300 |
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11282497 |
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09641652 |
Aug 18, 2000 |
6514733 |
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10277249 |
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60149534 |
Aug 18, 1999 |
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Current U.S.
Class: |
435/252.31 ;
435/252.3; 435/252.33; 435/252.34; 435/252.35 |
Current CPC
Class: |
C12N 15/52 20130101;
C12N 9/1205 20130101; C12P 7/18 20130101; C12N 9/0006 20130101;
C12N 9/90 20130101 |
Class at
Publication: |
435/252.31 ;
435/252.3; 435/252.34; 435/252.35; 435/252.33 |
International
Class: |
C12N 1/21 20060101
C12N001/21 |
Claims
1-6. (canceled)
7. A recombinant microorganism comprising genes encoding a G3PDH, a
G3P phosphtase, a dehydratase, and a dehydratase reactivation
factor wherein no functional dhaT gene encoding a 1,3 propanediol
oxidoreductase activity is present in the recombinant microorganism
and the microorganism is selected from the group consisting of
Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter,
Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces,
Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula,
Debaryomyces, Mucor, Torulopsis, Methylobacter, Salmonella,
Bacillus, Aerobacter, Streptomyces and Pseudomonas.
8-10. (canceled)
11. The recombinant microorganism of claim 7, further comprising a
set of endogenous genes, each having a mutation inactivating the
gene, the set consisting of: (a) a first gene encoding a
polypeptide having glycerol kinase activity; (b) a second gene
encoding a polypeptide having glycerol dehydrogenase activity; and
(c) a third gene encoding a polypeptide having triosephosphate
isomerase activity.
12-17. (canceled)
18. A recombinant E. coli comprising: a set of exogenous genes
consisting of: (i) at least one gene encoding a polypeptide having
a dehydratase activity; (ii) at least one gene encoding a
polypeptide having glycerol 3-phosphate dehydrogenase activity;
(iii) at least one gene encoding a polypeptide having glycerol
3-phosphatase activity; and (iv) at least one gene encoding a
dehydratase reactivation factor; wherein no functional dhaT gene
encoding a 1,3 propanediol oxidoreductase activity is present in
the recombinant E. coli.
19. A recombinant E. coli comprising: a set of exogenous genes
consisting of (i) at least one gene encoding a polypeptide having
glycerol 3-phosphate dehydrogenase activity; (ii) at least one gene
encoding a polypeptide having glycerol 3-phosphatase activity; and
(iii) at least one subset of genes encoding the gene products of
dhaR, orfY, orfX, orfW, dhaB1, dhaB2, dhaB3 and orfZ, wherein no
functional dhaT gene encoding a 1,3 propanediol oxidoreductase
activity is present in the recombinant E. coli.
20. The recombinant E. coli of claim 19 further comprising a set of
endogenous genes, each gene having a mutation inactivating the
gene, the set consisting of: (a) a gene encoding a polypeptide
having glycerol kinase activity; (b) a gene encoding a polypeptide
having glycerol dehydrogenase activity; and (c) a gene encoding a
polypeptide having triosephosphate isomerase activity.
21-29. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This is a divisional of allowed U.S. patent application Ser.
No. 11/282,497, filed Jan. 16, 2006, which is a divisional of U.S.
Pat. No. 7,067,300, which is a divisional of U.S. Pat. No.
6,514,733, the disclosures of which are hereby incorporated by
reference.
FIELD OF INVENTION
[0002] This invention comprises process for the bioconversion of a
fermentable carbon source to 1,3-propanediol by a single
microorganism.
BACKGROUND
[0003] 1,3-Propanediol is a monomer having potential utility in the
production of polyester fibers and the manufacture of polyurethanes
and cyclic compounds.
[0004] A variety of chemical routes to 1,3-propanediol are known.
For example ethylene oxide may be converted to 1,3-propanediol over
a catalyst in the presence of phosphine, water, carbon monoxide,
hydrogen and an acid, by the catalytic solution phase hydration of
acrolein followed by reduction, or from compounds such as glycerol,
reacted in the presence of carbon monoxide and hydrogen over
catalysts having atoms from group VIII of the periodic table.
Although it is possible to generate 1,3-propanediol by these
methods, they are expensive and generate waste streams containing
environmental pollutants.
[0005] It has been known for over a century that 1,3-propanediol
can be produced from the fermentation of glycerol. Bacterial
strains able to produce 1,3-propanediol have been found, for
example, in the groups Citrobacter, Clostridium, Enterobacter,
Ilyobacter, Klebsiella, Lactobacillus, and Pelobacter. In each case
studied, glycerol is converted to 1,3-propanediol in a two step,
enzyme catalyzed reaction sequence. In the first step, a
dehydratase catalyzes the conversion of glycerol to
3-hydroxypropionaldehyde (3-HPA) and water, Equation 1. In the
second step, 3-HPA is reduced to 1,3-propanediol by a
NAD.sup.+-linked oxidoreductase, Equation 2. The 1,3-propanediol is
not metabolized further and, as a result,
Glycerol.fwdarw.3-HPA+H.sub.2O (Equation 1)
3-HPA+NADH+H.sup.+.fwdarw.1,3-Propanediol+NAD.sup.+ (Equation
2)
accumulates in the media. The overall reaction consumes a reducing
equivalent in the form of a cofactor, reduced .beta.-nicotinamide
adenine dinucleotide (NADH), which is oxidized to nicotinamide
adenine dinucleotide (NAD.sup.+).
[0006] In Klebsiella pneumonia, Citrobacter freundii, and
Clostridium pasteurianum, the genes encoding the three structural
subunits of glycerol dehydratase (dhaB1-3 or dhaB, C and E) are
located adjacent to a gene encoding a specific 1,3-propanediol
oxidoreductase (dhaT) (see FIG. 1). Although the genetic
organization differs somewhat among these microorganisms, these
genes are clustered in a group which also comprises orfX and orfZ
(genes encoding a dehydratase reactivation factor for glycerol
dehydratase), as well as orfY and orfW (genes of unknown function).
The specific 1,3-propanediol oxidoreductases (dhaTs) of these
microorganisms are known to belong to the family of type III
alcohol dehydrogenases; each exhibits a conserved iron-binding
motif and has a preference for the NAD.sup.+/NADH linked
interconversion of 1,3-propandiol and 3-HPA. However, the
NAD.sup.+/NADH linked interconversion of 1,3-propandiol and 3-HPA
is also catalyzed by alcohol dehydrogenases which are not
specifically linked to dehydratase enzymes (for example, horse
liver and baker's yeast alcohol dehydrogenases (E.C. 1.1.1.1)),
albeit with less efficient kinetic parameters. Glycerol dehydratase
(E.C. 4.2.1.30) and diol[1,2-propanediol]dehydratase (E.C.
4.2.1.28) are related but distinct enzymes that are encoded by
distinct genes. Diol dehydratase genes from Klebsiella oxytoca and
Salmonella typhimurium are similar to glycerol dehydratase genes
and are clustered in a group which comprises genes analogous to
orfX and orfZ (Daniel et al., FEMS Microbiol. Rev. 22, 553 (1999);
Toraya and Mori, J. Biol. Chem. 274, 3372 (1999); GenBank
AF026270).
[0007] The production of 1,3-propanediol from glycerol is generally
performed under anaerobic conditions using glycerol as the sole
carbon source and in the absence of other exogenous reducing
equivalent acceptors. Under these conditions, in e.g., strains of
Citrobacter, Clostridium, and Klebsiella, a parallel pathway for
glycerol operates which first involves oxidation of glycerol to
dihydroxyacetone (DHA) by a NAD.sup.+- (or NADP.sup.+-) linked
glycerol dehydrogenase, Equation 3. The DHA, following
phosphorylation to dihydroxyacetone phosphate (DHAP) by a DHA
kinase (Equation 4),
Glycerol+NAD.sup.+.fwdarw.DHA+NADH+H.sup.+ (Equation 3)
DHA+ATP.fwdarw.DHAP+ADP (Equation 4)
becomes available for biosynthesis and for supporting ATP
generation via e.g., glycolysis. In contrast to the 1,3-propanediol
pathway, this pathway may provide carbon and energy to the cell and
produces rather than consumes NADH.
[0008] In Klebsiella pneumoniae and Citrobacter freundii, the genes
encoding the functionally linked activities of glycerol dehydratase
(dhaB), 1,3-propanediol oxidoreductase (dhaT), glycerol
dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK) are
encompassed by the dha regulon. The dha regulon, in Klebsiella
pneumoniae and Citrobacter freundii, also encompasses a gene
encoding a transcriptional activator protein (dhaR). The dha
regulons from Citrobacter and Klebsiella have been expressed in
Escherichia coli and have been shown to convert glycerol to
1,3-propanediol.
[0009] Neither the chemical nor biological methods described above
for the production of 1,3-propanediol are well suited for
industrial scale production since the chemical processes are energy
intensive and the biological processes are limited to relatively
low titer from the expensive starting material, glycerol. These
drawbacks could be overcome with a method requiring low energy
input and an inexpensive starting material such as carbohydrates or
sugars, or by increasing the metabolic efficiency of a glycerol
process. Development of either method will require the ability to
manipulate the genetic machinery responsible for the conversion of
sugars to glycerol and glycerol to 1,3-propanediol.
[0010] Biological processes for the preparation of glycerol are
known. The overwhelming majority of glycerol producers are yeasts
but some bacteria, other fungi and algae are also known. Both
bacteria and yeasts produce glycerol by converting glucose or other
carbohydrates through the fructose-1,6-bisphosphate pathway in
glycolysis or the Embden Meyerhof Parnas pathway, whereas, certain
algae convert dissolved carbon dioxide or bicarbonate in the
chloroplasts into the 3-carbon intermediates of the Calvin cycle.
In a series of steps, the 3-carbon intermediate, phosphoglyceric
acid, is converted to glyceraldehyde 3-phosphate which can be
readily interconverted to its keto isomer dihydroxyacetone
phosphate and ultimately to glycerol.
[0011] Specifically, the bacteria Bacillus licheniformis and
Lactobacillus lycopersica synthesize glycerol, and glycerol
production is found in the halotolerant algae Dunaliella sp. and
Asteromonas gracilis for protection against high external salt
concentrations. Similarly, various osmotolerant yeasts synthesize
glycerol as a protective measure. Most strains of Saccharomyces
produce some glycerol during alcoholic fermentation, and this can
be increased physiologically by the application of osmotic stress.
Earlier this century commercial glycerol production was achieved by
the use of Saccharomyces cultures to which "steering reagents" were
added such as sulfites or alkalis. Through the formation of an
inactive complex, the steering agents block or inhibit the
conversion of acetaldehyde to ethanol; thus, excess reducing
equivalents (NADH) are available to or "steered" towards DHAP for
reduction to produce glycerol. This method is limited by the
partial inhibition of yeast growth that is due to the sulfites.
This limitation can be partially overcome by the use of alkalis
that create excess NADH equivalents by a different mechanism. In
this practice, the alkalis initiated a Cannizarro
disproportionation to yield ethanol and acetic acid from two
equivalents of acetaldehyde.
[0012] The gene encoding glycerol-3-phosphate dehydrogenase (DAR1,
GPD1) has been cloned and sequenced from S. diastaticus (Wang et
al., J. Bact. 176, 7091-7095 (1994)). The DAR1 gene was cloned into
a shuttle vector and used to transform E. coli where expression
produced active enzyme. Wang et al. (supra) recognize that DAR1 is
regulated by the cellular osmotic environment but do not suggest
how the gene might be used to enhance 1,3-propanediol production in
a recombinant microorganism.
[0013] Other glycerol-3-phosphate dehydrogenase enzymes have been
isolated: for example, sn-glycerol-3-phosphate dehydrogenase has
been cloned and sequenced from Saccharomyces cerevisiae (Larason et
al., Mol. Microbiol. 10, 1101 (1993)) and Albertyn et al. (Mol.
Cell. Biol. 14, 4135 (1994)) teach the cloning of GPD1 encoding a
glycerol-3-phosphate dehydrogenase from Saccharomyces cerevisiae.
Like Wang et al. (supra), both Albertyn et al. and Larason et al.
recognize the osmo-sensitivity of the regulation of this gene but
do not suggest how the gene might be used in the production of
1,3-propanediol in a recombinant microorganism.
[0014] As with G3PDH, glycerol-3-phosphatase has been isolated from
Saccharomyces cerevisiae and the protein identified as being
encoded by the GPP1 and GPP2 genes (Norbeck et al., J. Biol. Chem.
271, 13875 (1996)). Like the genes encoding G3PDH, it appears that
GPP2 is osmosensitive.
[0015] Although a single microorganism conversion of fermentable
carbon source other than glycerol or dihydroxyacetone to
1,3-propanediol is desirable, it has been documented that there are
significant difficulties to overcome in such an endeavor. For
example, Gottschalk et al. (EP 373 230) teach that the growth of
most strains useful for the production of 1,3-propanediol,
including Citrobacter freundii, Clostridium autobutylicum,
Clostridium butylicum, and Klebsiella pneumoniae, is disturbed by
the presence of a hydrogen donor such as fructose or glucose.
Strains of Lactobacillus brevis and Lactobacillus buchner, which
produce 1,3-propanediol in co-fermentations of glycerol and
fructose or glucose, do not grow when glycerol is provided as the
sole carbon source, and, although it has been shown that resting
cells can metabolize glucose or fructose, they do not produce
1,3-propanediol (Veiga DA Cunha et al., J. Bacteriol., 174, 1013
(1992)). Similarly, it has been shown that a strain of Ilyobacter
polytropus, which produces 1,3-propanediol when glycerol and
acetate are provided, will not produce 1,3-propanediol from carbon
substrates other than glycerol, including fructose and glucose
(Steib et al., Arch. Microbiol. 140, 139 (1984)). Finally, Tong et
al. (Appl. Biochem. Biotech. 34, 149 (1992)) taught that
recombinant Escherichia coli transformed with the dha regulon
encoding glycerol dehydratase does not produce 1,3-propanediol from
either glucose or xylose in the absence of exogenous glycerol.
[0016] Attempts to improve the yield of 1,3-propanediol from
glycerol have been reported where co-substrates capable of
providing reducing equivalents, typically fermentable sugars, are
included in the process. Improvements in yield have been claimed
for resting cells of Citrobacter freundii and Klebsiella
pneumoniae. DSM 4270 co-fermenting glycerol and glucose (Gottschalk
et al., supra.; and Tran-Dinh et al., DE 3734 764); but not for
growing cells of Klebsiella pneumoniae. ATCC 25955 co-fermenting
glycerol and glucose, which produced no 1,3-propanediol (1-T. Tong,
Ph.D. Thesis, University of Wisconsin-Madison (1992)). Increased
yields have been reported for the cofermentation of glycerol and
glucose or fructose by a recombinant Escherichia coli; however, no
1,3-propanediol is produced in the absence of glycerol (Tong et
al., supra.). In these systems, single microorganisms use the
carbohydrate as a source of generating NADH while providing energy
and carbon for cell maintenance or growth. These disclosures
suggest that sugars do not enter the carbon stream that produces
1,3-propanediol.
[0017] Recently, however, the conversion of carbon substrates,
other than glycerol or dihydroxyacetone, to 1,3-propanediol by a
single microorganism that expresses a dehydratase enzyme has been
described (U.S. Pat. No. 5,686,276; WO 9821339; WO 9928480; and WO
9821341 (U.S. Pat. No. 6,013,494)). A specific deficiency in the
biological processes leading to the production of 1,3-propanediol
from either glycerol or glucose has been the low titer of the
product achieved via fermentation; thus, an energy-intensive
separation process to obtain 1,3-propanediol from the aqueous
fermentation broth is required. Fed batch or batch fermentations of
glycerol to 1,3-propanediol have led to final titers of 65 g/L by
Clostridium butyricum (Saint-Amans et al., Biotechnology Letters
16, 831 (1994)), 71 g/L by Clostridium butyricum mutants
(Abbad-Andaloussi et al., Appl. Environ. Microbiol. 61, 4413
(1995)), 61 g/L by Klebsiella pneumoniae (Homann et al., Appl.
Bicrobiol. Biotechnol. 33, 121 (1990)), and 35 g/L by Citrobacter
freundii (Homann et al., supra). Fermentations of glucose to
1,3-propanediol that exceed the titer obtained from glycerol
fermentations have not yet been disclosed.
[0018] The problem that remains to be solved is how to biologically
produce 1,3-propanediol, with high titer and by a single
microorganism, from an inexpensive carbon substrate such as glucose
or other sugars. The biological production of 1,3-propanediol
requires glycerol as a substrate for a two-step sequential reaction
in which a dehydratase enzyme (typically a coenzyme
B.sub.12-dependent dehydratase) converts glycerol to an
intermediate, 3-hydroxypropionaldehyde, which is then reduced to
1,3-propanediol by a NADH- (or NADPH) dependent oxidoreductase. The
complexity of the cofactor requirements necessitates the use of a
whole cell catalyst for an industrial process that utilizes this
reaction sequence for the production of 1,3-propanediol.
SUMMARY OF THE INVENTION
[0019] Applicants have solved the stated problem and the present
invention provides for bioconverting a fermentable carbon source
directly to 1,3-propanediol at significantly higher titer than
previously obtained and with the use of a single microorganism.
Glucose is used as a model substrate and E. coli is used as the
model host. In one aspect of this invention, recombinant E. coli
expressing a group of genes (comprising genes that encode a
dehydratase activity, a dehydratase reactivation factor, a
1,3-propanediol oxidoreductase (dhaT), a glycerol-3-phosphate
dehydrogenase, and a glycerol-3-phosphatase) convert glucose to
1,3-propanediol at titer that approaches that of glycerol to
1,3-propanediol fermentations.
[0020] In another aspect of this invention, the elimination of the
functional dhaT gene in this recombinant E. coli results in a
significantly higher titer of 1,3-propanediol from glucose. This
unexpected increase in titer results in improved economics, and
thus, an improved process for the production of 1,3-propanediol
from glucose.
[0021] Furthermore, the present invention may be generally applied
to include any carbon substrate that is readily converted to 1)
glycerol, 2) dihydroxyacetone, 3) C.sub.3 compounds at the
oxidation state of glycerol (e.g., glycerol 3-phosphate), or 4)
C.sub.3 compounds at the oxidation state of dihydroxyacetone (e.g.,
dihydroxyacetone phosphate or glyceraldehyde 3-phosphate). The
production of 1,3-propanediol in the dhaT minus strain requires a
non-specific catalytic activity that converts 3-HPA to
1,3-propanediol. Identification of the enzyme(s) and/or gene(s)
responsible for the non-specific catalytic activity that converts
3-HPA to 1,3-propanediol will lead to production of 1,3-propanediol
in a wide range of host microorganisms with substrates from a wide
range of carbon-containing substrates. It is also anticipated that
the use of this non-specific catalytic activity that converts 3-HPA
to 1,3-propanediol will lead to an improved process for the
production of 1,3-propanediol from glycerol or dihydroxyacetone, by
virtue of an improved titer and the resulting improved
economics.
[0022] This activity has been isolated from E. coli as a nucleic
acid fragment encoding a non-specific catalytic activity for the
conversion of 3-hydroxypropionaldehyde to 1,3-propanediol, as set
out in SEQ ID NO:58 or as selected from the group consisting of:
[0023] (a) an isolated nucleic acid fragment encoding all or a
substantial portion of the amino acid sequence of SEQ ID NO:57;
[0024] (b) an isolated nucleic acid fragment that is substantially
similar to an isolated nucleic acid fragment encoding all or a
substantial portion of the amino acid sequence of SEQ ID NO:57;
[0025] (c) an isolated nucleic acid fragment encoding a polypeptide
of at least 387 amino acids having at least 80% with the amino acid
sequence of SEQ ID NO:57; [0026] (d) an isolated nucleic acid
fragment that hybridizes with (a) under hybridization conditions of
0.1.times.SSC, 0.1% SDS, 65.degree. C. and washed with 2.times.SSC,
0.1% SDS followed by 0.1.times.SSC, 0.1% SDS; and [0027] (d) an
isolated nucleic acid fragment that is complementary to (a), (b),
(c), or (d). Alternatively, the nonspecific catalytic activity is
embodied in the polypeptide as set out in SEQ ID NO:57.
[0028] A chimeric gene may be constructed comprising the isolated
nucleic acid fragment described above operably linked to suitable
regulatory sequences. This chimeric gene can be used to transform
microorganisms selected from the group consisting of Citrobacter,
Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus,
Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces,
Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor,
Torulopsis, Methylobacter, Salmonella, Bacillus, Aerobacter,
Streptomyces, Escherichia, and Pseudomonas. E. coli is the
preferred host.
[0029] Accordingly, the present invention provides a recombinant
microorganism, useful for the production of 1,3-propanediol
comprising: (a) at least one gene encoding a polypeptide having
glycerol-3-phosphate dehydrogenase activity; (b) at least one gene
encoding a polypeptide having glycerol-3-phosphatase activity; (c)
at least one gene encoding a polypeptide having a dehydratase
activity; (d) at least one gene encoding a dehydratase reactivation
factor; (e) at least one endogenous gene encoding an non-specific
catalytic activity sufficient to convert 3-hydroxypropionaldehyde
to 1,3-propanediol, wherein no functional dhaT gene encoding a
1,3-propanediol oxidoreductase is present. The preferred embodiment
is a recombinant microorganism (preferably E. coli) where no dhaT
gene is present. Optionally, the recombinant microorganism may
comprise mutations (e.g., deletion mutations or point mutations) in
endogenous genes selected from the group consisting of: (a) a gene
encoding a polypeptide having glycerol kinase activity; (b) a gene
encoding a polypeptide having glycerol dehydrogenase activity; and
(c) gene encoding a polypeptide having triosephosphate isomerase
activity.
[0030] In another embodiment the invention includes a process for
the production of 1,3-propanediol comprising: (a) contacting, under
suitable conditions, a recombinant E. coli comprising a dha regulon
and lacking a functional dhaT gene encoding a 1,3-propanediol
oxidoreductase activity with at least one carbon source, wherein
the carbon source is selected from the group consisting of
monosaccharides, oligosaccharides, polysaccharides, and
single-carbon substrates; and (b) optionally recovering the
1,3-propanediol produced in (a).
[0031] The invention also provides a process for the production of
1,3-propanediol from a recombinant microorganism comprising: (a)
contacting the recombinant microorganism of the present invention
with at least one carbon source selected from the group consisting
of monosaccharides, oligosaccharides, polysaccharides, and
single-carbon substrates whereby 1,3-propanediol is produced; and
(b) optionally recovering the 1,3-propanediol produced in (a).
[0032] Similarly the invention intends to provide a process for the
production of 1,3-propanediol from a recombinant microorganism
comprising:
[0033] (a) contacting a recombinant microorganism with at least one
carbon source, said recombinant microorganism comprising: [0034]
(i) at least one gene encoding a polypeptide having a dehydratase
activity; [0035] (ii) at least one gene encoding a dehydratase
reactivation factor; [0036] (iii) at least one endogenous gene
encoding a non-specific catalytic activity sufficient to convert
3-hydroxypropionaldehyde to 1,3-propanediol; wherein no functional
dhaT gene encoding a 1,3-propanediol oxidoreductase is present;
[0037] said carbon source selected from the group consisting of
glycerol and dihydroxyacetone, wherein 1,3-propanediol is produced
and;
[0038] (b) optionally recovering the 1,3-propanediol produced in
(a).
[0039] Yet another aspect of the invention provides for the
co-feeding of the carbon substrate. In this embodiment for the
production of 1,3-propanediol, the steps are: (a) contacting a
recombinant E. coli with a first source of carbon and with a second
source of carbon, said recombinant E. coli comprising: (i) at least
one exogenous gene encoding a polypeptide having a dehydratase
activity; (ii) at least one exogenous gene encoding a dehydratase
reactivation factor; (iii) at least one exogenous gene encoding a
non-specific catalytic activity sufficient to convert
3-hydroxypropionaldehyde to 1,3-propanediol, wherein no functional
dhaT gene encoding a 1,3-propanediol oxidoreductase activity is
present in the recombinant E. coli and wherein said first carbon
source is selected from the group consisting of glycerol and
dihydroxyacetone, and said second carbon source is selected from
the group consisting of monosaccharides, oligosaccharides,
polysaccharides, and single-carbon substrates, and (b) the
1,3-propanediol produced in (a) is optionally recovered. The
co-feed may be sequential or simultaneous. The recombinant E. coli
used in a co-feeding embodiment may further comprise: (a) a set of
exogenous genes consisting of (i) at least one gene encoding a
polypeptide having glycerol-3-phosphate dehydrogenase activity;
(ii) at least one gene encoding a polypeptide having
glycerol-3-phosphatase activity; and (iii) at least one subset of
genes encoding the gene products of dhaR, orfY, orfX, orfW, dhaB1,
dhaB2, dhaB3 and orfZ, and (b) a set of endogenous genes, each gene
having a mutation inactivating the gene, the set consisting of: (i)
a gene encoding a polypeptide having glycerol kinase activity; (ii)
a gene encoding a polypeptide having glycerol dehydrogenase
activity; and (iii) a gene encoding a polypeptide having
triosephosphate isomerase activity.
[0040] Useful recombinant E. coli strains include recombinant E.
coli strain KLP23 comprising: (a) a set of two endogenous genes,
each gene having a mutation inactivating the gene, the set
consisting of: (i) a gene encoding a polypeptide having a glycerol
kinase activity; and (ii) a gene encoding a polypeptide having a
glycerol dehydrogenase activity; (b) at least one exogenous gene
encoding a polypeptide having glycerol-3-phosphate dehydrogenase
activity; (c) at least one exogenous gene encoding a polypeptide
having glycerol-3-phosphatase activity; and (d) a plasmid pKP32 and
a recombinant E. coli strain RJ8 comprising: (a) set of three
endogenous genes, each gene having a mutation inactivating the
gene, the set consisting of: (i) a gene encoding a polypeptide
having a glycerol kinase activity; (ii) a gene encoding a
polypeptide having a glycerol dehydrogenase activity; and (iii) a
gene encoding a polypeptide having a triosephosphate isomerase
activity.
[0041] Other useful embodiments include recombinant E. coli
comprising: (a) a set of exogenous genes consisting of: (i) at
least one gene encoding a polypeptide having a dehydratase
activity; (ii) at least one gene encoding a polypeptide having
glycerol-3-phosphate dehydrogenase activity; (iii) at least one
gene encoding a polypeptide having glycerol-3-phosphatase activity;
and (iv) at least one gene encoding a dehydratase reactivation
factor; and (b) at least one endogenous gene encoding a
non-specific catalytic activity to convert 3-hydroxypropionaldehyde
to 1,3-propanediol; wherein no functional dhaT gene encoding a
1,3-propanediol oxidoreductase activity is present in the
recombinant E. coli.
[0042] Another embodiment is a recombinant E. coli comprising: (a)
a set of exogenous genes consisting of (i) at least one gene
encoding a polypeptide having glycerol-3-phosphate dehydrogenase
activity; (ii) at least one gene encoding a polypeptide having
glycerol-3-phosphatase activity; and (iii) at least one subset of
genes encoding the gene products of dhaR, orfY, orfX, orfW, dhaB1,
dhaB2, dhaB3 and orfZ, and (b) at least one endogenous gene
encoding a non-specific catalytic activity to convert
3-hydroxypropionaldehyde to 1,3-propanediol, wherein no functional
dhaT gene encoding a 1,3-propanediol oxidoreductase activity is
present in the recombinant E. coli. This embodiment also includes a
process using a recombinant E. coli further comprising a set of
endogenous genes, each gene having a mutation inactivating the
gene, the set consisting of: (a) a gene encoding a polypeptide
having glycerol kinase activity; (b) a gene encoding a polypeptide
having glycerol dehydrogenase activity; and (c) a gene encoding a
polypeptide having triosephosphate isomerase activity.
[0043] This embodiment still further includes a process for the
bioproduction of 1,3-propanediol comprising: (a) contacting under
suitable conditions the immediately disclosed recombinant E. coli
with at least one carbon source selected from the group consisting
of monosaccharides, oligosaccharides, polysaccharides, and
single-carbon substrates whereby 1,3-propanediol is produced; and
(b) optionally recovering the 1,3-propanediol produced in (a).
[0044] And also includes a further process for the bioproduction of
1,3-propanediol comprising: (a) contacting the recombinant E. coli
of the immediately disclosed embodiments that further comprise: (i)
at least one exogenous gene encoding a polypeptide having a
dehydratase activity; (ii) at least one exogenous gene encoding a
dehydratase reactivation factor; (iii) at least one endogenous gene
encoding a non-specific catalytic activity to convert
3-hydroxy-propionaldehyde to 1,3-propanediol, with at least one
carbon source selected from the group consisting of glycerol and
dihydroxyacetone, and (b) optionally recovering the 1,3-propanediol
produced in (a).
BRIEF DESCRIPTION OF THE DRAWINGS, SEQUENCE DESCRIPTIONS, AND
BIOLOGICAL DEPOSITS
[0045] The invention can be more fully understood from the
following detailed description, Figures, the accompanying sequence
descriptions, and biological deposits that form parts of this
application.
[0046] FIG. 1 presents the gene organization within the sequence of
the dha regulon subclone pHK28-26.
[0047] FIG. 2 presents a graph of the extracellular soluble protein
(g/L) compared between two fermentations runs essentially as
described in Example 7 using a constant feed of vitamin B.sub.12.
In one case, solid lines, the strain used was KLP23/pAH48/pKP32. In
the other case, dashed lines, the strain used was
KLP23/pAH48/pDT29.
[0048] FIG. 3 presents a graph of the cell viability [(viable
cells/mL)/OD550] compared between two fermentations runs
essentially as described in Example 7 using a constant feed of
vitamin B.sub.12. In one case (solid lines), the strain used was
KLP23/pAH48/pKP32. In the other case (dashed lines), the strain
used was KLP23/pAH48/pDT29.
[0049] FIG. 4 presents a graph of the yield of glycerol from
glucose compared between two fermentations runs essentially as
described in Example 7, but in the absence of vitamin B.sub.12 or
coenzyme B.sub.12. In one case (solid lines), the strain used was
KLP23/pAH48/pKP32. In the other case (dashed lines), the strain
used was KLP23/pAH48/pDT29.
[0050] FIG. 5 is a flow diagram illustrating the metabolic
conversion of glucose to 1,3-propanediol.
[0051] FIG. 6 is a 2D-PAGE membrane blot with the soluble protein
fraction extracted from a band showing endogenous E. coli
oxidoreductase activity (non-specific catalytic activity) on a
native gel.
[0052] The 68 sequence descriptions and the sequence listing
attached hereto will comply with the rules governing nucleotide
and/or amino acid sequence disclosures in patent applications as
set forth in 37 C.F.R. .sctn.1.821-1.825 ("Requirements for Patent
Applications Containing Nucleotide Sequences and/or Amino Acid
Sequence Disclosures--the Sequence Rules") and will be consistent
with World Intellectual Property Organization (WIPO) Standard ST2.5
(1998) and the sequence listing requirements of the EPO and PCT
(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the
Administration Instructions). The Sequence Descriptions contain the
one letter code for nucleotide sequence characters and the three
letter codes for amino acids as defined in conformity with the
IUPAC-IYUB standards described in Nucleic Acids Res. 13, 3021-3030
(1985) and in the Biochemical Journal 219, 345-373 (1984) which are
herein incorporated by reference.
[0053] SEQ ID NO:1 contains the nucleotide sequence determined from
a 12.1 kb EcoRI-SalI fragment from pKP1 (cosmid containing DNA from
Klebsiella pneumoniae), subcloned into pIBI31 (IBI Biosystem, New
Haven, Conn.), and termed pHK28-26. Table 1 further details genes,
corresponding base pairs identified within SEQ ID NO:1, and
associated functionality. See also Example 1.
[0054] SEQ ID NO:57 contains the amino acid sequence determined for
YqhD.
[0055] SEQ ID NO:58 contains the nucleotide sequence determined for
yqhD.
[0056] Applicants have made the following biological deposits under
the terms of the Budapest Treaty on the International Recognition
of the Deposit of Micro-organisms for the Purposes of Patent
Procedure:
TABLE-US-00001 Int'l Depository Date of Depositor Identification
Reference Designation Deposit E. coli DH5.alpha.; transformed ATCC
69789 18 Apr. 1995 with plasmid pKP1comprising a portion of the
Klebsiella genome encoding the glycerol dehydratase enzyme E. coli
DH5.alpha. transformed with ATCC 69790 18 Apr. 1995 pKP4 comprising
a portion of Klebsiella genome encoding a diol dehydratase enzyme
E. coli MSP33.6 comprising a ATCC 98598 25 Nov. 1997 Deletion in
gldA E. coli RJF10m ATCC 98597 25 Nov. 1997 comprising a deletion
in glpK
[0057] The deposit(s) will be maintained in the indicated
international depository for at least 30 years and will be made
available to the public upon the grant of a patent disclosing it.
The availability of a deposit does not constitute a license to
practice the subject invention in derogation of patent rights
granted by government action.
[0058] As used herein, "ATCC" refers to the American Type Culture
Collection international depository located 10801 University Blvd.,
Manassas, Va. 20110-2209 U.S.A. The "ATCC No." is the accession
number to cultures on deposit with the ATCC.
DETAILED DESCRIPTION OF THE INVENTION
[0059] The present invention provides for an improved process for
bioconverting a fermentable carbon source directly to
1,3-propanediol using a single microorganism. The method is
characterized by improved titer, yield, and cell viability as well
as a decrease in cell lysis during fermentation.
[0060] The present invention is based, in part, upon the
observation that 1,3-propanediol fermentation processes comprising
1,3-propanediol oxidoreductase (dhaT) are characterized by high
levels of 3HPA and other aldehydes and ketones in the medium, which
is correlated to a decrease in cell viability. The present
invention is also based, in part, upon the unexpected finding that
the model host, E. coli, is capable of converting 3-HPA to
1,3-propanediol by an endogenous non-specific catalytic activity
capable of converting 3-hydroxypropionaldehyde to 1,3-propanediol.
The present invention is further based, in part, upon the
unexpected finding that an E. coli fermentation process comprising
this non-specific catalytic activity and lacking a functional dhaT
results in increased cell viability during fermentation and
provides for higher titers and/or yields of 1,3-propanediol than a
fermentation process comprising a functional dhaT.
[0061] In one aspect, glycerol is a model substrate, the host
microorganism has a mutation in wild-type dhaT such that there is
no 1,3-propanediol oxidoreductase activity and comprises a
non-specific catalytic activity sufficient to convert
3-hydroxypropionaldehyde to 1,3-propanediol. In another aspect,
glucose is a model substrate and recombinant E. coli is a model
host. In this aspect, E. coli comprises an endogenous non-specific
catalytic activity sufficient to convert 3-hydroxypropionaldehyde
to 1,3-propanediol. In one embodiment, the non-specific catalytic
activity is an alcohol dehydrogenase.
[0062] In one aspect, the present invention provides a recombinant
E. coli expressing a group of genes comprising (a) at least one
gene encoding a polypeptide having glycerol-3-phosphate
dehydrogenase activity; (b) at least one gene encoding a
polypeptide having glycerol-3-phosphatase activity; (c) at least
one gene encoding a polypeptide having a dehydratase activity; (d)
at least one gene encoding a dehydratase reactivation factor; and
(e) at least one endogenous gene encoding an non-specific catalytic
activity sufficient to convert 3-hydroxy-propionaldehyde to
1,3-propanediol; use of this microorganism converts glucose to
1,3-propanediol at a high titer. In another aspect of this
invention, the elimination of the functional dhaT gene in this
recombinant E. coli provides an unexpectedly higher titer of
1,3-propanediol from glucose than previously attained.
[0063] The present invention provides an improved method for the
biological production of 1,3-propanediol from a fermentable carbon
source in a single microorganism. In one aspect of the present
invention, an improved process for the conversion of glucose to
1,3-propanediol is achieved by the use of a recombinant
microorganism comprising a host E. coli transformed with the
Klebsiella pneumoniae dha regulon genes dhaR, orfY, dhaT, orfX,
orfW, dhaB1, dhaB2, dhaB3, and orfZ, all these genes arranged in
the same genetic organization as found in wild type Klebsiella
pneumoniae. The titer obtained for the fermentation process is
significantly higher than any titer previously reported for a
similar fermentation. This improvement relies on the use of the
plasmid pDT29 as described in Example 6 and Example 7.
[0064] In another aspect of the present invention, a further
improved process for the production of 1,3-propanediol from glucose
is achieved using a recombinant E. coli containing genes encoding a
G3PDH, a G3P phosphatase, a dehydratase, and a dehydratase
reactivation factor compared to a process using a recombinant E.
coli containing genes encoding a G3PDH, a G3P phosphatase, a
dehydratase, a dehydratase reactivation factor, and also a
functional dhaT. The dramatically improved process relies on an
endogenous gene encoding a non-specific catalytic activity,
expected to be an alcohol dehydrogenase, which is present in E.
coli.
[0065] The dramatic improvement in the process is evident as an
increase in 1,3-propanediol titer as illustrated in Examples 7 and
9. The improvement in the process is also evident as a decrease in
cell lysis as determined by the extracellular soluble protein
concentration in the fermentation broth. This aspect of the
invention is illustrated in FIG. 2. Additionally, the improvement
in the process is evident as prolonged cell viability over the
course of the fermentation. This aspect of the invention is
illustrated in FIG. 3. Furthermore, the improvement in the process
is also evident as an increase in yield. In E. coli expressing a
1,3-propanediol oxidoreductase (dhaT) (for example, E. coli KLP23
transformed with the plasmid pDT29), glycerol can be metabolized to
a product other than 3-HPA. In direct contrast, in E. coli not
expressing a 1,3-propanediol oxidoreductase (dhaT) (for example, E.
coli KLP23 transformed with the plasmid pKP32), glycerol is not
metabolized to a product other than 3-HPA. That this cryptic
pathway is attributable to the presence or absence of a functional
dhaT is demonstrated by the lower yield of glycerol from glucose as
illustrated in FIG. 4.
[0066] As used herein the following terms may be used for
interpretation of the claims and specification.
[0067] The terms "glycerol-3-phosphate dehydrogenase" and "G3PDH"
refer to a polypeptide responsible for an enzyme activity that
catalyzes the conversion of dihydroxyacetone phosphate (DHAP) to
glycerol-3-phosphate (G3P). In vivo G3PDH may be NADH; NADPH; or
FAD-dependent. When specifically referring to a cofactor specific
glycerol-3-phosphate dehydrogenase, the terms "NADH-dependent
glycerol-3-phosphate dehydrogenase", "NADPH-dependent
glycerol-3-phosphate dehydrogenase" and "FAD-dependent
glycerol-3-phosphate dehydrogenase" will be used. As it is
generally the case that NADH-dependent and NADPH-dependent
glycerol-3-phosphate dehydrogenases are able to use NADH and NADPH
interchangeably (for example by the gene encoded by gpsA), the
terms NADH-dependent and NADPH-dependent glycerol-3-phosphate
dehydrogenase will be used interchangeably. The NADH-dependent
enzyme (EC 1.1.1.8) is encoded, for example, by several genes
including GPD1 (GenBank Z74071.times.2), or GPD2 (GenBank
Z35169.times.1), or GPD3 (GenBank G984182), or DAR1 (GenBank
Z74071.times.2). The NADPH-dependent enzyme (EC 1.1.1.94) is
encoded by gpsA (GenBank U321643, (cds 197911-196892) G466746 and
L45246). The FAD-dependent enzyme (EC 1.1.99.5) is encoded by GUT2
(GenBank Z47047.times.23), or glpD (GenBank G147838), or glpABC
(GenBank M20938) (see WO 9928480 and references therein, which are
herein incorporated by reference).
[0068] The terms "glycerol-3-phosphatase",
"sn-glycerol-3-phosphatase", or "d,1-glycerol phosphatase", and
"G3P phosphatase" refer to a polypeptide responsible for an enzyme
activity that catalyzes the conversion of glycerol-3-phosphate and
water to glycerol and inorganic phosphate. G3P phosphatase is
encoded, for example, by GPP1 (GenBank Z47047.times.125), or GPP2
(GenBank U18813.times.11) (see WO 9928480 and references therein,
which are herein incorporated by reference).
[0069] The term "glycerol kinase" refers to a polypeptide
responsible for an enzyme activity that catalyzes the conversion of
glycerol and ATP to glycerol-3-phosphate and ADP. The high-energy
phosphate donor ATP may be replaced by physiological substitutes
(e.g., phosphoenolpyruvate). Glycerol kinase is encoded, for
example, by GUT1 (GenBank U11583.times.19) and glpK (GenBank
L19201) (see WO 9928480 and references therein, which are herein
incorporated by reference).
[0070] The term "glycerol dehydrogenase" refers to a polypeptide
responsible for an enzyme activity that catalyzes the conversion of
glycerol to dihydroxyacetone (E.C. 1.1.1.6) or glycerol to
glyceraldehyde (E.C. 1.1.1.72). A polypeptide responsible for an
enzyme activity that catalyzes the conversion of glycerol to
dihydroxyacetone is also referred to as a "dihydroxyacetone
reductase". Glycerol dehydrogenase may be dependent upon NADH (E.C.
1.1.1.6), NADPH (E.C. 1.1.1.72), or other cofactors (e.g., E.C.
1.1.99.22). A NADH-dependent glycerol dehydrogenase is encoded, for
example, by gldA (GenBank U00006) (see WO 9928480 and references
therein, which are herein incorporated by reference).
[0071] The term "dehydratase enzyme" or "dehydratase" will refer to
any enzyme activity that catalyzes the conversion of a glycerol
molecule to the product 3-hydroxypropionaldehyde. For the purposes
of the present invention the dehydratase enzymes include a glycerol
dehydratase (E.C. 4.2.1.30) and a diol dehydratase (E.C. 4.2.1.28)
having preferred substrates of glycerol and 1,2-propanediol,
respectively. Genes for dehydratase enzymes have been identified in
Klebsiella pneumoniae, Citrobacter freundii, Clostridium
pasteurianum, Salmonella typhimurium, and Klebsiella oxytoca. In
each case, the dehydratase is composed of three subunits: the large
or ".alpha." subunit, the medium or ".beta." subunit, and the small
or ".gamma." subunit. Due to the wide variation in gene
nomenclature used in the literature, a comparative chart is given
in Table 1 to facilitate identification. The genes are also
described in, for example, Daniel et al. (FEMS Microbiol. Rev. 22,
553 (1999)) and Toraya and Mori (J. Biol. Chem. 274, 3372 (1999)).
Referring to Table 1, genes encoding the large or ".alpha." subunit
of glycerol dehydratase include dhaB1, gldA and dhaB; genes
encoding the medium or ".beta." subunit include dhaB2, gldB, and
dhaC; genes encoding the small or ".gamma." subunit include dhaB3,
gldC, and dhaE. Also referring to Table 1, genes encoding the large
or ".alpha." subunit of diol dehydratase include pduC and pddA;
genes encoding the medium or ".beta." subunit include pduD and
pddB; genes encoding the small or ".gamma." subunit include pduE
and pddC.
TABLE-US-00002 TABLE 1 Comparative chart of gene names and GenBank
references for dehydratase and dehydratase linked functions. GENE
FUNCTION: 1,3-PD regulatory unknown reactivation dehydrogenase
unknown ORGANISM (GenBank Reference) gene base pairs gene base
pairs geme base pairs gene base pairs gene base pairs K. pneumoniae
(SEQ ID NO: 1) dhaR 2209-4134 orfW 4112-4642 orfX 4643-4996 dhaT
5017-6108 orfY 6202-6630 K. pneumoniae (U30903) orf2c 7116-7646
orf2b 6762-7115 dhaT 5578-6741 orf2a 5125-5556 K. pneumoniae
(U60992) gdrB C. freundii (U09771) dhaR 3746-5671 orfW 5649-6179
orfX 6180-6533 dhaT 6550-7713 orfY 7736-8164 C. pasteurianum
(AF051373) C. pasteurianum (AF006034) orfW 210-731 orfX 1-196 dhaT
1232-2389 orfY 746-1177 S. typhimurium (AF026270) pduH 8274-8645 K.
oxytoca (AF017781) ddrB 2063-2440 K. oxytoca (AF051373) GENE
FUNCTION: dehydratase, .alpha. dehydratase, .beta. dehydratase,
.gamma. reactivation ORGANISM (GenBank Reference) gene base pairs
gene base pairs gene base pairs gene base pairs K. pneumoniae (SEQ
ID NO: 1) dhaB1 7044-8711 dhaB2 8724-9308 dhaB3 9311-9736 orfZ
9749-11572 K. pneumoniae (U30903) dhaB1 3047-4714 dhaB2 2450-2890
dhaB3 2022-2447 dhaB4 186-2009 K. pneumoniae (U60992) gldA 121-1788
gldB 1801-2385 gldC 2388-2813 gdrA C. freundii (U09771) dhaB
8556-10223 dhaC 10235-10819 dhaE 10822-11250 orfZ 11261-13072 C.
pasteurianum (AF051373) dhaB 84-1748 dhaC 1779-2318 dhaE 2333-2773
orfZ 2790-4598 C. pasteurianum (AF006034) S. typhimurium (AF026270)
pduC 3557-5221 pduD 5232-5906 pduE 5921-6442 pduG 6452-8284 K.
oxytoca (AF017781) ddrA 241-2073 K. oxytoca (AF051373) pddA
121-1785 pddB 1796-2470 pddC 2485-3006
[0072] Glycerol and diol dehydratases are subject to
mechanism-based suicide inactivation by glycerol and some other
substrates (Daniel et al., FEMS Microbiol. Rev. 22, 553 (1999)).
The term "dehydratase reactivation factor" refers to those proteins
responsible for reactivating the dehydratase activity. The terms
"dehydratase reactivating activity", "reactivating the dehydratase
activity" or "regenerating the dehydratase activity" refers to the
phenomenon of converting a dehydratase not capable of catalysis of
a substrate to one capable of catalysis of a substrate or to the
phenomenon of inhibiting the inactivation of a dehydratase or the
phenomenon of extending the useful half-life of the dehydratase
enzyme in vivo. Two proteins have been identified as being involved
as the dehydratase reactivation factor (see WO 9821341 (U.S. Pat.
No. 6,013,494) and references therein, which are herein
incorporated by reference; Daniel et al., supra; Toraya and Mori,
J. Biol. Chem. 274, 3372 (1999); and Tobimatsu et al., J.
Bacteriol. 181, 4110 (1999)). Referring to Table 1, genes encoding
one of the proteins include orfZ, dhaB4, gdrA, pduG and ddrA. Also
referring to Table 1, genes encoding the second of the two proteins
include orfX, orf2b, gdrB, pduH and ddrB.
[0073] The terms "1,3-propanediol oxidoreductase", "1,3-propanediol
dehydrogenase" or "DhaT" refer to the polypeptide(s) responsible
for an enzyme activity that is capable of catalyzing the
interconversion of 3-HPA and 1,3-propanediol provided the gene(s)
encoding such activity is found to be physically or
transcriptionally linked to a dehydratase enzyme in its natural
(i.e., wild type) setting; for example, the gene is found within a
dha regulon as is the case with dhaT from Klebsiella pneumonia.
Referring to Table 1, genes encoding a 1,3-propanediol
oxidoreductase include dhaT from Klebsiella pneumoniae, Citrobacter
freundii, and Clostridium pasteurianum. Each of these genes encode
a polypeptide belonging to the family of type III alcohol
dehydrogenases, exhibits a conserved iron-binding motif, and has a
preference for the NAD.sup.+/NADH linked interconversion of 3-HPA
and 1,3-propanediol (Johnson and Lin, J. Bacteriol. 169, 2050
(1987); Daniel et al., J. Bacteriol. 177, 2151 (1995); and Leurs et
al., FEMS Microbiol. Lett. 154, 337 (1997)). Enzymes with similar
physical properties have been isolated from Lactobacillus brevis
and Lactobacillus buchneri (Veiga da Dunha and Foster, Appl.
Environ. Microbiol. 58, 2005 (1992)).
[0074] The term "dha regulon" refers to a set of associated genes
or open reading frames encoding various biological activities,
including but not limited to a dehydratase activity, a reactivation
activity, and a 1,3-propanediol oxidoreductase. Typically a dha
regulon comprises the open reading frames dhaR, orfY, dhaT, orfX,
orfW, dhaB1, dhaB2, dhaB3, and orfZ as described herein.
[0075] The term "non-specific catalytic activity" refers to the
polypeptide(s) responsible for an enzyme activity that is
sufficient to catalyze the interconversion of 3-HPA and
1,3-propanediol and specifically excludes 1,3-propanediol
oxidoreductase(s). Typically these enzymes are alcohol
dehydrogenases. Such enzymes may utilize cofactors other than
NAD.sup.+/NADH, including but not limited to flavins such as FAD or
FMN. A gene(s) for a non-specific alcohol dehydrogenase(s) is
found, for example, to be endogenously encoded and functionally
expressed within the microorganism E. coli KLP23.
[0076] The terms "function" or "enzyme function" refer to the
catalytic activity of an enzyme in altering the energy required to
perform a specific chemical reaction. It is understood that such an
activity may apply to a reaction in equilibrium where the
production of either product or substrate may be accomplished under
suitable conditions.
[0077] The terms "polypeptide" and "protein" are used
interchangeably.
[0078] The terms "carbon substrate" and "carbon source" refer to a
carbon source capable of being metabolized by host microorganisms
of the present invention and particularly carbon sources selected
from the group consisting of monosaccharides, oligosaccharides,
polysaccharides, and one-carbon substrates or mixtures thereof.
[0079] The terms "host cell" or "host microorganism" refer to a
microorganism capable of receiving foreign or heterologous genes
and of expressing those genes to produce an active gene
product.
[0080] The terms "foreign gene", "foreign DNA", "heterologous gene"
and "heterologous DNA" refer to genetic material native to one
organism that has been placed within a host microorganism by
various means. The gene of interest may be a naturally occurring
gene, a mutated gene, or a synthetic gene.
[0081] The terms "transformation" and "transfection" refer to the
acquisition of new genes in a cell after the incorporation of
nucleic acid. The acquired genes may be integrated into chromosomal
DNA or introduced as extrachromosomal replicating sequences. The
term "transformant" refers to the product of a transformation.
[0082] The term "genetically altered" refers to the process of
changing hereditary material by transformation or mutation.
[0083] The terms "recombinant microorganism" and "transformed host"
refer to any microorganism having been transformed with
heterologous or foreign genes or extra copies of homologous genes.
The recombinant microorganisms of the present invention express
foreign genes encoding glycerol-3-phosphate dehydrogenase (GPD1),
glycerol-3-phosphatase (GPP2), glycerol dehydratase (dhaB1, dhaB2
and dhaB3), dehydratase reactivation factor (orfZ and orfX), and
optionally 1,3-propanediol oxidoreductase (dhaT) for the production
of 1,3-propanediol from suitable carbon substrates. A preferred
embodiment is an E. coli transformed with these genes but lacking a
functional dhaT. A host microorganism, other than E. coli, may also
be transformed to contain the disclosed genes and the gene for the
non-specific catalytic activity for the interconversion of 3-HPA
and 1,3-propanediol, specifically excluding 1,3-propanediol
oxidoreductase(s) (dhaT).
[0084] "Gene" refers to a nucleic acid fragment that expresses a
specific protein, including regulatory sequences preceding (5'
non-coding) and following (3' non-coding) the coding region. The
terms "native" and "wild-type" refer to a gene as found in nature
with its own regulatory sequences.
[0085] The terms "encoding" and "coding" refer to the process by
which a gene, through the mechanisms of transcription and
translation, produces an amino acid sequence. It is understood that
the process of encoding a specific amino acid sequence includes DNA
sequences that may involve base changes that do not cause a change
in the encoded amino acid, or which involve base changes which may
alter one or more amino acids, but do not affect the functional
properties of the protein encoded by the DNA sequence. It is
therefore understood that the invention encompasses more than the
specific exemplary sequences.
[0086] The term "isolated" refers to a protein or DNA sequence that
is removed from at least one component with which it is naturally
associated.
[0087] An "isolated nucleic acid molecule" is a polymer of RNA or
DNA that is single- or double-stranded, optionally containing
synthetic, non-natural or altered nucleotide bases. An isolated
nucleic acid molecule in the form of a polymer of DNA may be
comprised of one or more segments of cDNA, genomic DNA or synthetic
DNA.
[0088] "Substantially similar" refers to nucleic acid molecules
wherein changes in one or more nucleotide bases result in
substitution of one or more amino acids, but do not affect the
functional properties of the protein encoded by the DNA sequence.
"Substantially similar" also refers to nucleic acid molecules
wherein changes in one or more nucleotide bases do not affect the
ability of the nucleic acid molecule to mediate alteration of gene
expression by antisense or co-suppression technology.
"Substantially similar" also refers to modifications of the nucleic
acid molecules of the instant invention (such as deletion or
insertion of one or more nucleotide bases) that do not
substantially affect the functional properties of the resulting
transcript vis-a-vis the ability to mediate alteration of gene
expression by antisense or co-suppression technology or alteration
of the functional properties of the resulting protein molecule. The
invention encompasses more than the specific exemplary
sequences.
[0089] For example, it is well known in the art that alterations in
a gene which result in the production of a chemically equivalent
amino acid at a given site, but do not effect the functional
properties of the encoded protein are common. For the purposes of
the present invention substitutions are defined as exchanges within
one of the following five groups: [0090] 1. Small aliphatic,
nonpolar or slightly polar residues: Ala, Ser, Thr (Pro, Gly);
[0091] 2. Polar, negatively charged residues and their amides: Asp,
Asn, Glu, Gln; [0092] 3. Polar, positively charged residues: His,
Arg, Lys; [0093] 4. Large aliphatic, nonpolar residues: Met, Leu,
Ile, Val (Cys); and [0094] 5. Large aromatic residues: Phe, Tyr,
Trp.
[0095] Thus, a codon for the amino acid alanine, a hydrophobic
amino acid, may be substituted by a codon encoding another less
hydrophobic residue (such as glycine) or a more hydrophobic residue
(such as valine, leucine, or isoleucine). Similarly, changes which
result in substitution of one negatively charged residue for
another (such as aspartic acid for glutamic acid) or one positively
charged residue for another (such as lysine for arginine) can also
be expected to produce a functionally equivalent product.
[0096] In many cases, nucleotide changes which result in alteration
of the N-terminal and C-terminal portions of the protein molecule
would also not be expected to alter the activity of the
protein.
[0097] Each of the proposed modifications is well within the
routine skill in the art, as is determination of retention of
biological activity of the encoded products. Moreover, the skilled
artisan recognizes that substantially similar sequences encompassed
by this invention are also defined by their ability to hybridize,
under stringent conditions (0.1.times.SSC, 0.1% SDS, 65.degree. C.
and washed with 2.times.SSC, 0.1% SDS followed by 0.1.times.SSC,
0.1% SDS), with the sequences exemplified herein. Preferred
substantially similar nucleic acid fragments of the instant
invention are those nucleic acid fragments whose DNA sequences are
at least 80% identical to the DNA sequence of the nucleic acid
fragments reported herein. More preferred nucleic acid fragments
are at least 90% identical to the DNA sequence of the nucleic acid
fragments reported herein. Most preferred are nucleic acid
fragments that are at least 95% identical to the DNA sequence of
the nucleic acid fragments reported herein.
[0098] A nucleic acid fragment is "hybridizable" to another nucleic
acid fragment, such as a cDNA, genomic DNA, or RNA, when a single
stranded form of the nucleic acid fragment can anneal to the other
nucleic acid fragment under the appropriate conditions of
temperature and solution ionic strength. Hybridization and washing
conditions are well known and exemplified in Sambrook, J., Fritsch,
E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual,
Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor (1989), particularly Chapter 11 and Table 11.1 therein
(entirely incorporated herein by reference). The conditions of
temperature and ionic strength determine the "stringency" of the
hybridization. For preliminary screening for homologous nucleic
acids, low stringency hybridization conditions, corresponding to a
Tm of 55.degree., can be used, e.g., 5.times.SSC, 0.1% SDS, 0.25%
milk, and no formamide; or 30% formamide, 5.times.SSC, 0.5% SDS.
Moderate stringency hybridization conditions correspond to a higher
Tm, e.g., 40% formamide, with 5.times. or 6.times.SSC.
Hybridization requires that the two nucleic acids contain
complementary sequences, although depending on the stringency of
the hybridization, mismatches between bases are possible. The
appropriate stringency for hybridizing nucleic acids depends on the
length of the nucleic acids and the degree of complementation,
variables well known in the art. The greater the degree of
similarity or homology between two nucleotide sequences, the
greater the value of Tm for hybrids of nucleic acids having those
sequences. The relative stability (corresponding to higher Tm) of
nucleic acid hybridization decreases in the following order:
RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100
nucleotides in length, equations for calculating Tm have been
derived (see Sambrook et al., supra, 9.50-9.51). For hybridization
with shorter nucleic acids, i.e., oligonucleotides, the position of
mismatches becomes more important, and the length of the
oligonucleotide determines its specificity (see Sambrook et al.,
supra, 11.7-11.8). In one embodiment the length for a hybridizable
nucleic acid is at least about 10 nucleotides. Preferable a minimum
length for a hybridizable nucleic acid is at least about 15
nucleotides; more preferably at least about 20 nucleotides; and
most preferably the length is at least 30 nucleotides. Furthermore,
the skilled artisan will recognize that the temperature and wash
solution salt concentration may be adjusted as necessary according
to factors such as length of the probe.
[0099] A "substantial portion" refers to an amino acid or
nucleotide sequence which comprises enough of the amino acid
sequence of a polypeptide or the nucleotide sequence of a gene to
afford putative identification of that polypeptide or gene, either
by manual evaluation of the sequence by one skilled in the art, or
by computer-automated sequence comparison and identification using
algorithms such as BLAST (Basic Local Alignment Search Tool;
Altschul et al., J. Mol. Biol. 215:403-410 (1993); see also
www.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or more
contiguous amino acids or thirty or more nucleotides is necessary
in order to putatively identify a polypeptide or nucleic acid
sequence as homologous to a known protein or gene. Moreover, with
respect to nucleotide sequences, gene-specific oligonucleotide
probes comprising 20-30 contiguous nucleotides may be used in
sequence-dependent methods of gene identification (e.g., Southern
hybridization) and isolation (e.g., in situ hybridization of
bacterial colonies or bacteriophage plaques). In addition, short
oligonucleotides of 12-15 bases may be used as amplification
primers in PCR in order to obtain a particular nucleic acid
molecule comprising the primers. Accordingly, a "substantial
portion" of a nucleotide sequence comprises enough of the sequence
to afford specific identification and/or isolation of a nucleic
acid molecule comprising the sequence. The instant specification
teaches partial or complete amino acid and nucleotide sequences
encoding one or more particular proteins. The skilled artisan,
having the benefit of the sequences as reported herein, may now use
all or a substantial portion of the disclosed sequences for the
purpose known to those skilled in the art. Accordingly, the instant
invention comprises the complete sequences as reported in the
accompanying Sequence Listing, as well as substantial portions of
those sequences as defined above.
[0100] The term "complementary" describes the relationship between
nucleotide bases that are capable to hybridizing to one another.
For example, with respect to DNA, adenosine is complementary to
thymine and cytosine is complementary to guanine. Accordingly, the
instant invention also includes isolated nucleic acid molecules
that are complementary to the complete sequences as reported in the
accompanying Sequence Listing as well as those substantially
similar nucleic acid sequences.
[0101] The term "percent identity", as known in the art, is a
relationship between two or more polypeptide sequences or two or
more polynucleotide sequences, as determined by comparing the
sequences. In the art, "identity" also means the degree of sequence
relatedness between polypeptide or polynucleotide sequences, as the
case may be, as determined by the match between strings of such
sequences. "Identity" and "similarity" can be readily calculated by
known methods, including but not limited to those described in:
Computational Molecular Biology; Lesk, A. M., Ed.; Oxford
University Press: New York, 1988; Biocomputing: Informatics and
Genome Projects; Smith, D. W., Ed.; Academic Press: New York, 1993;
Computer Analysis of Sequence Data, Part I; Griffin, A. M. and
Griffin, H. G., Eds.; Humana Press: New Jersey, 1994; Sequence
Analysis in Molecular Biology; von Heinje, G., Ed.; Academic Press:
New York, 1987; and Sequence Analysis Primer; Gribskov, M. and
Devereux, J., Eds.; Stockton Press: New York, 1991. Preferred
methods to determine identity are designed to give the largest
match between the sequences tested.
[0102] Methods to determine identity and similarity are codified in
publicly available computer programs. Preferred computer program
methods to determine identity and similarity between two sequences
include, but are not limited to, the GCG Pileup program found in
the GCG program package, using the Needleman and Wunsch algorithm
with their standard default values of gap creation penalty=12 and
gap extension penalty=4 (Devereux et al., Nucleic Acids Res.
12:387-395 (1984)), BLASTP, BLASTN, and FASTA (Pearson et al.,
Proc. Natl. Acad. Sci. USA 85:2444-2448 (1988). The BLASTX program
is publicly available from NCBI and other sources (BLAST Manual,
Altschul et al., Natl. Cent. Biotechnol. Inf., Natl. Library Med.
(NCBI NLM) NIH, Bethesda, Md. 20894; Altschul et al., J. Mol. Biol.
215:403-410 (1990); Altschul et al., "Gapped BLAST and PSI-BLAST: a
new generation of protein database search programs", Nucleic Acids
Res. 25:3389-3402 (1997)). Another preferred method to determine
percent identity, is by the method of DNASTAR protein alignment
protocol using the Jotun-Hein algorithm (Hein et al., Methods
Enzymol. 183:626-645 (1990)). Default parameters for the Jotun-Hein
method for alignments are: for multiple alignments, gap penalty=11,
gap length penalty=3; for pairwise alignments ktuple=6. As an
illustration, by a polynucleotide having a nucleotide sequence
having at least, for example, 95% "identity" to a reference
nucleotide sequence it is intended that the nucleotide sequence of
the polynucleotide is identical to the reference sequence except
that the polynucleotide sequence may include up to five point
mutations per each 100 nucleotides of the reference nucleotide
sequence. In other words, to obtain a polynucleotide having a
nucleotide sequence at least 95% identical to a reference
nucleotide sequence, up to 5% of the nucleotides in the reference
sequence may be deleted or substituted with another nucleotide, or
a number of nucleotides up to 5% of the total nucleotides in the
reference sequence may be inserted into the reference sequence.
These mutations of the reference sequence may occur at the 5' or 3'
terminal positions of the reference nucleotide sequence or anywhere
between those terminal positions, interspersed either individually
among nucleotides in the reference sequence or in one or more
contiguous groups within the reference sequence. Analogously, by a
polypeptide having an amino acid sequence having at least, for
example, 95% identity to a reference amino acid sequence is
intended that the amino acid sequence of the polypeptide is
identical to the reference sequence except that the polypeptide
sequence may include up to five amino acid alterations per each 100
amino acids of the reference amino acid. In other words, to obtain
a polypeptide having an amino acid sequence at least 95% identical
to a reference amino acid sequence, up to 5% of the amino acid
residues in the reference sequence may be deleted or substituted
with another amino acid, or a number of amino acids up to 5% of the
total amino acid residues in the reference sequence may be inserted
into the reference sequence. These alterations of the reference
sequence may occur at the amino or carboxy terminal positions of
the reference amino acid sequence or anywhere between those
terminal positions, interspersed either individually among residues
in the reference sequence or in one or more contiguous groups
within the reference sequence.
[0103] The term "homologous" refers to a protein or polypeptide
native or naturally occurring in a given host cell. The invention
includes microorganisms producing homologous proteins via
recombinant DNA technology.
[0104] The term "percent homology" refers to the extent of amino
acid sequence identity between polypeptides. When a first amino
acid sequence is identical to a second amino acid sequence, then
the first and second amino acid sequences exhibit 100% homology.
The homology between any two polypeptides is a direct function of
the total number of matching amino acids at a given position in
either sequence, e.g., if half of the total number of amino acids
in either of the two sequences are the same then the two sequences
are said to exhibit 50% homology.
[0105] "Codon degeneracy" refers to divergence in the genetic code
permitting variation of the nucleotide sequence without effecting
the amino acid sequence of an encoded polypeptide. Accordingly, the
instant invention relates to any nucleic acid molecule that encodes
all or a substantial portion of the amino acid sequence as set
forth in SEQ ID NO:57. The skilled artisan is well aware of the
"codon-bias" exhibited by a specific host cell in usage of
nucleotide codons to specify a given amino acid. Therefore, when
synthesizing a gene for improved expression in a host cell, it is
desirable to design the gene such that its frequency of codon usage
approaches the frequency of preferred codon usage of the host
cell.
[0106] Modifications to the sequence, such as deletions,
insertions, or substitutions in the sequence which produce silent
changes that do not substantially affect the functional properties
of the resulting protein molecule are also contemplated. For
example, alteration in the gene sequence which reflect the
degeneracy of the genetic code, or which result in the production
of a chemically equivalent amino acid at a given site, are
contemplated. Thus, a codon for the amino acid alanine, a
hydrophobic amino acid, may be substituted by a codon encoding
another less hydrophobic residue, such as glycine, or a more
hydrophobic residue, such as valine, leucine, or isoleucine.
Similarly, changes which result in substitution of one negatively
charged residue for another, such as aspartic acid for glutamic
acid, or one positively charged residue for another, such as lysine
for arginine, can also be expected to produce a biologically
equivalent product. Nucleotide changes which result in alteration
of the N-terminal and C-terminal portions of the protein molecule
would also not be expected to alter the activity of the protein. In
some cases, it may in fact be desirable to make mutants of the
sequence in order to study the effect of alteration on the
biological activity of the protein. Each of the proposed
modifications is well within the routine skill in the art, as is
determination of retention of biological activity in the encoded
products. Moreover, the skilled artisan recognizes that sequences
encompassed by this invention are also defined by their ability to
hybridize, under stringent conditions (0.1.times.SSC, 0.1% SDS,
65.degree. C.), with the sequences exemplified herein.
[0107] The term "expression" refers to the transcription and
translation to gene product from a gene coding for the sequence of
the gene product.
[0108] The terms "plasmid", "vector", and "cassette" refer to an
extra chromosomal element often carrying genes which are not part
of the central metabolism of the cell, and usually in the form of
circular double-stranded DNA molecules. Such elements may be
autonomously replicating sequences, genome integrating sequences,
phage or nucleotide sequences, linear or circular, of a single- or
double-stranded DNA or RNA, derived from any source, in which a
number of nucleotide sequences have been joined or recombined into
a unique construction which is capable of introducing a promoter
fragment and DNA sequence for a selected gene product along with
appropriate 3' untranslated sequence into a cell. "Transformation
cassette" refers to a specific vector containing a foreign gene and
having elements in addition to the foreign gene that facilitates
transformation of a particular host cell. "Expression cassette"
refers to a specific vector containing a foreign gene and having
elements in addition to the foreign gene that allow for enhanced
expression of that gene in a foreign host.
Construction of Recombinant Organisms
[0109] Recombinant organisms containing the necessary genes that
will encode the enzymatic pathway for the conversion of a carbon
substrate to 1,3-propanediol may be constructed using techniques
well known in the art. Genes encoding glycerol-3-phosphate
dehydrogenase (GPD1), glycerol-3-phosphatase (GPP2), glycerol
dehydratase (dhaB1, dhaB2, and dhaB3), dehydratase reactivation
factor (orfZ and orfX) and 1,3-propanediol oxidoreductase (dhaT)
were isolated from a native host such as Klebsiella or
Saccharomyces and used to transform host strains such as E. coli
DH5.alpha., ECL707, AA200, or KLP23.
Isolation of Genes
[0110] Methods of obtaining desired genes from a bacterial genome
are common and well known in the art of molecular biology. For
example, if the sequence of the gene is known, suitable genomic
libraries may be created by restriction endonuclease digestion and
may be screened with probes complementary to the desired gene
sequence. Once the sequence is isolated, the DNA may be amplified
using standard primer directed amplification methods such as
polymerase chain reaction (PCR) (U.S. Pat. No. 4,683,202) to obtain
amounts of DNA suitable for transformation using appropriate
vectors.
[0111] Alternatively, cosmid libraries may be created where large
segments of genomic DNA (35-45 kb) may be packaged into vectors and
used to transform appropriate hosts. Cosmid vectors are unique in
being able to accommodate large quantities of DNA. Generally cosmid
vectors have at least one copy of the cos DNA sequence which is
needed for packaging and subsequent circularization of the foreign
DNA. In addition to the cos sequence these vectors will also
contain an origin of replication such as ColE1 and drug resistance
markers such as a gene resistant to ampicillin or neomycin. Methods
of using cosmid vectors for the transformation of suitable
bacterial hosts are well described in Sambrook, J. et al.,
Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold
Spring Harbor Laboratory Press, herein incorporated by
reference.
[0112] Typically to clone cosmids, foreign DNA is isolated and
ligated, using the appropriate restriction endonucleases, adjacent
to the cos region of the cosmid vector. Cosmid vectors containing
the linearized foreign DNA are then reacted with a DNA packaging
vehicle such as bacteriophage. During the packaging process the cos
sites are cleaved and the foreign DNA is packaged into the head
portion of the bacterial viral particle. These particles are then
used to transfect suitable host cells such as E. coli. Once
injected into the cell, the foreign DNA circularizes under the
influence of the cos sticky ends. In this manner large segments of
foreign DNA can be introduced and expressed in recombinant host
cells.
Isolation and Cloning of Genes Encoding Glycerol Dehydratase
(dhaB1, dhaB2, and dhaB3) Dehydratase Reactivating Factors (orfZ
and orfX), and 1,3-Propanediol Dehydrogenase (dhaT)
[0113] Cosmid vectors and cosmid transformation methods were used
within the context of the present invention to clone large segments
of genomic DNA from bacterial genera known to possess genes capable
of processing glycerol to 1,3-propanediol. Specifically, genomic
DNA from K. pneumoniae was isolated by methods well known in the
art and digested with the restriction enzyme Sau3A for insertion
into a cosmid vector Supercos 1 and packaged using GigapackII
packaging extracts. Following construction of the vector E. coli
XL1-Blue MR cells were transformed with the cosmid DNA.
Transformants were screened for the ability to convert glycerol to
1,3-propanediol by growing the cells in the presence of glycerol
and analyzing the media for 1,3-propanediol formation.
[0114] Two of the 1,3-propanediol positive transformants were
analyzed and the cosmids were named pKP1 and pKP2. DNA sequencing
revealed extensive homology to the glycerol dehydratase gene from
C. freundii, demonstrating that these transformants contained DNA
encoding the glycerol dehydratase gene. Other 1,3-propanediol
positive transformants were analyzed and the cosmids were named
pKP4 and pKP5. DNA sequencing revealed that these cosmids carried
DNA encoding a diol dehydratase gene.
[0115] Although the instant invention utilizes the isolated genes
from within a Klebsiella cosmid, alternate sources of dehydratase
genes and dehydratase reactivation factor genes include, but are
not limited to, Citrobacter, Clostridia and Salmonella (see Table
1).
Genes Encoding G3PDH and G3P Phosphatase
[0116] The present invention provides genes suitable for the
expression of G3PDH and G3P phosphatase activities in a host
cell.
[0117] Genes encoding G3PDH are known. For example, GPD1 has been
isolated from Saccharomyces and has the base sequence given by SEQ
ID NO:53, encoding the amino acid sequence given in SEQ ID NO:54
(Wang et al., supra). Similarly, G3PDH activity has also been
isolated from Saccharomyces encoded by GPD2 (Eriksson et al., Mol.
Microbiol. 17, 95 (1995)).
[0118] For the purposes of the present invention it is contemplated
that any gene encoding a polypeptide responsible for NADH-dependent
G3PDH activity is suitable wherein that activity is capable of
catalyzing the conversion of dihydroxyacetone phosphate (DHAP) to
glycerol-3-phosphate (G3P). Further, it is contemplated that any
gene encoding the amino acid sequence of NADH-dependent G3PDH's
corresponding to the genes DAR1, GPD1, GPD2, GPD3, and gpsA will be
functional in the present invention wherein that amino acid
sequence may encompass amino acid substitutions, deletions or
additions that do not alter the function of the enzyme. The skilled
person will appreciate that genes encoding G3PDH isolated from
other sources will also be suitable for use in the present
invention. Genes encoding G3P phosphatase are known. For example,
GPP2 has been isolated from Saccharomyces cerevisiae and has the
base sequence given by SEQ ID NO:55, which encodes the amino acid
sequence given in SEQ ID NO:56 (Norbeck et al., J. Biol. Chem. 271,
13875 (1996)).
[0119] For the purposes of the present invention, any gene encoding
a G3P phosphatase activity is suitable for use in the method
wherein that activity is capable of catalyzing the conversion of
glycerol-3-phosphate plus H.sub.2O to glycerol plus inorganic
phosphate. Further, any gene encoding the amino acid sequence of
G3P phosphatase corresponding to the genes GPP2 and GPP1 will be
functional in the present invention including any amino acid
sequence that encompasses amino acid substitutions, deletions or
additions that do not alter the function of the G3P phosphatase
enzyme. The skilled person will appreciate that genes encoding G3P
phosphatase isolated from other sources will also be suitable for
use in the present invention.
Host Cells
[0120] Suitable host cells for the recombinant production of
1,3-propanediol may be either prokaryotic or eukaryotic and will be
limited only by the host cell ability to express the active enzymes
for the 1,3-propanediol pathway. Suitable host cells will be
bacteria such as Citrobacter, Enterobacter, Clostridium,
Klebsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces,
Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces,
Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter,
Escherichia, Salmonella, Bacillus, Streptomyces, and Pseudomonas.
Preferred in the present invention are E. coli, E. blattae,
Klebsiella, Citrobacter, and Aerobacter.
[0121] Microorganisms can be converted to a high titer
1,3-propanediol producer by using the following general protocol.
[0122] 1. Determine the presence in a potential host organism of an
endogenous dhaT-like activity that allows for the steady state
concentration of a toxic or inhibitory level of 3-HPA in the
presence of 1-2 M 1,3-propanediol. [0123] 2. If such an activity
exists in the potential host organism, perform suitable mutagenisis
to delete or inactivate this activity. Confirmation of a
non-functional or deleted dhaT-like activity can be detected by the
lack of 3-HPA accumulation in the presence of 1-2 M
1,3-propanediol. [0124] 3. Express appropriate genes for a)
glycerol production, if glycerol is not the carbon source, b)
glycerol dehydratase and the associated maintenance system, and c)
yqhD.
[0125] Considerations which would need to be taken with respect to
certain microorganisms concern the expression or repression of
endogenous dhaT-like enzymes under the conditions for
1,3-propanediol production. These might also include the presence
of glycerol, glucose or anaerobisis.
Vectors and Expression Cassettes
[0126] The present invention provides a variety of vectors and
transformation and expression cassettes suitable for the cloning,
transformation and expression of G3PDH, G3P phosphatase,
dehydratase, and dehydratase reactivation factor into a suitable
host cell. Suitable vectors will be those which are compatible with
the microorganism employed. Suitable vectors can be derived, for
example, from a bacteria, a virus (such as bacteriophage T7 or a
M-13 derived phage), a cosmid, a yeast or a plant. Protocols for
obtaining and using such vectors are known to those in the art
(Sambrook et al., Molecular Cloning: A Laboratory Manual--volumes
1, 2, 3 (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.,
1989)).
[0127] Typically, the vector or cassette contains sequences
directing transcription and translation of the appropriate gene, a
selectable marker, and sequences allowing autonomous replication or
chromosomal integration. Suitable vectors comprise a region 5' of
the gene, which harbors transcriptional initiation controls, and a
region 3' of the DNA fragment which controls transcriptional
termination. It is most preferred when both control regions are
derived from genes homologous to the transformed host cell. Such
control regions need not be derived from the genes native to the
specific species chosen as a production host.
[0128] Initiation control regions, or promoters, which are useful
to drive expression of the G3PDH and G3P phosphatase genes (DAR1
and GPP2, respectively) in the desired host cell are numerous and
familiar to those skilled in the art. Virtually any promoter
capable of driving these genes is suitable for the present
invention including but not limited to CYC1, HIS3, GAL1, GAL10,
ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, and TPI
(useful for expression in Saccharomyces); AOX1 (useful for
expression in Pichia); and lac, trp, .lamda.P.sub.L,
.lamda.P.sub.R, T7, tac, and trc (useful for expression in E.
coli).
[0129] Termination control regions may also be derived from various
genes native to the preferred hosts. Optionally, a termination site
may be unnecessary; however, it is most preferred if included.
[0130] For effective expression of the instant enzymes, DNA
encoding the enzymes are linked operably through initiation codons
to selected expression control regions such that expression results
in the formation of the appropriate messenger RNA.
[0131] Particularly useful in the present invention are the vectors
pDT29 and pKP32 which are designed to be used in conjunction with
pAH48. The essential elements of pDT29 and pKP32 are derived from
the dha regulon isolated from Klebsiella pneumoniae pDT29 contains
the open reading frames dhaR, orfY, dhaT, orfX, orfW, dhaB1, dhaB2,
and dhaB3, nucleotide the sequences of which are contained within
SEQ ID NO:1. pKP32 contains the same set of open reading frames as
found on pDT29, from the same source, with the difference that
pKP32 lacks the dhaT. pAH48 is the vehicle used for the
introduction of DAR1 and GPP2 genes into the host cell and more
specifically comprises the DAR1 and GPP2 genes isolated from
Saccharomyces cerevisiae.
Transformation of Suitable Hosts and Expression of Genes for the
Production of 1,3-propanediol
[0132] Once suitable cassettes are constructed they are used to
transform appropriate host cells. Introduction of the cassette
containing the genes encoding G3PDH, G3P phosphatase, dehydratase,
and dehydratase reactivation factor into the host cell may be
accomplished by known procedures such as by transformation (e.g.,
using calcium-permeabilized cells, electroporation), or by
transfection using a recombinant phage virus (Sambrook et al.,
supra).
[0133] In the present invention cassettes were used to transform
the E. coli as fully described in the GENERAL METHODS and
EXAMPLES.
Mutants
[0134] In addition to the cells exemplified, it is contemplated
that the present method will be able to make use of cells having
single or multiple mutations specifically designed to enhance the
production of 1,3-propanediol. Cells that normally divert a carbon
feed stock into non-productive pathways, or that exhibit
significant catabolite repression could be mutated to avoid these
phenotypic deficiencies. For example, many wild type cells are
subject to catabolite repression from glucose and by-products in
the media and it is contemplated that mutant strains of these wild
type organisms, capable of 1,3-propanediol production that are
resistant to glucose repression, would be particularly useful in
the present invention.
[0135] Methods of creating mutants are common and well known in the
art. For example, wild type cells may be exposed to a variety of
agents such as radiation or chemical mutagens and then screened for
the desired phenotype. When creating mutations through radiation
either ultraviolet (UV) or ionizing radiation may be used. Suitable
short wave UV wavelengths for genetic mutations will fall within
the range of 200 nm to 300 nm where 254 nm is preferred. UV
radiation in this wavelength principally causes changes within
nucleic acid sequence from guanidine and cytosine to adenine and
thymidine. Since all cells have DNA repair mechanisms that would
repair most UV induced mutations, agents such as caffeine and other
inhibitors may be added to interrupt the repair process and
maximize the number of effective mutations. Long wave UV mutations
using light in the 300 nm to 400 nm range are also possible but are
generally not as effective as the short wave UV light unless used
in conjunction with various activators such as psoralen dyes that
interact with the DNA.
[0136] Mutagenesis with chemical agents is also effective for
generating mutants and commonly used substances include chemicals
that affect nonreplicating DNA such as HNO.sub.2 and NH.sub.2OH, as
well as agents that affect replicating DNA such as acridine dyes,
notable for causing frameshift mutations. Specific methods for
creating mutants using radiation or chemical agents are well
documented in the art. See for example Thomas D. Brock in
Biotechnology: A Textbook of Industrial Microbiology, Second
Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., or
Deshpande, Mukund V., Appl. Biochem. Biotechnol. 36, 227 (1992),
herein incorporated by reference.
[0137] After mutagenesis has occurred, mutants having the desired
phenotype may be selected by a variety of methods. Random screening
is most common where the mutagenized cells are selected for the
ability to produce the desired product or intermediate.
Alternatively, selective isolation of mutants can be performed by
growing a mutagenized population on selective media where only
resistant colonies can develop. Methods of mutant selection are
highly developed and well known in the art of industrial
microbiology. See for example Brock, Supra; DeMancilha et al., Food
Chem. 14, 313 (1984).
[0138] The elimination of an undesired enzyme activity may be also
accomplished by disruption of the gene encoding the enzyme. Such
methods are known to those skilled in the art and are exemplified
in Example 4 and Example 8.
Alterations in the 1,3-propanediol Production Pathway
[0139] Representative Enzyme Pathway. The production of
1,3-propanediol from glucose can be accomplished by the following
series of steps. This series is representative of a number of
pathways known to those skilled in the art and is illustrated in
FIG. 5. Glucose is converted in a series of steps by enzymes of the
glycolytic pathway to dihydroxyacetone phosphate (DHAP) and
3-phospho-glyceraldehyde (3-PG). Glycerol is then formed by either
hydrolysis of DHAP to dihydroxyacetone (DHA) followed by reduction,
or reduction of DHAP to glycerol 3-phosphate (G3P) followed by
hydrolysis. The hydrolysis step can be catalyzed by any number of
cellular phosphatases, which are known to be non-specific with
respect to their substrates, or the activity can be introduced into
the host by recombination. The reduction step can be catalyzed by a
NAD.sup.+ (or NADP.sup.+) linked host enzyme or the activity can be
introduced into the host by recombination. It is notable that the
dha regulon contains a glycerol dehydrogenase (E.C. 1.1.1.6) that
catalyzes the reversible reaction of Equation 3.
Glycerol.fwdarw.3-HPA+H.sub.2O (Equation 1)
3-HPA+NADH+H.sup.+.fwdarw.1,3-Propanediol+NAD.sup.+ (Equation
2)
Glycerol+NAD.sup.+.fwdarw.DHA+NADH+H.sup.+ (Equation 3)
Glycerol is converted to 1,3-propanediol via the intermediate
3-hydroxy-propionaldehye (3-HPA) as has been described in detail
above. The intermediate 3-HPA is produced from glycerol, Equation
1, by a dehydratase enzyme that can be encoded by the host or can
be introduced into the host by recombination. This dehydratase can
be glycerol dehydratase (E.C. 4.2.1.30), diol dehydratase (E.C.
4.2.1.28) or any other enzyme able to catalyze this transformation.
Glycerol dehydratase, but not diol dehydratase, is encoded by the
dha regulon. 1,3-Propanediol is produced from 3-HPA, Equation 2, by
a NAD.sup.+- (or NADP.sup.+) linked host enzyme or the activity can
be introduced into the host by recombination. This final reaction
in the production of 1,3-propanediol can be catalyzed by
1,3-propanediol dehydrogenase (E.C. 1.1.1.202) or other alcohol
dehydrogenases.
[0140] Mutations and transformations that affect carbon channeling.
A variety of mutant microorganisms comprising variations in the
1,3-propanediol production pathway will be useful in the present
invention. For example the introduction of a triosephosphate
isomerase mutation (tpi-) into the microorganism of the present
invention is an example of the use of a mutation to improve the
performance by carbon channeling. Triosephosphate isomerase is the
enzyme responsible for the conversion of DAHP to
3-phosphoglyceraldehyde, and as such allows the diversion of carbon
flow from the main pathway form glucose to glycerol and
1,3-propanediol (FIG. 5). Thus, the deletion mutation (tpi-)
enhances the overall metabolic efficiency of the desired pathway
over that described in the art. Similarly, mutations which block
alternate pathways for intermediates of the 1,3-propanediol
production pathway would also be useful to the present invention.
For example, the elimination of glycerol kinase prevents glycerol,
formed from G3P by the action of G3P phosphatase, from being
re-converted to G3P at the expense of ATP (FIG. 5). Also, the
elimination of glycerol dehydrogenase (for example, gldA) prevents
glycerol, formed from DHAP by the action of NADH-dependent
glycerol-3-phosphate dehydrogenase, from being converted to
dihydroxyacetone (FIG. 5). Mutations can be directed toward a
structural gene so as to impair or improve the activity of an
enzymatic activity or can be directed toward a regulatory gene,
including promoter regions and ribosome binding sites, so as to
modulate the expression level of an enzymatic activity.
[0141] It is thus contemplated that transformations and mutations
can be combined so as to control particular enzyme activities for
the enhancement of 1,3-propanediol production. Thus, it is within
the scope of the present invention to anticipate modifications of a
whole cell catalyst which lead to an increased production of
1,3-propanediol.
[0142] The present invention utilizes a preferred pathway for the
production of 1,3-propanediol from a sugar substrate where the
carbon flow moves from glucose to DHAP, G3P, Glycerol, 3-HPA and
finally to 1,3-propanediol. The present production strains have
been engineered to maximize the metabolic efficiency of the pathway
by incorporating various deletion mutations that prevent the
diversion of carbon to non-productive compounds. Glycerol may be
diverted from conversion to 3HPA by transformation to either DHA or
G3P via glycerol dehydrogenase or glycerol kinase as discussed
above (FIG. 5). Accordingly, the present production strains contain
deletion mutations in the gldA and glpK genes. Similarly DHAP may
be diverted to 3-PG by triosephosphate isomerase, thus the present
production microorganism also contains a deletion mutation in this
gene. The present method additionally incorporates a dehydratase
enzyme for the conversion of glycerol to 3HPA, which functions in
concert with the reactivation factor, encoded by orfX and orfZ of
the dha regulon (FIG. 5). Although conversion of the 3HPA to
1,3-propanediol is typically accomplished via a 1,3-propanediol
oxidoreductase, the present method utilizes a non-specific
catalytic activity that produces greater titers and yields of the
final product, 1,3-propanediol (FIG. 5). In such a process, titers
of 1,3-propanediol of at least 10 g/L are achieved, where titers of
200 g/L are expected.
[0143] Alternatively, an improved process for 1,3-propanediol
production may utilize glycerol or dihydroxyacetone as a substrate
where the pathway comprises only the last three substrates,
glycerol .fwdarw.3HPA.fwdarw.1,3-propanediol. In such a process,
the oxidoreductase is again eliminated in favor of the non-specific
catalytic activity, (expected to be an alcohol dehydrogenase),
however the need for deletion mutations are nullified by the energy
considerations of adding glycerol to the culture. In such as
process titers of 1,3-propanediol of at least 71 g/L are achieved
where titers of 200 g/L are expected.
[0144] Similarly it is within the scope of the invention to provide
mutants of wildtype microorganisms that have been modified by the
deletion or mutation of the dhaT activity to create improved
1,3-propandiol producers. For example, microorganisms, which
naturally contain all the elements of the dha regulon, may be
manipulated so as to inactivate the dhaT gene encoding the
1,3-propandiol oxidoreductase activity. These microorganisms will
be expected to produce higher yields and titers of 1,3-propanediol,
mediated by the presence of an endogenous catalytic activity,
expected to be an alcohol dehydrogenase. Examples of such
microorganisms include but are not limited to Klebsiella sp.,
Citrobacter sp., and Clostridium sp.
Media and Carbon Substrates
[0145] Fermentation media in the present invention must contain
suitable carbon substrates. Suitable substrates may include but are
not limited to monosaccharides such as glucose and fructose,
oligosaccharides such as lactose or sucrose, polysaccharides such
as starch or cellulose or mixtures thereof and unpurified mixtures
from renewable feedstocks such as cheese whey permeate, cornsteep
liquor, sugar beet molasses, and barley malt. Additionally the
carbon substrate may also be one-carbon substrates such as carbon
dioxide, or methanol for which metabolic conversion into key
biochemical intermediates has been demonstrated. Glycerol
production from single carbon sources (e.g., methanol, formaldehyde
or formate) has been reported in methylotrophic yeasts (K. Yamada
et al., Agric. Biol. Chem. 53(2), 541-543 (1989)) and in bacteria
(Hunter et. al., Biochemistry 24, 4148-4155 (1985)). These
microorganisms can assimilate single carbon compounds, ranging in
oxidation state from methane to formate, and produce glycerol. The
pathway of carbon assimilation can be through ribulose
monophosphate, through serine, or through xylulose-momophosphate
(Gottschalk, Bacterial Metabolism, Second Edition, Springer-Verlag:
New York (1986)). The ribulose monophosphate pathway involves the
condensation of formate with ribulose-5-phosphate to form a
6-carbon sugar that becomes fructose and eventually the
three-carbon product glyceraldehyde-3-phosphate. Likewise, the
serine pathway assimilates the one-carbon compound into the
glycolytic pathway via methylenetetrahydrofolate.
[0146] In addition to one and two carbon substrates, methylotrophic
microorganisms are also known to utilize a number of other
carbon-containing compounds such as methylamine, glucosamine and a
variety of amino acids for metabolic activity. For example,
methylotrophic yeast are known to utilize the carbon from
methylamine to form trehalose or glycerol (Bellion et al., Microb.
Growth C1 Compd., [Int. Symp.], 7th (1993), 415-32. Editor(s):
Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover,
UK). Similarly, various species of Candida will metabolize alanine
or oleic acid (Sulter et al., Arch. Microbiol. 153(5), 485-489
(1990)). Hence, it is contemplated that the source of carbon
utilized in the present invention may encompass a wide variety of
carbon-containing substrates and will only be limited by the choice
of microorganism or process.
[0147] Although it is contemplated that all of the above mentioned
carbon substrates and mixtures (co-feed) thereof are suitable in
the present invention, preferred carbon substrates are glucose,
fructose, sucrose, or methanol where the process intends to produce
an endogenous glycerol, and glycerol or dihydroxyacetone where the
process anticipates a glycerol or dihydroxyacetone feed.
[0148] In addition to an appropriate carbon source, fermentation
media must contain suitable minerals, salts, cofactors, buffers and
other components, known to those skilled in the art, suitable for
the growth of the cultures and promotion of the enzymatic pathway
necessary for 1,3-propanediol production. Particular attention is
given to Co(II) salts and/or vitamin B.sub.12 or precursors
thereof.
[0149] Adenosyl-cobalamin (coenzyme B.sub.12) is an essential
cofactor for dehydratase activity. Synthesis of coenzyme B.sub.12
is found in prokaryotes, some of which are able to synthesize the
compound de novo, for example, Escherichia blattae, Klebsiella
species, Citrobacter species, and Clostridium species, while others
can perform partial reactions. E. coli, for example, cannot
fabricate the corrin ring structure, but is able to catalyze the
conversion of cobinamide to corrinoid and can introduce the
5'-deoxyadenosyl group. Thus, it is known in the art that a
coenzyme B.sub.12 precursor, such as vitamin B.sub.12, need be
provided in E. coli fermentations.
[0150] Vitamin B 2 additions to E. coli fermentations may be added
continuously, at a constant rate or staged as to coincide with the
generation of cell mass, or may be added in single or multiple
bolus additions. Preferred ratios of vitamin B.sub.12 (mg) fed to
cell mass (OD550) are from 0.06 to 0.60. Most preferred ratios of
vitamin B.sub.12 (mg) fed to cell mass (OD550) are from 0.12 to
0.48.
[0151] Although vitamin B.sub.12 is added to the transformed E.
coli of the present invention it is contemplated that other
microorganisms, capable of de novo B.sub.12 biosynthesis will also
be suitable production cells and the addition of B.sub.12 to these
microorganisms will be unnecessary.
Culture Conditions:
[0152] Typically cells are grown at 35.degree. C. in appropriate
media. Preferred growth media in the present invention are common
commercially prepared media such as Luria Bertani (LB) broth,
Sabouraud Dextrose (SD) broth or Yeast medium (YM) broth. Other
defined or synthetic growth media may also be used and the
appropriate medium for growth of the particular microorganism will
be known by someone skilled in the art of microbiology or
fermentation science. The use of agents known to modulate
catabolite repression directly or indirectly, e.g., cyclic
adenosine 2':3'-monophosphate, may also be incorporated into the
reaction media. Similarly, the use of agents known to modulate
enzymatic activities (e.g., methyl viologen) that lead to
enhancement of 1,3-propanediol production may be used in
conjunction with or as an alternative to genetic manipulations.
[0153] Suitable pH ranges for the fermentation are between pH 5.0
to pH 9.0, where pH 6.0 to pH 8.0 is preferred as the initial
condition.
[0154] Reactions may be performed under aerobic or anaerobic
conditions where anaerobic or microaerobic conditions are
preferred.
[0155] Fed-batch fermentations may be performed with carbon feed,
for example, glucose, limited or excess.
Batch and Continuous Fermentations:
[0156] The present process employs a batch method of fermentation.
Classical batch fermentation is a closed system where the
composition of the media is set at the beginning of the
fermentation and is not subject to artificial alterations during
the fermentation. Thus, at the beginning of the fermentation the
media is inoculated with the desired microorganism or
microorganisms and fermentation is permitted to occur adding
nothing to the system. Typically, however, "batch" fermentation is
batch with respect to the addition of carbon source and attempts
are often made at controlling factors such as pH and oxygen
concentration. In batch systems the metabolite and biomass
compositions of the system change constantly up to the time the
fermentation is stopped. Within batch cultures cells moderate
through a static lag phase to a high growth log phase and finally
to a stationary phase where growth rate is diminished or halted. If
untreated, cells in the stationary phase will eventually die. Cells
in log phase generally are responsible for the bulk of production
of end product or intermediate. A variation on the standard batch
system is the Fed-Batch system.
[0157] Fed-Batch fermentation processes are also suitable in the
present invention and comprise a typical batch system with the
exception that the substrate is added in increments as the
fermentation progresses. Fed-Batch systems are useful when
catabolite repression is apt to inhibit the metabolism of the cells
and where it is desirable to have limited amounts of substrate in
the media. Measurement of the actual substrate concentration in
Fed-Batch systems is difficult and is therefore estimated on the
basis of the changes of measurable factors such as pH, dissolved
oxygen and the partial pressure of waste gases such as CO.sub.2.
Batch and Fed-Batch fermentations are common and well known in the
art and examples may be found in Brock, supra.
[0158] Although the present invention is performed in batch mode it
is contemplated that the method would be adaptable to continuous
fermentation methods. Continuous fermentation is an open system
where a defined fermentation media is added continuously to a
bioreactor and an equal amount of conditioned media is removed
simultaneously for processing. Continuous fermentation generally
maintains the cultures at a constant high density where cells are
primarily in log phase growth.
[0159] Continuous fermentation allows for the modulation of one
factor or any number of factors that affect cell growth or end
product concentration. For example, one method will maintain a
limiting nutrient such as the carbon source or nitrogen level at a
fixed rate and allow all other parameters to moderate. In other
systems a number of factors affecting growth can be altered
continuously while the cell concentration, measured by media
turbidity, is kept constant. Continuous systems strive to maintain
steady state growth conditions and thus the cell loss due to media
being drawn off must be balanced against the cell growth rate in
the fermentation. Methods of modulating nutrients and growth
factors for continuous fermentation processes as well as techniques
for maximizing the rate of product formation are well known in the
art of industrial microbiology and a variety of methods are
detailed by Brock, supra.
[0160] It is contemplated that the present invention may be
practiced using batch, fed-batch or continuous processes and that
any known mode of fermentation would be suitable. Additionally, it
is contemplated that cells may be immobilized on a substrate as
whole cell catalysts and subjected to fermentation conditions for
1,3-propanediol production.
Identification and purification of 1,3-propanediol:
[0161] Methods for the purification of 1,3-propanediol from
fermentation media are known in the art. For example, propanediols
can be obtained from cell media by subjecting the reaction mixture
to extraction with an organic solvent, distillation, and column
chromatography (U.S. Pat. No. 5,356,812). A particularly good
organic solvent for this process is cyclohexane (U.S. Pat. No.
5,008,473).
[0162] 1,3-Propanediol may be identified directly by submitting the
media to high pressure liquid chromatography (HPLC) analysis.
Preferred in the present invention is a method where fermentation
media is analyzed on an analytical ion exchange column using a
mobile phase of 0.01N sulfuric acid in an isocratic fashion.
EXAMPLES
General Methods
[0163] Procedures for phosphorylations, ligations and
transformations are well known in the art. Techniques suitable for
use in the following examples may be found in Sambrook, J. et al.,
Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring
Harbor Laboratory Press (1989).
[0164] Materials and methods suitable for the maintenance and
growth of bacterial cultures are well known in the art. Techniques
suitable for use in the following examples may be found in Manual
of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E.
Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel
R. Krieg and G. Briggs Phillips, eds), American Society for
Microbiology, Washington, D.C. (1994) or Thomas D. Brock in
Biotechnology: A Textbook of Industrial Microbiology, Second
Edition (1989) Sinauer Associates, Inc., Sunderland, Mass. All
reagents and materials used for the growth and maintenance of
bacterial cells were obtained from Aldrich Chemicals (Milwaukee,
Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL
(Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.)
unless otherwise specified.
[0165] The meaning of abbreviations is as follows: "h" means
hour(s), "min" means minute(s), "sec" means second(s), "d" means
day(s), "mL" means milliliters, "L" means liters, 50 amp is 50
.mu.g/mL ampicillin, and LB-50 amp is Luria-Bertani broth
containing 50 .mu.g/mL ampicillin.
[0166] Within the tables the following abbreviations are used.
"Con." is conversion, "Sel." is selectivity based on carbon, and
"nd" is not detected.
[0167] Strains and vectors used and constructed in the following
examples are listed in the chart below:
TABLE-US-00003 STRAIN/PLASMID DELETION ORF/GENE KLP23 gldA glpK
RJ8m gldA glpK Tpi pAH48 GPP2 DAR1 pDT29 dhaR orfY dhaT orfX orfW
dhaB1 dhaB2 dhaB3 orfZ pKP32 dhaR orfY orfX orfW dhaB1 dhaB2 dhaB3
orfZ
Enzyme Assays
Assays for Dehydratase Enzymes:
[0168] Dehydratase activity in cell-free extracts was determined
using either glycerol or 1,2-propanediol as substrate. Typically,
cell-free extracts were prepared by cell disruption using a french
press followed by centrifugation of the cellular debris. The assay,
based on the reaction of aldehydes with methylbenzo-2-thiazolone
hydrazone, has been described by Forage and Foster (Biochim.
Biophys. Acta 569, 249 (1979)).
[0169] Honda et al. (J. Bacteriol. 143, 1458 (1980)) disclose an
assay that measures the reactivation of dehydratases. Dehydratase
activity was determined in toluenized whole cells, with and without
ATP, using either glycerol or 1,2-propanediol as substrate.
Reactivation was determined by the ratio of product formation with
versus without the ATP addition. Product formation (3-HPA or
propionaldehyde when glycerol or 1,2-propanediol is used as
substrate, respectively) was measured directly, using HPLC, or
indirectly, using the methylbenzo-2-thiazolone hydrazone reagent.
Alternatively, product formation was determined by coupling the
conversion of the aldehyde to its respective alcohol using a NADH
linked alcohol dehydrogenase and monitoring the disappearance of
NADH.
Assays for 1,3-propanediol Oxidoreductase:
[0170] The activity of 1,3-propanediol oxidoreductase, sometimes
referred to as 1,3-propanediol dehydrogenase, was determined for
cell-free extracts in solution or in slab gels using
1,3-propanediol and NAD.sup.+ as substrates has been described
(Johnson and Lin, J. Bacteriol. 169, 2050 (1987)). Alternatively,
the conversion of 3-HPA and NADH to 1,3-propanediol and NAD.sup.+
was determined by the disappearance of NADH. The slab gel assay has
the potential advantage of separating the activity of
1,3-propanediol oxidoreductase (dhaT) from that of non-specific
alcohol dehydrogenases by virtue of size separation. The native
molecular weights of 1,3-propanediol oxidoreductases (dhaT) from
Citrobacter freundii, Klebsiella pneumoniae, and Clostridium
pasteurianum are unusually large, on the order of 330,000 to
440,000 daltons. Lactobacillus brevis and Lactobacillus buchneri
contain dehydratase associated 1,3-propanediol oxidoreductases with
properties similar to those of known 1,3-propanediol
oxidoreductases (dhaT).
Assays for Glycerol 3-phosphate Dehydrogenase Activity:
[0171] A procedure was used as modified below from a method
published by Bell et al. (J. Biol. Chem. 250, 7153 (1975)). This
method involved incubating a cell-free extract sample in a cuvette
that contained 0.2 mM NADH, 2.0 mM dihydroxyacetone phosphate
(DHAP), and enzyme in 0.1 M Tris/HCl, pH 7.5 buffer with 5 mM DTT,
in a total volume of 1.0 mL at 30.degree. C. A background rate of
the reaction of enzyme and NADH was first determined at 340 nm for
at least 3 min. The second substrate, DHAP, was subsequently added
and the absorbance change over time was further monitored for at
least 3 min. G3PDH activity was defined by subtracting the
background rate from the gross rate.
Assay for Glycerol-3-phosphatase Activity:
[0172] The assay for enzyme activity was performed by incubating
the extract with an organic phosphate substrate in a bis-Tris or
MES and magnesium buffer, pH 6.5. The substrate used was either
1-.alpha.-glycerol phosphate, or d,1-.alpha.-glycerol phosphate.
The final concentrations of the reagents in the assay are: buffer
(20 mM, bis-Tris or 50 mM MES); MgCl.sub.2 (10 mM); and substrate
(20 mM). If the total protein in the sample was low and no visible
precipitation occurs with an acid quench, the sample was
conveniently assayed in the cuvette. This method involved
incubating an enzyme sample in a cuvette that contained 20 mM
substrate (50 .mu.L, 200 mM), 50 mM MES, 10 mM MgCl.sub.2, pH 6.5
buffer. The final phosphatase assay volume was 0.5 mL. The
enzyme-containing sample was added to the reaction mixture; the
contents of the cuvette were mixed and then the cuvette was placed
in a circulating water bath at T=37.degree. C. for 5 to 120 min,
the length of time depending on whether the phosphatase activity in
the enzyme sample ranged from 2 to 0.02 U/mL. The enzymatic
reaction was quenched by the addition of the acid molybdate reagent
(0.4 mL). After the Fiske SubbaRow reagent (0.1 mL) and distilled
water (1.5 mL) were added, the solution was mixed and allowed to
develop. After 10 min, to allow full color development, the
absorbance of the samples was read at 660 nm using a Cary 219
UV/vis spectrophotometer. The amount of inorganic phosphate
released was compared to a standard curve that was prepared by
using a stock inorganic phosphate solution (0.65 mM) and preparing
6 standards with final inorganic phosphate concentrations ranging
from 0.026 to 0.130 .mu.mol/mL.
Assay for Glycerol Kinase Activity:
[0173] An appropriate amount of enzyme, typically a cell-free crude
extract, was added to a reaction mixture containing 40 mM ATP, 20
mM MgSO.sub.4, 21 mM uniformly .sup.13C labelled glycerol (99%,
Cambridge Isotope Laboratories), and 0.1 M Tris-HCl, pH 9 for 75
min at 25.degree. C. The conversion of glycerol to glycerol
3-phosphate was detected by .sup.13C-NMR (125 MHz): glycerol (63.11
ppm, .delta., J=41 Hz and 72.66 ppm, t, J=41 Hz); glycerol
3-phosphate (62.93 ppm, .delta., J=41 Hz; 65.31 ppm, br d, J=43 Hz;
and 72.66 ppm, dt, J=6.41 Hz).
NADH-Linked Glycerol Dehydrogenase Assay:
[0174] NADH-linked glycerol dehydrogenase activity (gldA) in
cell-free extracts from E. coli strains was determined after
protein separation by non-denaturing polyacrylamide gel
electrophoresis. The conversion of glycerol plus NAD.sup.+ to
dihydroxyacetone plus NADH was coupled with the conversion of
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
to a deeply colored formazan, using phenazine methosulfate (PMS) as
mediator (Tang et al., J. Bacteriol. 140, 182 (1997)).
[0175] Electrophoresis was performed in duplicate by standard
procedures using native gels (8-16% TG, 1.5 mm, 15 lane gels from
Novex, San Diego, Calif.). Residual glycerol was removed from the
gels by washing 3.times. with 50 mM Tris or potassium carbonate
buffer, pH 9 for 10 min. The duplicate gels were developed, with
and without glycerol (approximately 0.16 M final concentration), in
15 mL of assay solution containing 50 mM Tris or potassium
carbonate, pH 9, 60 mg ammonium sulfate, 75 mg NAD.sup.+, 1.5 mg
MTT, and 0.5 mg PMS.
[0176] The presence or absence of NADH-linked glycerol
dehydrogenase activity in E. coli strains (gldA) was also
determined, following polyacrylamide gel electrophoresis, by
reaction with polyclonal antibodies raised to purified K.
pneumoniae glycerol dehydrogenase (dhaD).
Isolation and identification of 1,3-propanediol:
[0177] The conversion of glycerol to 1,3-propanediol was monitored
by HPLC. Analyses were performed using standard techniques and
materials available to one of skill in the art of chromatography.
One suitable method utilized a Waters Maxima 820 HPLC system using
UV (210 nm) and RI detection. Samples were injected onto a Shodex
SH-1011 column (8 mm.times.300 mm, purchased from Waters, Milford,
Mass.) equipped with a Shodex SH-1011P precolumn (6 mm.times.50
mm), temperature controlled at 50.degree. C., using 0.01
NH.sub.2SO.sub.4 as mobile phase at a flow rate of 0.5 mL/min. When
quantitative analysis was desired, samples were prepared with a
known amount of trimethylacetic acid as external standard.
Typically, the retention times of glucose (RI detection), glycerol,
1,3-propanediol (RI detection), and trimethylacetic acid (UV and RI
detection) were 15.27 min, 20.67 min, 26.08 min, and 35.03 min,
respectively.
[0178] Production of 1,3-propanediol was confirmed by GC/MS.
Analyses were performed using standard techniques and materials
available to one of skill in the art of GC/MS. One suitable method
utilized a Hewlett Packard 5890 Series II gas chromatograph coupled
to a Hewlett Packard 5971 Series mass selective detector (EI) and a
HP-INNOWax column (30 m length, 0.25 mm i.d., 0.25 micron film
thickness). The retention time and mass spectrum of 1,3-propanediol
generated were compared to that of authentic 1,3-propanediol (m/e:
57, 58).
[0179] An alternative method for GC/MS involved derivatization of
the sample. To 1.0 mL of sample (e.g., culture supernatant) was
added 30 .mu.L of concentrated (70% v/v) perchloric acid. After
mixing, the sample was frozen and lyophilized. A 1:1 mixture of
bis(trimethylsilyl)trifluoroacetamide:pyridine (300 .mu.L) was
added to the lyophilized material, mixed vigorously and placed at
65.degree. C. for one h. The sample was clarified of insoluble
material by centrifugation. The resulting liquid partitioned into
two phases, the upper of which was used for analysis. The sample
was chromatographed on a DB-5 column (48 m, 0.25 mm I.D., 0.25
.mu.m film thickness; from J&W Scientific) and the retention
time and mass spectrum of the 1,3-propanediol derivative obtained
from culture supernatants were compared to that obtained from
authentic standards. The mass spectra of TMS-derivatized
1,3-propanediol contains the characteristic ions of 205, 177, 130
and 115 AMU.
Cell Lysis:
[0180] Cell lysis was estimated by measuring the extracellular
soluble protein concentration in the fermentation broth. Fermenter
samples were centrifuged in a desktop centrifuge (typically, 3-5
min at 12,000 rpm in an Eppendorf, Model 5415C micro centrifuge) in
order to separate cells. The resulting supernatant was analyzed for
protein concentration by the Bradford method using a commercially
available reagent (Bio-Rad Protein Assay, Bio-Rad, Hercules,
Calif.).
Viability:
[0181] Cell viability was determined by plating, at appropriate
dilutions, cells obtained from the fermenter on non-selective LB
agar plates. Cell viability between fermenter experiments is
compared by using the ratio of viable cells per mL of fermenter
broth divided by OD550 (AU).
Example 1
Cloning and Transformation of E. Coli Host Cells with Cosmid DNA
for the Expression of 1,3-Propanediol
Media:
[0182] Synthetic S12 medium was used in the screening of bacterial
transformants for the ability to make 1,3-propanediol. S12 medium
contains: 10 mM ammonium sulfate, 50 mM potassium phosphate buffer,
pH 7.0, 2 mM MgCl.sub.2, 0.7 mM CaCl.sub.2, 50 .mu.M MnCl.sub.2, 1
.mu.M FeCl.sub.3, 1 M ZnCl, 1.7 .mu.M CuSO.sub.4, 2.5 .mu.M
CoCl.sub.2, 2.4 .mu.M Na.sub.2MoO.sub.4, and 2 .mu.M thiamine
hydrochloride.
[0183] Medium A used for growth and fermentation consisted of: 10
mM ammonium sulfate; 50 mM MOPS/KOH buffer, pH 7.5; 5 mM potassium
phosphate buffer, pH 7.5; 2 mM MgCl.sub.2; 0.7 mM CaCl.sub.2; 50
.mu.M MnCl.sub.2; 1 .mu.M FeCl.sub.3; 1 .mu.M ZnCl; 1.72 .mu.M
CuSO.sub.4; 2.53 .mu.M CoCl.sub.2; 2.42 .mu.M Na.sub.2MoO.sub.4; 2
.mu.M thiamine hydrochloride; 0.01% yeast extract; 0.01% casamino
acids; 0.8 .mu.g/mL vitamin B.sub.12; and 50 .mu.g/mL amp. Medium A
was supplemented with either 0.2% glycerol or 0.2% glycerol plus
0.2% D-glucose as required.
Cells:
[0184] Klebsiella pneumoniae. ECL2106 (Ruch et al., J. Bacteriol.
124, 348 (1975)), also known in the literature as K. aerogenes or
Aerobacter aerogenes, was obtained from E. C. C. Lin (Harvard
Medical School, Cambridge, Mass.) and was maintained as a
laboratory culture.
[0185] Klebsiella pneumoniae. ATCC 25955 was purchased from
American Type Culture Collection (Manassas, Va.).
[0186] E. coli DH5.alpha. was purchased from Gibco/BRL and was
transformed with the cosmid DNA isolated from Klebsiella
pneumoniae. ATCC 25955 containing a gene coding for either a
glycerol or diol dehydratase enzyme. Cosmids containing the
glycerol dehydratase were identified as pKP1 and pKP2 and cosmid
containing the diol dehydratase enzyme were identified as pKP4.
Transformed DH5.alpha. cells were identified as DH5.alpha.-pKP1,
DH5.alpha.-pKP2, and DH5.alpha.-pKP4.
[0187] E. coli ECL707 (Sprenger et al., J. Gen. Microbiol. 135,
1255 (1989)) was obtained from E. C. C. Lin (Harvard Medical
School, Cambridge, Mass.) and was similarly transformed with cosmid
DNA from Klebsiella pneumoniae. These transformants were identified
as ECL707-pKP1 and ECL707-pKP2, containing the glycerol dehydratase
gene and ECL707-pKP4 containing the diol dehydratase gene.
[0188] E. coli AA200 containing a mutation in the tpi gene
(Anderson et al., J. Gen. Microbiol. 62, 329 (1970)) was purchased
from the E. coli Genetic Stock Center, Yale University (New Haven,
Conn.) and was transformed with Klebsiella cosmid DNA to give the
recombinant microorganisms AA200-pKP1 and AA200-pKP2, containing
the glycerol dehydratase gene, and AA200-pKP4, containing the diol
dehydratase gene.
DH5.alpha.:
[0189] Six transformation plates containing approximately 1,000
colonies of E. coli XL1-Blue MR transfected with K. pneumoniae. DNA
were washed with 5 mL LB medium and centrifuged. The bacteria were
pelleted and resuspended in 5 mL LB medium+glycerol. An aliquot (50
.mu.L) was inoculated into a 15 mL tube containing S12 synthetic
medium with 0.2% glycerol+400 ng per mL of vitamin B.sub.12+0.001%
yeast extract+50 amp. The tube was filled with the medium to the
top and wrapped with parafilm and incubated at 30.degree. C. A
slight turbidity was observed after 48 h. Aliquots, analyzed for
product distribution as described above at 78 h and 132 h, were
positive for 1,3-propanediol, the later time points containing
increased amounts of 1,3-propanediol.
[0190] The bacteria, testing positive for 1,3-propanediol
production, were serially diluted and plated onto LB-50 amp plates
in order to isolate single colonies. Forty-eight single colonies
were isolated and checked again for the production of
1,3-propanediol. Cosmid DNA was isolated from 6 independent clones
and transformed into E. coli strain DH5.alpha.. The transformants
were again checked for the production of 1,3-propanediol. Two
transformants were characterized further and designated as
DH5.alpha.-pKP1 and DH5.alpha.-pKP2.
[0191] A 12.1 kb EcoRI-SalI fragment from pKP1, subcloned into
pIBI31 (IBI Biosystem, New Haven, Conn.), was sequenced and termed
pHK28-26 (SEQ ID NO:1). Sequencing revealed the loci of the
relevant open reading frames of the dha operon encoding glycerol
dehydratase and genes necessary for regulation. Referring to SEQ ID
NO:1, a fragment of the open reading frame for dhaK1 encoding
dihydroxyacetone kinase is found at bases 1-399 (complement); the
open reading frame dhaD encoding glycerol dehydrogenase is found at
bases 1010-2107; the open reading frame dhaR encoding the repressor
is found at bases 2209-4134; the open reading frame orfW, encoding
a protein of unknown function is found at bases 4112-4642
(complement); the open reading frame orfX encoding a dehydratase
reactivation protein is found at bases 4643-4996 (complement); the
open reading frame dhaT encoding 1,3-propanediol oxidoreductase is
found at bases 5017-6180 (complement); the open reading frame orfY,
encoding a protein of unknown function is found at bases 6202-6630
(complement); the open reading frame dhaB1 encoding the alpha
subunit glycerol dehydratase is found at bases 7044-8711; the open
reading frame dhaB2 encoding the beta subunit glycerol dehydratase
is found at bases 8724-9308; the open reading frame dhaB3 encoding
the gamma subunit glycerol dehydratase is found at bases 9311-9736;
the open reading frame dhaBX, encoding a dehydratase reactivation
protein is found at bases 9749-11572; and a fragment of the open
reading frame for glpF encoding a glycerol uptake facilitator
protein is found at bases 11626-12145.
[0192] Single colonies of E. coli XL1-Blue MR transfected with
packaged cosmid DNA from K. pneumoniae were inoculated into
microtiter wells containing 200 .mu.L of S15 medium (ammonium
sulfate, 10 mM; potassium phosphate buffer, pH 7.0, 1 mM; MOPS/KOH
buffer, pH 7.0, 50 mM; MgCl.sub.2, 2 mM; CaCl.sub.2, 0.7 mM;
MnCl.sub.2, 50 .mu.M; FeCl.sub.3, 1 .mu.M; ZnCl, 1 .mu.M;
CuSO.sub.4, 1.72 .mu.M; CoCl.sub.2, 2.53 .mu.M; Na.sub.2MoO.sub.4,
2.42 .mu.M; and thiamine hydrochloride, 2 .mu.M)+0.2% glycerol+400
ng/mL of vitamin B.sub.12+0.001% yeast extract+50 .mu.g/mL
ampicillin. In addition to the microtiter wells, a master plate
containing LB-50 amp was also inoculated. After 96 h, 100 .mu.L was
withdrawn and centrifuged in a Rainin microfuge tube containing a
0.2 micron nylon membrane filter. Bacteria were retained and the
filtrate was processed for HPLC analysis. Positive clones
demonstrating 1,3-propanediol production were identified after
screening approximately 240 colonies. Three positive clones were
identified, two of which had grown on LB-50 amp and one of which
had not. A single colony, isolated from one of the two positive
clones grown on LB-50 amp and verified for the production of
1,3-propanediol, was designated as pKP4. Cosmid DNA was isolated
from E. coli strains containing pKP4 and E. coli strain DH5.alpha.
was transformed. An independent transformant, designated as
DH5.alpha.-pKP4, was verified for the production of
1,3-propanediol.
ECL707:
[0193] E. coli strain ECL707 was transformed with cosmid K.
pneumoniae. DNA corresponding to one of pKP1, pKP2, pKP4 or the
Supercos vector alone and named ECL707-pKP1, ECL707-pKP2,
ECL707-pKP4, and ECL707-sc, respectively. ECL707 is defective in
glpK, gld, and ptsD which encode the ATP-dependent glycerol kinase,
NAD.sup.+-linked glycerol dehydrogenase, and enzyme II for
dihydroxyacetone of the phosphoenolpyruvate-dependent
phosphotransferase system, respectively.
[0194] Twenty single colonies of each cosmid transformation and
five of the Supercos vector alone (negative control)
transformation, isolated from LB-50 amp plates, were transferred to
a master LB-50 amp plate. These isolates were also tested for their
ability to convert glycerol to 1,3-propanediol in order to
determine if they contained dehydratase activity. The transformants
were transferred with a sterile toothpick to microtiter plates
containing 200 .mu.L of Medium A supplemented with either 0.2%
glycerol or 0.2% glycerol plus 0.2% D-glucose. After incubation for
48 h at 30.degree. C., the contents of the microtiter plate wells
were filtered through a 0.45 micron nylon filter and
chromatographed by HPLC. The results of these tests are given in
Table 2.
TABLE-US-00004 TABLE 2 Conversion of glycerol to 1,3-propanediol by
transformed ECL707 transformant glycerol* glycerol plus glucose*
ECL707-pKP1 19/20 19/20 ECL707-pKP2 18/20 20/20 ECL707-pKP4 0/20
20/20 ECL707-sc 0/5 0/5 *(Number of positive isolates/number of
isolates tested)
[0195] AA200:
[0196] E. coli strain AA200 was transformed with cosmid K.
pneumoniae. DNA corresponding to one of pKP1, pKP2, pKP4 and the
Supercos vector alone and named AA200-pKP1, AA200-pKP2, AA200-pKP4,
and AA200-sc, respectively. Strain AA200 is defective in
triosephosphate isomerase (tpi-).
[0197] Twenty single colonies of each cosmid transformation and
five of the empty vector transformation were isolated and tested
for their ability to convert glycerol to 1,3-propanediol as
described for E. coli strain ECL707. The results of these tests are
given in Table 3.
TABLE-US-00005 TABLE 3 Conversion of glycerol to 1,3-propanediol by
transformed AA200 transformant glycerol* glycerol plus glucose*
AA200-pKP1 17/20 17/20 AA200-pKP2 17/20 17/20 AA200-pKP4 2/20 16/20
AA200-sc 0/5 0/5 *(Number of positive isolates/number of isolates
tested)
Example 2
Engineering of Glycerol Kinase Mutants of E. Coli FM5 for
Production of Glycerol from Glucose
[0198] Construction of Integration Plasmid for Glycerol Kinase Gene
Replacement in E. coli FM5:
[0199] E. coli FM5 (ATCC 53911) genomic DNA was prepared using the
Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, Minn.). A
1.0 kb DNA fragment containing partial glpF and glycerol kinase
(glpK) genes was amplified by PCR (Mullis and Faloona, Methods
Enzymol. 155, 335 (1987)) from FM5 genomic DNA using primers SEQ ID
NO:2 and SEQ ID NO:3. A 1.1 kb DNA fragment containing partial glpK
and glpX genes was amplified by PCR from FM5 genomic DNA using
primers SEQ ID NO:4 and SEQ ID NO:5. A MunI site was incorporated
into primer SEQ ID NO:4. The 5' end of primer SEQ ID NO:4 was the
reverse complement of primer SEQ ID NO:3 to enable subsequent
overlap extension PCR. The gene splicing by overlap extension
technique (Horton et al., BioTechniques 8, 528 (1990)) was used to
generate a 2.1 kb fragment by PCR using the above two PCR fragments
as templates and primers SEQ ID NO:2 and SEQ ID NO:5. This fragment
represented a deletion of 0.8 kb from the central region of the 1.5
kb glpK gene. Overall, this fragment had 1.0 kb and 1.1 kb flanking
regions on either side of the MunI cloning site (within the partial
glpK) to allow for chromosomal gene replacement by homologous
recombination.
[0200] The above 2.1 kb PCR fragment was blunt-ended (using mung
bean nuclease) and cloned into the pCR-Blunt vector using the Zero
Blunt PCR Cloning Kit (Invitrogen, San Diego, Calif.) to yield the
5.6 kb plasmid pRN100 containing kanamycin and Zeocin resistance
genes. The 1.2 kb HincII fragment from pLoxCat1 (unpublished
results), containing a chloramphenicol-resistance gene flanked by
bacteriophage P1 loxP sites (Snaith et al., Gene 166, 173 (1995)),
was used to interrupt the glpK fragment in plasmid pRN100 by
ligating it to MunI-digested (and blunt-ended) plasmid pRN100 to
yield the 6.9 kb plasmid pRN101-1. A 376 bp fragment containing the
R6K origin was amplified by PCR from the vector pGP704 (Miller and
Mekalanos, J. Bacteriol. 170, 2575-2583 (1988)) using primers SEQ
ID NO:6 and SEQ ID NO:7, blunt-ended, and ligated to the 5.3 kb
Asp718-AatII fragment (which was blunt-ended) from pRN101-1 to
yield the 5.7 kb plasmid pRN102-1 containing kanamycin and
chloramphenicol resistance genes. Substitution of the ColE1 origin
region in pRN101-1 with the R6K origin to generate pRN102-1 also
involved deletion of most of the Zeocin resistance gene. The host
for pRN102-1 replication was E. coli SY327 (Miller and Mekalanos,
J. Bacteriol. 170, 2575-2583 (1988)) which contains the pir gene
necessary for the function of the R6K origin.
Engineering of Glycerol Kinase Mutant RJF10m with Chloramphenicol
Resistance Gene Interrupt:
[0201] E. coli FM5 was electrotransformed with the non-replicative
integration plasmid pRN102-1 and transformants that were
chloramphenicol-resistant (12.5 .mu.g/mL) and kanamycin-sensitive
(30 .mu.g/mL) were further screened for glycerol non-utilization on
M9 minimal medium containing 1 mM glycerol. An EcoRI digest of
genomic DNA from one such mutant, RJF10m, when probed with the
intact glpK gene via Southern analysis (Southern, J. Mol. Biol. 98,
503-517 (1975)) indicated that it was a double-crossover integrant
(glpK gene replacement) since the two expected 7.9 kb and 2.0 kb
bands were observed, owing to the presence of an additional EcoRI
site within the chloramphenicol resistance gene. The wild-type
control yielded the single expected 9.4 kb band. A .sup.13C NMR
analysis of mutant RJF10m confirmed that it was incapable of
converting .sup.13C-labeled glycerol and ATP to
glycerol-3-phosphate. This glpK mutant was further analyzed by
genomic PCR using primer combinations SEQ ID NO:8 and SEQ ID NO:9,
SEQ ID NO:10 and SEQ ID NO:11, and SEQ ID NO:8 and SEQ ID NO:11
which yielded the expected 2.3 kb, 2.4 kb, and 4.0 kb PCR fragments
respectively. The wild-type control yielded the expected 3.5 kb
band with primers SEQ ID NO:8 and SEQ ID NO:11. The glpK mutant
RJF10m was electrotransformed with plasmid pAH48 to allow glycerol
production from glucose. The glpK mutant E. coli RJF10m has been
deposited with ATCC under the terms of the Budapest Treaty on 24
Nov. 1997.
Engineering of Glycerol Kinase Mutant RJF10 with Chloramphenicol
Resistance Gene Interrupt Removed:
[0202] After overnight growth on YENB medium (0.75% yeast extract,
0.8% nutrient broth) at 37.degree. C., E. coli RJF10m in a water
suspension was electrotransformed with plasmid pJW168 (unpublished
results), which contained the bacteriophage P1 Cre recombinase gene
under the control of the IPTG-inducible lacUV5 promoter, a
temperature-sensitive pSC101 replicon, and an ampicillin resistance
gene. Upon outgrowth in SOC medium at 30.degree. C., transformants
were selected at 30.degree. C. (permissive temperature for pJW168
replication) on LB agar medium supplemented with carbenicillin (50
.mu.g/mL) and IPTG (1 mM). Two serial overnight transfers of pooled
colonies were carried out at 30.degree. C. on fresh LB agar medium
supplemented with carbenicillin and IPTG in order to allow excision
of the chromosomal chloramphenicol resistance gene via
recombination at the loxP sites mediated by the Cre recombinase
(Hoess and Abremski, J. Mol. Biol. 181, 351-362 (1985)). Resultant
colonies were replica-plated on to LB agar medium supplemented with
carbenicillin and IPTG and LB agar supplemented with
chloramphenicol (12.5 .mu.g/mL) to identify colonies that were
carbenicillin-resistant and chloramphenicol-sensitive indicating
marker gene removal. An overnight 30.degree. C. culture of one such
colony was used to inoculate 10 mL of LB medium. Upon growth at
30.degree. C. to OD (600 nm) of 0.6 AU, the culture was incubated
at 37.degree. C. overnight. Several dilutions were plated on
prewarmed LB agar medium and the plates incubated overnight at
42.degree. C. (the non-permissive temperature for pJW168
replication). Resultant colonies were replica-plated on to LB agar
medium and LB agar medium supplemented with carbenicillin (75
.mu.g/mL) to identify colonies that were carbenicillin-sensitive
indicating loss of plasmid pJW168. One such glpK mutant, RJF10, was
further analyzed by genomic PCR using primers SEQ ID NO:8 and SEQ
ID NO:11 and yielded the expected 3.0 kb band confirming marker
gene excision. Glycerol non-utilization by mutant RJF10 was
confirmed by lack of growth on M9 minimal medium containing 1 mM
glycerol. The glpK mutant RJF10 was electrotransformed with plasmid
pAH48 to allow glycerol production from glucose.
Example 3
Construction of E. Coli Strain with gldA Gene Knockout
[0203] The gldA gene was isolated from E. coli by PCR (K. B. Mullis
and F. A. Faloona, Meth. Enzymol. 155, 335-350 (1987)) using
primers SEQ ID NO:12 and SEQ ID NO:13, which incorporate terminal
Sph1 and Xba1 sites, respectively, and cloned (T. Maniatis (1982)
Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, Cold
Spring Harbor, N.Y.) between the Sph1 and Xba1 sites in pUC18, to
generate pKP8. pKP8 was cut at the unique Sal1 and Nco1 sites
within the gldA gene, the ends flushed with Klenow and religated,
resulting in a 109 bp deletion in the middle of gldA and
regeneration of a unique Sal1 site, to generate pKP9. A 1.4 kb DNA
fragment containing the gene conferring kanamycin resistance (kan),
and including about 400 bps of DNA upstream of the translational
start codon and about 100 bps of DNA downstream of the
translational stop codon, was isolated from pET-28a(+) (Novagen,
Madison, Wis.) by PCR using primers SEQ ID NO:14 and SEQ ID NO:15,
which incorporate terminal Sal1 sites, and subcloned into the
unique Sal1 site of pKP9, to generate pKP13. A 2.1 kb DNA fragment
beginning 204 bps downstream of the gldA translational start codon
and ending 178 bps upstream of the gldA translational stop codon,
and containing the kan insertion, was isolated from pKP13 by PCR
using primers SEQ ID NO:16 and SEQ ID NO:17, which incorporate
terminal Sph1 and Xba1 sites, respectively, was subcloned between
the Sph1 and Xba1 sites in pMAK705 (Genencor International, Palo
Alto, Calif.), to generate pMP33. E. coli FM5 was transformed with
pMP33 and selected on 20 .mu.g/mL kan at 30.degree. C., which is
the permissive temperature for pMAK705 replication. One colony was
expanded overnight at 30.degree. C. in liquid media supplemented
with 20 .mu.g/mL kan. Approximately 32,000 cells were plated on 20
.mu.g/mL kan and incubated for 16 h at 44.degree. C., which is the
restrictive temperature for pMAK705 replication. Transformants
growing at 44.degree. C. have plasmid integrated into the
chromosome, occurring at a frequency of approximately 0.0001. PCR
and Southern blot (E. M. Southern, J. Mol. Biol. 98, 503-517
(1975)) analyses were used to determine the nature of the
chromosomal integration events in the transformants. Western blot
analysis (Towbin et al., Proc. Natl. Acad. Sci. 76, 4350 (1979))
was used to determine whether glycerol dehydrogenase protein, the
product of gldA, is produced in the transformants. An activity
assay was used to determine whether glycerol dehydrogenase activity
remained in the transformants. Activity in glycerol dehydrogenase
bands on native gels was determined by coupling the conversion of
glycerol plus NAD.sup.+ to dihydroxyacetone plus NADH to the
conversion of a tetrazolium dye, MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to a
deeply colored formazan, with phenazine methosulfate as mediator.
Glycerol dehydrogenase also requires the presence of 30 mM ammonium
sulfate and 100 mM Tris, pH 9 (Tang et al., J. Bacteriol. 140, 182
(1997)). Of 8 transformants analyzed, 6 were determined to be gldA
knockouts. E. coli MSP33.6 has been deposited with ATCC under the
terms of the Budapest Treaty on 24 Nov. 1997.
Example 4
Construction of an E. Coli Strain with glpK and gldA Gene
Knockouts
[0204] A 1.6 kb DNA fragment containing the gldA gene and including
228 bps of DNA upstream of the translational start codon and 220
bps of DNA downstream of the translational stop codon was isolated
from E. coli by PCR using primers SEQ ID NO:18 and SEQ ID NO:19,
which incorporate terminal Sph1 and Xba1 sites, respectively, and
cloned between the Sph1 and Xba1 sites of pUC18, to generate pQN2.
pQN2 was cut at the unique Sal1 and Nco1 sites within the gldA
gene, the ends flushed with Klenow and religated, resulting in a
109 bps deletion in the middle of gldA and regeneration of a unique
Sal1 site, to generate pQN4. A 1.2 kb DNA fragment containing the
gene conferring kanamycin resistance (kan), and flanked by loxP
sites was isolated from pLoxKan2 (Genencor International, Palo
Alto, Calif.) as a Stu1/Xho1 fragment, the ends flushed with
Klenow, and subcloned into pQN4 at the Sal1 site after flushing
with Klenow, to generate pQN8. A 0.4 kb DNA fragment containing the
R6K origin of replication was isolated from pGP704 (Miller and
Mekalanos, J. Bacteriol. 170, 2575-2583 (1988)) by PCR using
primers SEQ ID NO:20 and SEQ ID NO:21, which incorporate terminal
Sph1 and Xba1 sites, respectively, and ligated to the 2.8 kb
Sph1/Xba1 DNA fragment containing the gldA::kan cassette from pQN8,
to generate pKP22. A 1.0 kb DNA fragment containing the gene
conferring chloramphenicol resistance (cam), and flanked by loxP
sites was isolated from pLoxCat2 (Genencor International, Palo
Alto, Calif.) as an Xba1 fragment, and subcloned into pKP22 at the
Xba1 site, to generate pKP23. E. coli strain RJF10 (see Example 2),
which is glpK-, was transformed with pKP23 and transformants with
the phenotype kanRcamS were isolated, indicating double crossover
integration, which was confirmed by southern blot analysis.
Glycerol dehydrogenase gel activity assays (as described in Example
3) demonstrated that active glycerol dehydrogenase was not present
in these transformants. The kan marker was removed from the
chromosome using the Cre-producing plasmid pJW168, as described in
Example 2, to produce strain KLP23. Several isolates with the
phenotype kanS demonstrated no glycerol dehydrogenase activity, and
southern blot analysis confirmed loss of the kan marker.
Example 5
Plasmid Construction and Strain Construction for the Expression of
Glycerol 3-Phosphate Dehydrogenase (DAR1) and/or Glycerol
3-Phosphatase (GPP2)
[0205] Construction of Expression Cassettes for Glycerol
3-phosphatase (GPP2):
[0206] The Saccharomyces cerevisiae chromosomeV lamda clone 6592
(GenBank, accession # U18813.times.11) was obtained from ATCC. The
glycerol 3-phosphate phosphatase gene (GPP2) was cloned by cloning
from the lamda clone as target DNA using synthetic primers (SEQ ID
NO:22 with SEQ ID NO:23) incorporating an BamHI-RBS-XbaI site at
the 5' end and a SmaI site at the 3' end. The product was subcloned
into pCR-Script (Stratagene, Madison, Wis.) at the SrfI site to
generate the plasmid pAH15 containing GPP2. The plasmid pAH15
contains the GPP2 gene in the inactive orientation for expression
from the lac promoter in pCR-Script SK+. The BamHI-SmaI fragment
from pAH15 containing the GPP2 gene was inserted into pBlueScriptII
SK+ to generate plasmid pAH19. The pAH19 contains the GPP2 gene in
the correct orientation for expression from the lac promoter. The
XbaI-PstI fragment from pAH19 containing the GPP2 gene was inserted
into pPHOX2 to create plasmid pAH21. The pAH21/DH5.alpha. is the
expression plasmid.
Construction of Expression Cassettes for Glycerol 3-phosphate
Dehydrogenase (DAR1):
[0207] DAR1 was isolated by PCR cloning from genomic S. cerevisiae
DNA using synthetic primers (SEQ ID NO:24 with SEQ ID NO:25).
Successful PCR cloning places an NcoI site at the 5' end of DAR1
where the ATG within NcoI is the DAR1 initiator methionine. At the
3' end of DAR1 a BamHI site is introduced following the translation
terminator. The PCR fragments were digested with NcoI+BamHI and
cloned into the same sites within the expression plasmid pTrc99A
(Pharmacia, Piscataway, N.J.) to give pDAR1A.
[0208] In order to create a better ribosome binding site at the 5'
end of DAR1, an SpeI-RBS-NcoI linker obtained by annealing
synthetic primers (SEQ ID NO:26 with SEQ ID NO:27) was inserted
into the NcoI site of pDAR1A to create pAH40. Plasmid pAH40
contains the new RBS and DAR1 gene in the correct orientation for
expression from the trc promoter of pTrc99A (Pharmacia, Piscataway,
N.J.). The NcoI-BamHI fragment from pDAR1A and an second set of
SpeI-RBS-NcoI linker obtained by annealing synthetic primers (SEQ
ID NO:28 with SEQ ID NO:29) was inserted into the SpeI-BamHI site
of pBC-SK+ (Stratagene, Madison, Wis.) to create plasmid pAH42. The
plasmid pAH42 contains a chloramphenicol resistant gene.
Construction of Expression Cassettes for DAR1 and GPP2:
[0209] Expression cassettes for DAR1 and GPP2 were assembled from
the individual DAR1 and GPP2 subclones described above using
standard molecular biology methods. The BamHI-PstI fragment from
pAH19 containing the ribosomal binding site (RBS) and GPP2 gene was
inserted into pAH40 to create pAH43. The BamHI-PstI fragment from
pAH19 containing the RBS and GPP2 gene was inserted into pAH42 to
create pAH45.
[0210] The ribosome binding site at the 5' end of GPP2 was modified
as follows. A BamHI-RBS-SpeI linker, obtained by annealing
synthetic primers GATCCAGGAAACAGA (SEQ ID NO:30) with
CTAGTCTGTTTCCTG (SEQ ID NO:31) to the XbaI-PstI fragment from pAH19
containing the GPP2 gene, was inserted into the BamHI-PstI site of
pAH40 to create pAH48. Plasmid pAH48 contains the DAR1 gene, the
modified RBS, and the GPP2 gene in the correct orientation for
expression from the trc promoter of pTrc99A (Pharmacia, Piscataway,
N.J.).
Transformation of E. coli:
[0211] The plasmids described here were transformed into E. coli
DH5.alpha., FM5 and KLP23 using standard molecular biology
techniques. The transformants were verified by their DNA RFLP
pattern.
Example 6
Construction of Expression Plasmids for Use in Transformation of
Escherichia Coli with Genes from the Klebsiella Pneumoniae dha
Regulon
[0212] Construction of the Expression Vector pTacIQ:
[0213] The E. coli expression vector pTacIQ was prepared by
inserting lacIq gene (Farabaugh, Nature 274(5673), 765-769 (1978))
and tac promoter (Amann et al., Gene 25, 167-178 (1983)) into the
restriction endonuclease site EcoRI of pBR322 (Sutcliffe, Cold
Spring Harb. Symp. Quant. Biol. 43, 77-90 (1979)). A multiple
cloning site and terminator sequence (SEQ ID NO:32) replaces the
pBR322 sequence from EcoRI to SphI.
Subcloning the Glycerol Dehydratase Genes (dhaB1,2,3, X):
[0214] The open reading frame for the dhaB3 gene was amplified from
pHK28-26 by PCR using primers (SEQ ID NO:33 and SEQ ID NO:34)
incorporating an EcoRI site at the 5' end and a Xba1 site at the 3'
end. The product was subcloned into pLitmus29 (New England Biolab,
Inc., Beverly, Mass.) to generate the plasmid pDHAB3 containing
dhaB3.
[0215] The region containing the entire coding region for dhaB1,
dhaB2, dhaB3 and dhaBX of the dhaB operon from pHK28-26 was cloned
into pBluescriptIIKS+ (Stratagene, La Jolla, Calif.) using the
restriction enzymes KpnI and EcoRI to create the plasmid pM7.
[0216] The dhaBX gene was removed by digesting plasmid pM7 with
ApaI and XbaI, purifying the 5.9 kb fragment and ligating it with
the 325-bp ApaI-XbaI fragment from plasmid pDHAB3 to create pM11
containing dhaB1, dhaB2 and dhaB3.
[0217] The open reading frame for the dhaB1 gene was amplified from
pHK28-26 by PCR using primers (SEQ ID NO:35 and SEQ ID NO:36)
incorporating a HindIII site and a consensus ribosome binding site
at the 5' end and a XbaI site at the 3' end. The product was
subcloned into pLitmus28 (New England Biolab, Inc., Beverly, Mass.)
to generate the plasmid pDT1 containing dhaB1.
[0218] A NotI-XbaI fragment from pM11 containing part of the dhaB1
gene, the dhaB2 gene and the dhaB3 gene was inserted into pDT1 to
create the dhaB expression plasmid, pDT2. The HindIII-XbaI fragment
containing the dhaB(1,2,3) genes from pDT2 was inserted into pTacIQ
to create pDT3.
Subcloning the 1,3-propanediol Dehydrogenase Gene (dhaT):
[0219] The KpnI-SacI fragment of pHK28-26, containing the
1,3-propanediol dehydrogenase (dhaT) gene, was subcloned into
pBluescriptII KS+ creating plasmid pAH1. The dhaT gene was
amplified by PCR from pAH1 as template DNA and synthetic primers
(SEQ ID NO:37 with SEQ ID NO:38) incorporating an Xba1 site at the
5' end and a BamHI site at the 3' end. The product was subcloned
into pCR-Script (Stratagene) at the SrfI site to generate the
plasmids pAH4 and pAH5 containing dhaT. The plasmid pAH4 contains
the dhaT gene in the right orientation for expression from the lac
promoter in pCR-Script and pAH5 contains dhaT gene in the opposite
orientation. The XbaI-BamHI fragment from pAH4 containing the dhaT
gene was inserted into pTacIQ to generate plasmid pAH8. The
HindIII-BamHI fragment from pAH8 containing the RBS and dhaT gene
was inserted into pBluescriptIIKS+ to create pAH11.
Construction of an Expression Cassette for dhaT and
dhaB(1,2,3):
[0220] An expression cassette for dhaT and dhaB(1,2,3) was
assembled from the individual dhaB(1,2,3) and dhaT subclones
described previously using standard molecular biology methods. A
SpeI-SacI fragment containing the dhaB(1,2,3) genes from pDT3 was
inserted into pAH11 at the SpeI-SacI sites to create pAH24. A
SalI-XbaI linker (SEQ ID NO:39 and SEQ ID NO:40) was inserted into
pAH5 that was digested with the restriction enzymes SalI-XbaI to
create pDT16. The linker destroys the Xba1 site. The 1 kb SalI-MluI
fragment from pDT16 was then inserted into pAH24 replacing the
existing SalI-MluI fragment to create pDT18. pDT21 was constructed
by inserting the SalI-NotI fragment from pDT 18 and the NotI-XbaI
fragment from pM7 into pCL1920 (SEQ ID NO:41). The glucose
isomerase promoter sequence from Streptomyces (SEQ ID NO:42) was
cloned by PCR and inserted into EcoRI-HinDIII sites of pLitmus28 to
construct pDT5. pCL1925 was constructed by inserting EcoRI-PvuII
fragment of pDT5 into the EcoRI-PvuII site of pCL1920. pDT24 was
constructed by cloning the HinDIII-MluII fragment of pDT21 and the
MluI-XbaI fragment of pDT21 into the HinDIII-XbaI sites of
pCL1925.
Construction of an Expression Cassette for dhaT and
dhaB(1,2,3,X):
[0221] pDT21 was constructed by inserting the SalI-NotI fragment
from pDT 18 and the NotI-XbaI fragment from pM7 into pCL1920 (SEQ
ID NO:41). The glucose isomerase promoter sequence from
Streptomyces (SEQ ID NO:42) was cloned by PCR and inserted into
EcoRI-HinDIII sites of pLitmus28 to construct pDT5. pCL1925 was
constructed by inserting EcoRI-PvuII fragment of pDT5 into the
EcoRI-PvuI site of pCL1920. pDT24 was constructed by cloning the
HinDIII-MluII fragment of pDT21 and the MluI-XbaI fragment of pDT21
into the HinDIII-XbaI sites of pCL1925.
Construction of an Expression Cassette for dhaR, orfY, dhaT, orfX,
orfW and dhaB(1,2,3,X):
[0222] pDT29 was constructed by inserting the SacI-EcoRI fragment
of pHK28-26 into SacI-EcoRI sites of pCL1925.
Construction of an expression cassette for dhaR, orfY, orfX, orfW
and dhaB(1,2,3,X):
[0223] A derivative of plasmid pDT29 was constructed in which all
except the first 5 and the last 5 codons (plus stop codon) of the
gene dhaT were deleted by a technique known as PCR-mediated overlap
extension. Using pDT29 as template, 2 primary PCR products were
generated using the following primers:
TABLE-US-00006 SEQ ID NO:43 = 5'GAC GCA ACA GTA TTC CGT CGC3'; SEQ
ID NO:44 = 5'ATG AGC TAT CGT ATG TTC CGC CAG GCA TTC TGA GTG TTA
ACG3'; SEQ ID NO:45 = 5'GCC TGG CGG AAC ATA CGA TAG CTC ATA ATA
TAC3'; SEQ ID NO:46 = 5'CGG GGC GCT GGG CCA GTA CTG3'.
[0224] SEQ ID NO:45 was paired with SEQ ID NO:46 to generate a
product of 931 bps and encompassing nucleic acid including 5' dhaB1
(to unique ScaI site), all of orfY, and the first five codons of
dhaT. SEQ ID NO:43 was paired with SEQ ID NO:44 to generate a
product of 1348 bps and encompassing nucleic acid including the
last five codons (plus stop codon) of dhaT, all of orfX, all of
orfW, and 5' dhaR (to unique SapI site). The 15 bases at the 5' end
of SEQ ID NO:44 constitute a tail that is the inverse complement of
a 15 base portion of SEQ ID NO:45. Similarly, the 11 bases at the
5' end of SEQ ID NO:45 constitute a tail that is the inverse
complement of an 11 base portion of SEQ ID NO:44. Thus, the 2
primary PCR products were joined together after annealing (via 26
bp tail overlap) and extending by PCR, to generate a third nucleic
acid product of 2253 bps. This third PCR product was digested with
SapI and ScaI and ligated into pDT29 which was also digested with
SapI and ScaI, to generate the plasmid pKP32, which is identical to
pDT29, except for the large, in-frame deletion within dhaT.
Example 7
Conversion of Glucose to 1,3-Propanediol Using E. Coli Strain
KLP23/pAH48/pDT29 and the Improved Process Using
KLP23/pAH48/pKP32
Pre-Culture:
[0225] KLP23/pAH48/pDT29 and KLP23/pAH48/pKP32 were pre-cultured
for seeding a fermenter in 2YT medium (10 g/L yeast extract, 16 g/L
tryptone, and 10 g/L NaCl) containing 200 mg/L carbenicillin (or
ampicillin) and 50 mg/L spectinomycin. KLP23/pAH48/pKP32 is
identical to KLP23/pAH48/pDT29 except that dhaT is deleted.
[0226] Cultures were started from frozen stocks (10% DMSO as
cryoprotectant) in 500 mL of medium in a 2-L Erlenmeyer flask,
grown at 35.degree. C. in a shaker at 250 rpm until an OD.sub.550
of approximately 1.0 AU was reached and used to seed the
fermenter.
Fermenter Medium:
[0227] The following components were sterilized together in the
fermenter vessel: 45 g KH.sub.2PO.sub.4, 12 g citric acid, 12 g
MgSO.sub.4.7H.sub.2O, 30 g yeast extract, 2.0 g ferric ammonium
citrate, 5 mL Mazu DF204 as antifoam, 1.2 g CaCl.sub.2.2H.sub.2O,
and 7.3 mL sulfuric acid. The pH was raised to 6.8 with 20-28%
NH.sub.4OH and the following components were added: 1.2 g
carbenicillin or ampicillin, 0.30 g spectinomycin, 60 mL of a
solution of trace elements and glucose (from a 60-67 weight %
feed). After inoculation, the volume was 6.0 L and the glucose
concentration was 10 g/L. The solution of trace elements contained
(g/L): citric acid. H.sub.2O (4.0), MnSO.sub.4.H.sub.2O (3.0), NaCl
(1.0), FeSO.sub.4.7H.sub.2O (0.10), CoCl.sub.2.6H.sub.2O (0.10),
ZnSO.sub.4.7H.sub.2O (0.10), CuSO.sub.4.5H.sub.2O (0.010),
H.sub.3BO.sub.3 (0.010), and Na.sub.2MoO.sub.4.2H.sub.2O
(0.010).
Fermentation Growth:
[0228] A 15 L stirred tank fermenter was prepared with the medium
described above. The temperature was controlled at 35.degree. C.
and aqueous ammonia (20-28 weight %) was used to control pH at 6.8.
Initial values for air flow rate (set to minimum values of between
6 and 12 standard liters per min) and agitator speed (set to
minimum values of between 350 and 690 rpm) were set so that
dissolved oxygen (DO) control was initiated when OUR values reached
approximately 140 mmol/L/h. Back pressure was controlled at 0.5
bar. DO control was set at 10%. Except for minor excursions,
glucose was maintained at between 0 g/L and 10 g/L with a 60% or
67% (wt) feed. Vitamin B.sub.12 or coenzyme B.sub.12 was added as
noted below.
Fermentation with KLP23/pAH48/pDT29:
[0229] A representative fermentation summary of the conversion of
glucose to 1,3-propanediol (1,3-PD) using E. coli strain
KLP23/pAH48/pDT29 is given in Table 4. Vitamin B.sub.12 (0.075 g/L,
500 mL) was fed, starting 3 h after inoculation, at a rate of 16
mL/h. The yield of 1,3-propanediol was 24 wt % (g 1,3-propanediol/g
glucose consumed) and a titer of 68 g/L 1,3-propanediol was
obtained.
TABLE-US-00007 TABLE 4 Representative fermentation summary of the
conversion of glucose to 1,3-propanediol (1,3-PD) using E. coli
strain KLP23/pAH48/pDT29 Time OD550 DO Glucose Glycerol 1,3-PD (h)
(AU) (%) (g/L) (g/L) (g/L) 0 0 150 12.9 0.0 0 6 17 80 8.3 3.1 1 12
42 53 2.8 12.5 9 18 98 9 5.7 12.6 32 24 136 11 32.8 12.0 51 30 148
10 12.3 13.3 62 32 152 11 12.5 14.3 65 38 159 11 1.5 17.2 68
[0230] Similar results were obtained with an identical vitamin
B.sub.12 feed at twice the concentration or bolus additions of
vitamin B.sub.12 across the time course of the fermentation. The
highest titer obtained was 77 g/L.
Improved Fermentation with KLP23/pAH48/pKP32:
[0231] A representative fermentation summary of the conversion of
glucose to 1,3-propanediol (1,3-PD) using E. coli strain
KLP23/pAH48/pKP32 is given in Table 5. Vitamin B.sub.12 (0.150 g/L,
500 mL) was fed, starting 3 h after inoculation, at a rate of 16
mL/h. After 36, h, approximately 2 L of fermentation broth was
purged in order to allow for the continued addition of glucose
feed. The yield of 1,3-propanediol was 26 wt % (g 1,3-propanediol/g
glucose consumed) and a titer of 112 g/L 1,3-propanediol was
obtained.
TABLE-US-00008 TABLE 5 Representative fermentation summary of the
improved conversion of glucose to 1,3-propanediol (1,3-PD) using E.
coli strain KLP23/pAH48/pKP32 Time OD550 DO Glucose Glycerol 1,3-PD
(h) (AU) (%) (g/L) (g/L) (g/L) 0 0 148 12.8 0.0 0 6 22 84 6.9 3.3 0
12 34 90 9.7 10.4 7 18 66 43 9.3 5.9 24 24 161 9 0.2 2.5 46 30 200
10 0.2 6.0 67 36 212 10 1.2 9.7 88 42 202 2 0.1 15.5 98 48 197 12
1.2 23.8 112
[0232] Similar results were obtained with an identical vitamin
B.sub.12 feed at half the concentration or bolus additions of
vitamin B.sub.12 across the time course of the fermentation. The
highest titer obtained was 114 g/L.
Example 8
Engineering of Triosephosphate Isomerase Mutant of E. Coli KLP23
for Enhanced Yield of 1,3-Propanediol from Glucose
[0233] Construction of Plasmid for Triosephosphate Isomerase Gene
Replacement in E. coli KLP23:
[0234] E. coli KLP23 genomic DNA was prepared using the Puregene
DNA Isolation Kit (Gentra Systems, Minneapolis, Minn.). A 1.0 kb
DNA fragment containing cdh and the 3' end of triosephosphate
isomerase (tpiA) genes was amplified by PCR (Mullis and Faloona,
Methods Enzymol. 155, 335-350 (1987)) from KLP23 genomic DNA using
primers SEQ ID NO:47 and SEQ ID NO:48. A 1.0 kb DNA fragment
containing the 5' end of tpiA, yiiQ, and the 5' end of yiiR genes
was amplified by PCR from KLP23 genomic DNA using primers SEQ ID
NO:49 and SEQ ID NO:50. A ScaI site was incorporated into primer
SEQ ID NO:49. The 5' end of primer SEQ ID NO:49 was the reverse
complement of primer SEQ ID NO:48 to enable subsequent overlap
extension PCR. The gene splicing by overlap extension technique
(Horton et al., BioTechniques 8, 528-535 (1990)) was used to
generate a 2.0 kb fragment by PCR using the above two PCR fragments
as templates and primers SEQ ID NO:47 and SEQ ID NO:50. This
fragment represented a deletion of 73% of the 768 bp tpiA
structural gene. Overall, this fragment had 1.0 kb flanking regions
on either side of the ScaI cloning site (within the partial tpiA)
to allow for chromosomal gene replacement by homologous
recombination.
[0235] The above blunt-ended 2.0 kb PCR fragment was cloned into
the pCR-Blunt vector using the Zero Blunt PCR Cloning Kit
(Invitrogen, San Diego, Calif.) to yield the 5.5 kb plasmid
pRN106-2 containing kanamycin and Zeocin resistance genes. The 1.2
kb HincII fragment from pLoxCat1 (unpublished results), containing
a chloramphenicol-resistance gene flanked by bacteriophage P1 loxP
sites (Snaith et al., Gene 166, 173-174 (1995)), was used to
interrupt the tpiA fragment in plasmid pRN106-2 by ligating it to
ScaI-digested plasmid pRN106-2 to yield the 6.8 kb plasmid
pRN107-1.
Engineering of Triosephosphate Isomerase Mutant RJ8m by Linear DNA
Transformation:
[0236] Using pRN107-1 as template and primers SEQ ID NO:47 and SEQ
ID NO:50, the 3.2 kb fragment containing tpiA flanking regions and
the loxP-CmR-loxP cassette was PCR amplified and gel-extracted. E.
coli KLP23 was electrotransformed with up to 1 .mu.g of this 3.2 kb
linear DNA fragment and transformants that were
chloramphenicol-resistant (12.5 .mu.g/mL) and kanamycin-sensitive
(30 .mu.g/mL) were further screened on M9 minimal media for poor
glucose utilization on 1 mM glucose, for normal gluconate
utilization on 1 mM gluconate, and to ensure the glycerol
non-utilization phenotype of host KLP23 on 1 mM glycerol. An EcoRI
digest of genomic DNA from one such mutant, RJ8m, when probed with
the intact tpiA gene via Southern analysis (Southern, J. Mol. Biol.
98, 503-517 (1975)) indicated that it was a double-crossover
integrant (tpiA gene replacement) since the two expected 6.6 kb and
3.0 kb bands were observed, owing to the presence of an additional
EcoRI site within the chloramphenicol resistance gene. As expected,
the host KLP23 and wild-type FM5 controls yielded single 8.9 kb and
9.4 kb bands respectively. This tpiA mutant was further analyzed by
genomic PCR using primers SEQ ID NO:51 and SEQ ID NO:52, which
yielded the expected 4.6 kb PCR fragment while for the same primer
pair the host KLP23 and wild-type FM5 strains both yielded the
expected 3.9 kb PCR fragment. When cell-free extracts from tpiA
mutant RJ8m and host KLP23 were tested for tpiA activity using
glyceraldehyde 3-phosphate as substrate, no activity was observed
with RJ8m. The tpiA mutant RJ8m was electrotransformed with plasmid
pAH48 to allow glycerol production from glucose and also with both
plasmids pAH48 and pDT29 or pKP32 to allow 1,3-propanediol
production from glucose. The chloramphenicol resistance marker was
eliminated from RJ8m to give RJ8.
Example 9
Conversion of Glucose to 1,3-Propanediol Using E. Coli Strain
RJ8/pAH48/pDT29 and the Improved Process Using RJ8/pAH48/pKP32
Pre-Culture:
[0237] RJ8/pAH48/pDT29 and RJ8/pAH48/pKP32 were pre-cultured for
seeding a fermenter as described in Example 7. RJ8/pAH48/pKP32 is
identical to RJ8/pAH48/pDT29 except that dhaT is deleted.
Fermenter Medium:
[0238] Fermenter medium was as described in Example 7.
Fermentation Growth:
[0239] Fermenter growth was as described in Example 7 except that
initial values for air flow rate (set to minimum values of between
5 and 6 standard liters per min) and agitator speed (set to minimum
values of between 300 and 690 rpm) were set so that dissolved
oxygen (DO) control was initiated when OUR values reached between
60 and 100 mmol/L/h. Vitamin B.sub.12 or coenzyme B.sub.12 was
added as noted below.
Fermentation with RJ8/pAH48/pDT29:
[0240] A representative fermentation summary of the conversion of
glucose to 1,3-propanediol (1,3-PD) using E. coli strain
RJ8/pAH48/pDT29 is given in Table 6. Vitamin B.sub.12 was provided
as bolus additions of 2, 16 and 16 mg at 2, 8, and 26 h,
respectively. The yield of 1,3-propanediol was 35 wt % (g
1,3-propanediol/g glucose consumed) and a titer of 50.1 g/L
1,3-propanediol was obtained.
TABLE-US-00009 TABLE 6 Representative fermentation summary of the
conversion of glucose to 1,3-propanediol (1,3-PD) using E. coli
strain RJ8/pAH48/pDT29 Time OD550 DO Glucose Glycerol 1,3-PD (h)
(AU) (%) (g/L) (g/L) (g/L) 0 0 140 10.6 0.1 0.0 6 5 107 11.1 0.5
0.4 10 16 90 8.5 1.7 1.3 14 25 86 1.8 2.4 5.9 19 38 53 3.5 5.9 15.4
25 53 38 0.1 9.2 26.7 31 54 10 4.5 7.4 39.0 37 37 23 17.2 6.0 45.0
43 21 13 9.9 7.7 50.1
Improved Fermentation with RJ8/pAH48/pKP32:
[0241] A representative fermentation summary of the conversion of
glucose to 1,3-propanediol (1,3-PD) using E. coli strain
RJ8/pAH48/pKP32 is given in Table 7. Vitamin B.sub.12 was provided
as bolus additions of 48 and 16 mg at approximately 26 and 44 hr,
respectively. The yield of 1,3-propanediol was 34 wt % (g
1,3-propanediol/g glucose consumed) and a titer of 129 g/L
1,3-propanediol was obtained.
TABLE-US-00010 TABLE 7 Representative fermentation summary of the
improved conversion of glucose to 1,3-propanediol (1,3-PD) using E.
coli strain RJ8/pAH48/pKP32. Time OD550 DO Glucose Glycerol 1,3-PD
(h) (AU) (%) (g/L) (g/L) (g/L) 0 0 150 12.6 0.1 0 6 12 113 6.0 2.6
0 12 24 99 0.0 10.6 0 18 51 76 2.4 28.9 0 24 78 82 2.4 44.2 5 30
114 70 3.8 26.9 33 36 111 72 0.0 20.0 57 42 139 65 0.1 21.9 69 48
157 36 0.1 22.4 79 55 158 25 0.2 21.4 94 64 169 14 0.1 15.8 113 72
169 12 0.1 13.4 119 74 162 14 0.1 14.8 129
Example 10
Identification of the E. Coli Non-Specific Catalytic Activity
(yqhD) in the Improved 1,3-Propanediol Process
[0242] Demonstration of Non-Specific Catalytic Activity in
1,3-Propanediol-Producing Fermentations with the Improved
Catalyst:
[0243] A whole cell assay for 1,3-propanediol dehydrogenase
activity was used to demonstrate that the non-specific catalytic
activity in E. coli is present under fermentative conditions after
the addition of vitamin B.sub.12 and the production of
3-hydroxypropionaldehyde (3-HPA), but not before. A recombinant E.
coli strain containing the glycerol-production and
1,3-propanediol-production plasmids, pAH48 and pKP32, respectively,
was grown in 10 L fermenters, essentially as described in Example
7, but in the absence of vitamin B.sub.12. A vitamin B.sub.12 bolus
(48 mg) was added when the tanks reached approximately 100
OD.sub.550. Aliquots of cells were taken from the tanks immediately
before and 2 h post-vitamin B.sub.12 addition. The cells were
recovered by centrifugation and resuspended to their original
volume in PBS buffer containing 150 .mu.g/mL chloramphenicol to
inhibit new protein synthesis. An appropriate volume of the
chloramphenicol treated cells was added to 250 mL baffled flasks
containing a reaction mixture (PBS buffer containing 10 g/L
glucose, 10 g/L glycerol, 1 mg/L coenzyme B.sub.12, and 150
.mu.g/mL chloramphenicol) so that the final volume was 50 mL at an
OD.sub.550 of approximately 10. The flasks, protected from light,
were shaken at 250 rpm at 35.degree. C. Aliquots for HPLC analysis
were taken over time. Time-dependent production of 3-HPA was
observed in flasks containing cells recovered from the fermenter
either pre- or post-vitamin B.sub.12 addition. In direct contrast,
significant levels of 1,3-propanediol were observed only in those
flasks containing cells recovered from the fermenter post-vitamin
B.sub.12 addition.
Detection of Non-Specific Catalytic Activity in Cell-Free
Extracts:
[0244] A native gel activity stain assay was used to demonstrate
non-specific catalytic activity in cell-free extracts. Cells were
recovered, pre- and post-vitamin B.sub.12 addition, from
representative 10-L fermentations employing recombinant E. coli
strains containing the glycerol-production and
1,3-propanediol-production plasmids, pAH48 and pKP32, respectively;
and cell-free extracts were prepared by cell disruption using a
French press. The cell-free extracts, a preparation of pure
Klebsiella pneumoniae. 1,3-propanediol dehydrogenase (dhaT), and
molecular weight standards were applied to and run out on native
gradient polyacrylamide gels. The gels were then exposed to either
the substrates 1,3-propanediol and NAD.sup.+ or ethanol and
NAD.sup.+. As expected in the gels where 1,3-propanediol was the
substrate, an activity stain for DhaT was observed which migrated
on the native gel at approximately 340 Kdal. This activity was
observed only in lanes where pure Klebsiella pneumoniae.
1,3-propanediol dehydrogenase was applied. In contrast, where
1,3-propanediol was the substrate and post-vitamin B.sub.12
cell-free extracts were applied, a non-specific catalytic activity
was observed at approximately 90 Kdal. When ethanol was used as a
substrate, neither the DhaT band nor the non-specific catalytic
activity band were visible, but a separate band was found pre- and
post-vitamin B.sub.12 addition at approximately 120 Kdal. This new
band most likely represents an alcohol dehydrogenase with
specificity towards ethanol as substrate as is typically found in
all organisms.
[0245] This native gel assay, where proteins are separated by
molecular weight prior to the enzymatic assay step, offered greater
sensitivity and accuracy in measuring the reduction of
1,3-propanediol in those constructs with low activity and where the
activity is likely to be distinct from the alcohol dehydrogenases
with specificity towards ethanol as substrate that have been well
characterized for E. coli and found in all organisms. The
dehydrogenase assay works on the principle that dehydrogenase
catalyzes the transfer of electrons from 1,3-propanediol (or other
alcohols) to NAD.sup.+. PMS (phenazine methosulfate) then couples
electron transfer between NADH and a tetrazolium bromide dye (MTT,
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) which
forms a precipitate in the gel. After a few hours to overnight
soaking in the substrates, the gels are washed to remove reagents
and soluble dye. At bands on the gel where there is an active
dehydrogenase, an insoluble blue dye forms. Various aspects of the
assay have been described by Johnson and Lin (J. Bacteriol.
169:2050 (1987)).
Purification and Identification of the Non-Specific Catalytic
Activity in E. coli:
[0246] A large scale, partial purification of non-specific
catalytic activity was performed on cells harvested from the end of
a typical 1,3-propanediol production run as described in the
improved process using KLP23/pAH48/pKP32 of Example 7. The cell
pellet (16 g) was washed and resuspended three times in 20 mL of 50
mM Hepes buffer, pH 7.5. The cells in the suspension were lysed by
sonication. The cell-free extract was obtained by centrifugation
(15 min, 20,000.times.g, 10.degree. C.) and the supernatant was
further clarified by addition of 250 mg of protamine sulfate with
stirring on ice. The supernatant obtained by centrifugation (20
min, 20,000.times.g, 10.degree. C.) was fractionated by passage
through a Superdex 200 preparative grade column (6.times.60 cm)
equilibrated with Hepes buffer. Fractions of 10 mL each were
collected and an aliquot of each was concentrated twenty five-fold
using 10,000 MW cutoff Centricon.RTM. membranes prior to assay by
the native gel activity stain. The non-specific catalytic activity
was identified in fractions 107-112, and the peak activity in
fractions 108-109. A larger aliquot (7 mL each) of fractions 108
and 109 were concentrated fifty-fold and loaded on all lanes of a
12-lane native gel. The gel was cut in half and one half was
stained for dehydrogenase activity where a dark blue band appeared
that represented the non-specific catalytic activity. The unstained
gel was aligned top to bottom with the stained gel and a band was
cut on the unstained gel that corresponded to the band of
non-specific catalytic activity. The gel strip was pulverized and
soluble protein was extracted by immersing the pulverized particles
in 0.5 mL of 2D-loading buffer, heating to 95.degree. C. for 5 min,
and centrifugation to remove the gel particles. The supernatant was
loaded onto an isoelectricfocusing (IEF) strip for 2-dimension
polyacrylamide gel electrophoresis (2D-PAGE) using conditions
described for 2D-PAGE of E. coli extracts in the Swiss 2D database
(http://www.expasy.ch/ch2d/; Tonella et al. Electrophoresis
19:1960-1971 (1998)). The gel was transferred to a PVDF membrane by
electroblotting. The membrane was stained for proteins using the
Colloidal blue gel stain. The stained blot used to obtain the
identity of the non-specific catalytic activity is shown in FIG. 6.
Spots were identified using standard techniques for amino terminus
peptide sequencing. Only a single spot (Spot A) encoded for an
oxidoreductase activity. Nineteen cycles of Spot A (FIG. 6) yielded
a 100% identity match by the FASTA search tool with the
amino-terminus of yqhD, an E. coli open reading frame with putative
oxidoreductase activity. Complete amino acid sequence for the
protein encoded by yqhD is given in SEQ ID NO:57; the corresponding
DNA sequence is given in SEQ ID NO:58. The yqhD gene has 40%
identity to the gene adhB in Clostridium, a probable NADH-dependent
butanol dehydrogenase 2.
Gene Disruption of yqhD in E. coli KLP23:
[0247] Biochemical assays and amino-terminal amino acid sequencing
suggested that non-specific catalytic activity may be encoded by
the E. coli yqhD gene. This gene of unknown function encodes a
hypothetical oxidoreductase and contains two alcohol dehydrogenase
signatures also found in the Citrobacter freundii and Klebsiella
pneumoniae. 1,3-propanediol dehydrogenase encoded by the dhaT
gene.
[0248] To disrupt this gene, yqhD and 830 bp of 5'-flanking DNA
sequence and 906 bp of 3'-flanking DNA sequence were amplified from
E. coli KLP23 (Example 4) genomic DNA in a PCR using Taq polymerase
and the following primers:
TABLE-US-00011 (SEQ ID NO:59)
5'-GCGGTACCGTTGCTCGACGCTCAGGTTTTCGG-3' (SEQ ID NO:60)
5'-GCGAGCTCGACGCTTGCCCTGATCGAGTTTTGC-3'
The reaction was run at 94.degree. C. for 1 min, 50.degree. C. for
1 min, and 72.degree. C. for 3 min for 35 cycles followed by a
final extension at 72.degree. C. for 5 min. The resulting 3.7 Kb
DNA fragment was purified, digested with SacI and KpnI and ligated
to similarly digested pBluescriptII KS(+) (Strategene) for 16 h at
16.degree. C. The ligated DNA was used to transform E. coli
DH5.alpha. (Gibco/BRL) and the expected plasmid, pJSP29, was
isolated from a transformant demonstrating white colony color on LB
agar (Difco) containing X-gal (40 .mu.g/mL) and ampicillin (100
.mu.g/mL). Plasmid pJSP29 was digested with AflII and NdeI to
liberate a 409 bp DNA fragment comprising 363 bp of the yqhD gene
and 46 bp of 3'-flanking DNA sequence. The remaining 5,350 bp DNA
fragment was purified and ligated to the 1,374 bp AflII/NdeI DNA
fragment containing the kanamycin resistance gene from pLoxKan2
(Genencor International, Palo Alto, Calif.) for 16 h at 16.degree.
C. The ligated DNA was used to transform E. coli DH5.alpha. and the
expected plasmid, pJSP32-Blue, was isolated from a transformant
selected on LB agar media containing kanamycin (50 .mu.g/mL).
Plasmid pJSP32-Blue was digested with KpnI and SacI and the 3,865
bp yqhD disruption cassette was purified and ligated to similarly
digested pGP704 (Miller and Mekalanos, J. Bacteriol. 170:2575-2583
(1988)) for 16 h at 16.degree. C. The ligated DNA was used to
transform E. coli SY327 (Miller and Mekalanos, J. Bacteriol.
170:2575-2583 (1988)) and the expected plasmid, pJSP32, was
isolated from a transformant selected on LB agar media containing
kanamycin (50 .mu.g/mL). Plasmid pJSP32 was transformed into E.
coli KLP23 and transformants were selected on LB agar containing
kanamycin (50 .mu.g/mL). Of the 200 kanamycin-resistant
transformants screened, two demonstrated the ampicillin-sensitive
phenotype expected for a double-crossover recombination event
resulting in replacement of the yqhD gene with the yqhD disruption
cassette.
[0249] The disruption of the yqhD gene was confirmed by PCR using
genomic DNA isolated from these two transformants as the template
and the following sets of primer pairs:
TABLE-US-00012 Set #1: (SEQ ID NO:61)
'-GCGAGCTCGACGCTTGCCCTGATCGAGTTTTGC-3' (SEQ ID NO:62)
5'-CAGCTGGCAATTCCGGTTCG-3' Set #2: (SEQ ID NO:63)
5'-CCCAGCTGGCAATTCCGGTTCGCTTGCTGT-3' (SEQ ID NO:64)
5'-GGCGACCCGACGCTCCAGACGGAAGCTGGT-3' Set #3: (SEQ ID NO:65)
5'-CCGCAAGATTCACGGATGCATCGTGAAGGG-3' (SEQ ID NO:66)
5'-CGCCTTCTTGACGAGTTCTGAGCGGGA-3' Set #4: (SEQ ID NO: 67)
5'-GGAATTCATGAACAACTTTAATCTGCACAC-3' (SEQ ID NO: 68)
5'-GTTTGAGGCGTAAAAAGCTTAGCGGGCGGC-3'
The reactions were run using either Expand High Fidelity Polymerase
(Boehringer Manheim) or Platinum PCR Supermix containing Taq
polymerase (Gibco/BRL) at 94.degree. C. for 1 min, 50.degree. C.
for 1 min, and 72.degree. C. for 2 min for 35 cycles followed by a
final extension at 72.degree. C. for 5 min. The resulting PCR
products were analyzed by gel electrophoresis in 1.0% (w/v)
agarose. The results summarized in Table 8 confirmed disruption of
the yqhD gene in both transformants.
TABLE-US-00013 TABLE 8 Expected Size (bp) Primer Set yqhD
disruption yqhD wild-type Observed Size (bp) 1 1,200 no product
~1,200 2 1,266 no product ~1,266 3 2,594 no product ~2,594 4 no
product 1,189 ~900
The yqhD disruption deletes the 3' end of yqhD, including 46 bp of
3'-flanking intergenic DNA sequence. The deletion removes 363 bp of
3' yqhD coding sequence corresponding to 121 amino acids. A stop
codon is present 15 bp downstream of the remaining yqhD coding
sequence in the kanamycin resistance cassette.
[0250] Plasmids pAH48 and pKP32 were co-transformed into E. coli
KLP23 (yqhD.sup.-) and transformants containing both plasmids were
selected on LB agar containing ampicillin (100 .mu.g/mL) and
spectinomycin (50 .mu.g/1 mL). A representative transformant was
tested for its ability to covert glucose to 1,3-propanediol in 10 L
fermentations either in the presence or absence of vitamin
B.sub.12.
Demonstration that yqhD is Required for Significant 1,3-propanediol
Production in E. coli Strain KLP23/pAH48/pKP32:
[0251] Fermentations for the production of 1,3-propanediol were
performed, essentially as described in Example 7, with the E. coli
strain KLP23 (yqhD.sup.-)/pAH48/pKP32 in order to test for the
effect of the yqhD disruption on 1,3-propanediol production.
[0252] A representative 10-L fermentation using the knockout of the
non-specific catalytic activity, E. coli strain KLP23
(yqhD.sup.-)/pAH48/pKP32, is shown in Table 9. The organism
steadily accumulated cell mass and glycerol until the addition of
vitamin B.sub.12 when the OD.sub.550 exceeded 30 A (10.4 h).
Vitamin B.sub.12 was added as a bolus addition of 8 mg at 10.4 h
and thereafter vitamin B.sub.12 was continuously fed at a rate of
1.32 mg/h. In the 4 h that followed B.sub.12 addition, glucose
consumption slowed, the oxygen utilization rate dropped and there
was no further increase in optical density. Fermentation of glucose
ceased and the glucose concentration in the tank accumulated. The
highest titer of 1,3-propanediol obtained was 0.41 g/L. The
organism was checked for its viability by plating a dilution series
of the cells on agar plates containing ampicillin and
spectinomycin. The plates were incubated for 24 h in a 30.degree.
C. incubator. There were no viable colonies on the plate from the
fermentation of E. coli KLP23 (yqhD.sup.-)/pAH48/pKP32, Table
11.
[0253] By contrast, the cell suspension from a control tank to
which no vitamin B.sub.12 was added continued to accrue cell mass
and glycerol until the 10-L tank was full due to the complete
addition of the glucose feed solution (Table 10). An agar plate
viability determination by dilution series of the cell suspension
at the end of this fermentation showed a viable cell count that was
consistent with the total cell number estimated by the optical
density value (Table 11).
TABLE-US-00014 TABLE 9 Representative fermentation summary of the
failed conversion of glucose to 1,3-propanediol (1,3-PD) using E.
coli strain KLP23 (yqhD-)/pAH48/pKP32. time OD.sub.550 DO glucose
glycerol 1,3-PD (h) (AU) (%) (g/L) (g/L) (g/L) 0 0.4 150 11.3 0.05
0 2.3 3.0 134 10.7 0.13 0 4.3 10.8 85.0 8.2 1.41 0 8.3 23.1 81.8
0.9 10.0 0 16.3 37.2 149 13.1 21.4 0.41 18.3 47.6 149 18.9 21.6
0.39 20.3 39.6 149 24.4 22.3 0.42 23.8 33.6 149 25.4 22.0 0.41
TABLE-US-00015 TABLE 10 Representative fermentation summary of the
conversion of glucose to glycerol using E. coli strain KLP23
(yqhD-)/pAH48/pKP32. Time (h) OD.sub.550 (AU) DO (%) glucose (g/L)
glycerol (g/L) 0 0.2 148 9.5 0.06 2.2 2.8 128 8.9 0.13 4.2 10.4
58.5 7.0 1.4 8.2 21.6 57.6 2.7 11.2 16.2 76.8 10.7 0 40.5 20.2 117
10.2 0 52.9 23.7 154 8.5 0 63.9 36.2 239 10.1 0.1 122
TABLE-US-00016 TABLE 11 Representative summary of viability plate
counts from endpoints of fermentations of glucose using E. coli
strain KLP23(yqhD.sup.-)/pAH48/pKP32 in the absence and presence of
vitamin B.sub.12. vitamin B.sub.12 time (h) at endpoint OD.sub.550
(AU) viable counts (cfu/mL) no 36.2 239 2.1E11 yes 23.8 33.6 0 yes
23.8 41.2 0
Sequence CWU 1
1
68112145DNAKlebsiella pneumoniae 1gtcgaccacc acggtggtga ctttaatgcc
gctctcatgc agcagctcgg tggcggtctc 60aaaattcagg atgtcgccgg tatagttttt
gataatcagc aagacgcctt cgccgccgtc 120aatttgcatc gcgcattcaa
acattttgtc cggcgtcggc gaggtgaata tttcccccgg 180acaggcgccg
gagagcatgc cctggccgat atagccgcag tgcatcggtt catgtccgct
240gccgccgccg gagagcaggg ccaccttgcc agccaccggc gcgtcggtgc
gggtcacata 300cagcgggtcc tgatgcaggg tcagctgcgg atgggcttta
gccagcccct gtaattgttc 360attcagtaca tcttcaacac ggttaatcag
ctttttcatt attcagtgct ccgttggaga 420aggttcgatg ccgcctctct
gctggcggag gcggtcatcg cgtaggggta tcgtctgacg 480gtggagcgtg
cctggcgata tgatgattct ggctgagcgg acgaaaaaaa gaatgccccg
540acgatcgggt ttcattacga aacattgctt cctgattttg tttctttatg
gaacgttttt 600gctgaggata tggtgaaaat gcgagctggc gcgctttttt
tcttctgcca taagcggcgg 660tcaggatagc cggcgaagcg ggtgggaaaa
aattttttgc tgattttctg ccgactgcgg 720gagaaaaggc ggtcaaacac
ggaggattgt aagggcatta tgcggcaaag gagcggatcg 780ggatcgcaat
cctgacagag actagggttt tttgttccaa tatggaacgt aaaaaattaa
840cctgtgtttc atatcagaac aaaaaggcga aagatttttt tgttccctgc
cggccctaca 900gtgatcgcac tgctccggta cgctccgttc aggccgcgct
tcactggccg gcgcggataa 960cgccagggct catcatgtct acatgcgcac
ttatttgagg gtgaaaggaa tgctaaaagt 1020tattcaatct ccagccaaat
atcttcaggg tcctgatgct gctgttctgt tcggtcaata 1080tgccaaaaac
ctggcggaga gcttcttcgt catcgctgac gatttcgtaa tgaagctggc
1140gggagagaaa gtggtgaatg gcctgcagag ccacgatatt cgctgccatg
cggaacggtt 1200taacggcgaa tgcagccatg cggaaatcaa ccgtctgatg
gcgattttgc aaaaacaggg 1260ctgccgcggc gtggtcggga tcggcggtgg
taaaaccctc gataccgcga aggcgatcgg 1320ttactaccag aagctgccgg
tggtggtgat cccgaccatc gcctcgaccg atgcgccaac 1380cagcgcgctg
tcggtgatct acaccgaagc gggcgagttt gaagagtatc tgatctatcc
1440gaaaaacccg gatatggtgg tgatggacac ggcgattatc gccaaagcgc
cggtacgcct 1500gctggtctcc ggcatgggcg atgcgctctc cacctggttc
gaggccaaag cttgctacga 1560tgcgcgcgcc accagcatgg ccggaggaca
gtccaccgag gcggcgctga gcctcgcccg 1620cctgtgctat gatacgctgc
tggcggaggg cgaaaaggcc cgtctggcgg cgcaggccgg 1680ggtagtgacc
gaagcgctgg agcgcatcat cgaggcgaac acttacctca gcggcattgg
1740ctttgaaagc agtggcctgg ccgctgccca tgcaatccac aacggtttca
ccattcttga 1800agagtgccat cacctgtatc acggtgagaa agtggccttc
ggtaccctgg cgcagctggt 1860gctgcagaac agcccgatgg acgagattga
aacggtgcag ggcttctgcc agcgcgtcgg 1920cctgccggtg acgctcgcgc
agatgggcgt caaagagggg atcgacgaga aaatcgccgc 1980ggtggcgaaa
gctacctgcg cggaagggga aaccatccat aatatgccgt ttgcggtgac
2040cccggagagc gtccatgccg ctatcctcac cgccgatctg ttaggccagc
agtggctggc 2100gcgttaattc gcggtggcta aaccgctggc ccaggtcagc
ggtttttctt tctcccctcc 2160ggcagtcgct gccggagggg ttctctatgg
tacaacgcgg aaaaggatat gactgttcag 2220actcaggata ccgggaaggc
ggtctcttcc gtcattgccc agtcatggca ccgctgcagc 2280aagtttatgc
agcgcgaaac ctggcaaacg ccgcaccagg cccagggcct gaccttcgac
2340tccatctgtc ggcgtaaaac cgcgctgctc accatcggcc aggcggcgct
ggaagacgcc 2400tgggagttta tggacggccg cccctgcgcg ctgtttattc
ttgatgagtc cgcctgcatc 2460ctgagccgtt gcggcgagcc gcaaaccctg
gcccagctgg ctgccctggg atttcgcgac 2520ggcagctatt gtgcggagag
cattatcggc acctgcgcgc tgtcgctggc cgcgatgcag 2580ggccagccga
tcaacaccgc cggcgatcgg cattttaagc aggcgctaca gccatggagt
2640ttttgctcga cgccggtgtt tgataaccac gggcggctgt tcggctctat
ctcgctttgc 2700tgtctggtcg agcaccagtc cagcgccgac ctctccctga
cgctggccat cgcccgcgag 2760gtgggtaact ccctgcttac cgacagcctg
ctggcggaat ccaaccgtca cctcaatcag 2820atgtacggcc tgctggagag
catggacgat ggggtgatgg cgtggaacga acagggcgtg 2880ctgcagtttc
tcaatgttca ggcggcgaga ctgctgcatc ttgatgctca ggccagccag
2940gggaaaaata tcgccgatct ggtgaccctc ccggcgctgc tgcgccgcgc
catcaaacac 3000gcccgcggcc tgaatcacgt cgaagtcacc tttgaaagtc
agcatcagtt tgtcgatgcg 3060gtgatcacct taaaaccgat tgtcgaggcg
caaggcaaca gttttattct gctgctgcat 3120ccggtggagc agatgcggca
gctgatgacc agccagctcg gtaaagtcag ccacaccttt 3180gagcagatgt
ctgccgacga tccggaaacc cgacgcctga tccactttgg ccgccaggcg
3240gcgcgcggcg gcttcccggt gctactgtgc ggcgaagagg gggtcgggaa
agagctgctg 3300agccaggcta ttcacaatga aagcgaacgg gcgggcggcc
cctacatctc cgtcaactgc 3360cagctatatg ccgacagcgt gctgggccag
gactttatgg gcagcgcccc taccgacgat 3420gaaaatggtc gcctgagccg
ccttgagctg gccaacggcg gcaccctgtt tctggaaaag 3480atcgagtatc
tggcgccgga gctgcagtcg gctctgctgc aggtgattaa gcagggcgtg
3540ctcacccgcc tcgacgcccg gcgcctgatc ccggtggatg tgaaggtgat
tgccaccacc 3600accgtcgatc tggccaatct ggtggaacag aaccgcttta
gccgccagct gtactatgcg 3660ctgcactcct ttgagatcgt catcccgccg
ctgcgcgccc gacgcaacag tattccgtcg 3720ctggtgcata accggttgaa
gagcctggag aagcgtttct cttcgcgact gaaagtggac 3780gatgacgcgc
tggcacagct ggtggcctac tcgtggccgg ggaatgattt tgagctcaac
3840agcgtcattg agaatatcgc catcagcagc gacaacggcc acattcgcct
gagtaatctg 3900ccggaatatc tcttttccga gcggccgggc ggggatagcg
cgtcatcgct gctgccggcc 3960agcctgactt ttagcgccat cgaaaaggaa
gctattattc acgccgcccg ggtgaccagc 4020gggcgggtgc aggagatgtc
gcagctgctc aatatcggcc gcaccaccct gtggcgcaaa 4080atgaagcagt
acgatattga cgccagccag ttcaagcgca agcatcaggc ctagtctctt
4140cgattcgcgc catggagaac agggcatccg acaggcgatt gctgtagcgt
ttgagcgcgt 4200cgcgcagcgg atgcgcgcgg tccatggccg tcagcaggcg
ttcgagccga cgggactggg 4260tgcgcgccac gtgcagctgg gcagaggcga
gattcctccc cgggatcacg aactgtttta 4320acgggccgct ctcggccata
ttgcggtcga taagccgctc cagggcggtg atctcctctt 4380cgccgatcgt
ctggctcagg cgggtcaggc cccgcgcatc gctggccagt tcagccccca
4440gcacgaacag cgtctgctga atatggtgca ggctttcccg cagcccggcg
tcgcgggtcg 4500tggcgtagca gacgcccagc tgggatatca gttcatcgac
ggtgccgtag gcctcgacgc 4560gaatatggtc tttctcgatg cggctgccgc
cgtacagggc ggtggtgcct ttatccccgg 4620tgcgggtata gatacgatac
attcagtttc tctcacttaa cggcaggact ttaaccagct 4680gcccggcgtt
ggcgccgagc gtacgcagtt gatcgtcgct atcggtgacg tgtccggtag
4740ccagcggcgc gtccgccggc agctgggcat gagtgagggc tatctcgccg
gacgcgctga 4800gcccgatacc cacccgcagg ggcgagcttc tggccgccag
ggcgcccagc gcagcggcgt 4860caccgcctcc gtcataggtt atggtctggc
aggggacccc ctgctcctcc agcccccagc 4920acagctcatt gatggcgccg
gcatggtgcc cgcgcggatc gtaaaacagg cgtacgcctg 4980gcggtgaaag
cgacatgacg gtcccctcgt taacactcag aatgcctggc ggaaaatcgc
5040ggcaatctcc tgctcgttgc ctttacgcgg gttcgagaac gcattgccgt
cttttagagc 5100catctccgcc atgtagggga agtcggcctc ttttaccccc
agatcgcgca gatgctgcgg 5160aataccgata tccatcgaca gacgcgtgat
agcggcgatg gctttttccg ccgcgtcgag 5220agtggacagt ccggtgatat
tttcgcccat cagttcagcg atatcggcga atttctccgg 5280gttggcgatc
aggttgtagc gcgccacatg cggcagcagg acagcgttgg ccacgccgtg
5340cggcatgtcg tacaggccgc ccagctggtg cgccatggcg tgcacgtagc
cgaggttggc 5400gttattgaaa gccatcccgg ccagcagaga agcataggcc
atgttttccc gcgcctgcag 5460attgctgccg agggccacgg cctggcgcag
gttgcgggcg atgaggcgga tcgcctgcat 5520ggcggcggcg tccgtcaccg
ggttagcgtc tttggagata taggcctcta cggcgtgggt 5580cagggcatcc
atcccggtcg ccgcggtcag ggcggccggt ttaccgatca tcagcagtgg
5640atcgttgata gagaccgacg gcagtttgcg ccagctgacg atcacaaact
tcactttggt 5700ttcggtgttg gtcaggacgc agtggcgggt gacctcgctg
gcggtgccgg cggtggtatt 5760gaccgcgacg ataggcggca gcgggttggt
cagggtctcg attccggcat actggtacag 5820atcgccctca tgggtggcgg
cgatgccgat gcctttgccg caatcgtgcg ggctgccgcc 5880gcccacggtg
acgatgatgt cgcactgttc gcggcgaaac acggcgaggc cgtcgcgcac
5940gttggtgtct ttcgggttcg gctcgacgcc gtcaaagatc gccacctcga
tcccggcctc 6000ccgcagataa tgcagggttt tgtccaccgc gccatcttta
attgcccgca ggcctttgtc 6060ggtgaccagc agggcttttt tcccccccag
cagctggcag cgttcgccga ctacggaaat 6120ggcgttgggg ccaaaaaagt
taacgtttgg caccagataa tcaaacatac gatagctcat 6180aatatacctt
ctcgcttcag gttataatgc ggaaaaacaa tccagggcgc actgggctaa
6240taattgatcc tgctcgaccg taccgccgct aacgccgacg gcgccaatta
cctgctcatt 6300aaaaataact ggcaggccgc cgccaaaaat aataattcgc
tgttggttgg ttagctgcag 6360accgtacaga gattgtcctg gctggaccgc
tgacgtaatt tcatgggtac cttgcttcag 6420gctgcaggcg ctccaggctt
tattcaggga aatatcgcag ctggagacga aggcctcgtc 6480catccgctgg
ataagcagcg tgttgcctcc gcggtcaact acggaaaaca ccaccgccac
6540gttgatctca gtggcttttt tttccaccgc cgccgccatt tgctgggcgg
cggccagggt 6600gattgtctga acttgttggc tcttgttcat cattctctcc
cgcaccagga taacgctggc 6660gcgaatagtc agtagggggc gatagtaaaa
aactattacc attcggttgg cttgctttat 6720ttttgtcagc gttattttgt
cgcccgccat gatttagtca atagggttaa aatagcgtcg 6780gaaaaacgta
attaagggcg ttttttatta attgatttat atcattgcgg gcgatcacat
6840tttttatttt tgccgccgga gtaaagtttc atagtgaaac tgtcggtaga
tttcgtgtgc 6900caaattgaaa cgaaattaaa tttatttttt tcaccactgg
ctcatttaaa gttccgctat 6960tgccggtaat ggccgggcgg caacgacgct
ggcccggcgt attcgctacc gtctgcggat 7020ttcacctttt gagccgatga
acaatgaaaa gatcaaaacg atttgcagta ctggcccagc 7080gccccgtcaa
tcaggacggg ctgattggcg agtggcctga agaggggctg atcgccatgg
7140acagcccctt tgacccggtc tcttcagtaa aagtggacaa cggtctgatc
gtcgaactgg 7200acggcaaacg ccgggaccag tttgacatga tcgaccgatt
tatcgccgat tacgcgatca 7260acgttgagcg cacagagcag gcaatgcgcc
tggaggcggt ggaaatagcc cgtatgctgg 7320tggatattca cgtcagccgg
gaggagatca ttgccatcac taccgccatc acgccggcca 7380aagcggtcga
ggtgatggcg cagatgaacg tggtggagat gatgatggcg ctgcagaaga
7440tgcgtgcccg ccggaccccc tccaaccagt gccacgtcac caatctcaaa
gataatccgg 7500tgcagattgc cgctgacgcc gccgaggccg ggatccgcgg
cttctcagaa caggagacca 7560cggtcggtat cgcgcgctac gcgccgttta
acgccctggc gctgttggtc ggttcgcagt 7620gcggccgccc cggcgtgttg
acgcagtgct cggtggaaga ggccaccgag ctggagctgg 7680gcatgcgtgg
cttaaccagc tacgccgaga cggtgtcggt ctacggcacc gaagcggtat
7740ttaccgacgg cgatgatacg ccgtggtcaa aggcgttcct cgcctcggcc
tacgcctccc 7800gcgggttgaa aatgcgctac acctccggca ccggatccga
agcgctgatg ggctattcgg 7860agagcaagtc gatgctctac ctcgaatcgc
gctgcatctt cattactaaa ggcgccgggg 7920ttcagggact gcaaaacggc
gcggtgagct gtatcggcat gaccggcgct gtgccgtcgg 7980gcattcgggc
ggtgctggcg gaaaacctga tcgcctctat gctcgacctc gaagtggcgt
8040ccgccaacga ccagactttc tcccactcgg atattcgccg caccgcgcgc
accctgatgc 8100agatgctgcc gggcaccgac tttattttct ccggctacag
cgcggtgccg aactacgaca 8160acatgttcgc cggctcgaac ttcgatgcgg
aagattttga tgattacaac atcctgcagc 8220gtgacctgat ggttgacggc
ggcctgcgtc cggtgaccga ggcggaaacc attgccattc 8280gccagaaagc
ggcgcgggcg atccaggcgg ttttccgcga gctggggctg ccgccaatcg
8340ccgacgagga ggtggaggcc gccacctacg cgcacggcag caacgagatg
ccgccgcgta 8400acgtggtgga ggatctgagt gcggtggaag agatgatgaa
gcgcaacatc accggcctcg 8460atattgtcgg cgcgctgagc cgcagcggct
ttgaggatat cgccagcaat attctcaata 8520tgctgcgcca gcgggtcacc
ggcgattacc tgcagacctc ggccattctc gatcggcagt 8580tcgaggtggt
gagtgcggtc aacgacatca atgactatca ggggccgggc accggctatc
8640gcatctctgc cgaacgctgg gcggagatca aaaatattcc gggcgtggtt
cagcccgaca 8700ccattgaata aggcggtatt cctgtgcaac agacaaccca
aattcagccc tcttttaccc 8760tgaaaacccg cgagggcggg gtagcttctg
ccgatgaacg cgccgatgaa gtggtgatcg 8820gcgtcggccc tgccttcgat
aaacaccagc atcacactct gatcgatatg ccccatggcg 8880cgatcctcaa
agagctgatt gccggggtgg aagaagaggg gcttcacgcc cgggtggtgc
8940gcattctgcg cacgtccgac gtctccttta tggcctggga tgcggccaac
ctgagcggct 9000cggggatcgg catcggtatc cagtcgaagg ggaccacggt
catccatcag cgcgatctgc 9060tgccgctcag caacctggag ctgttctccc
aggcgccgct gctgacgctg gagacctacc 9120ggcagattgg caaaaacgct
gcgcgctatg cgcgcaaaga gtcaccttcg ccggtgccgg 9180tggtgaacga
tcagatggtg cggccgaaat ttatggccaa agccgcgcta tttcatatca
9240aagagaccaa acatgtggtg caggacgccg agcccgtcac cctgcacatc
gacttagtaa 9300gggagtgacc atgagcgaga aaaccatgcg cgtgcaggat
tatccgttag ccacccgctg 9360cccggagcat atcctgacgc ctaccggcaa
accattgacc gatattaccc tcgagaaggt 9420gctctctggc gaggtgggcc
cgcaggatgt gcggatctcc cgccagaccc ttgagtacca 9480ggcgcagatt
gccgagcaga tgcagcgcca tgcggtggcg cgcaatttcc gccgcgcggc
9540ggagcttatc gccattcctg acgagcgcat tctggctatc tataacgcgc
tgcgcccgtt 9600ccgctcctcg caggcggagc tgctggcgat cgccgacgag
ctggagcaca cctggcatgc 9660gacagtgaat gccgcctttg tccgggagtc
ggcggaagtg tatcagcagc ggcataagct 9720gcgtaaagga agctaagcgg
aggtcagcat gccgttaata gccgggattg atatcggcaa 9780cgccaccacc
gaggtggcgc tggcgtccga ctacccgcag gcgagggcgt ttgttgccag
9840cgggatcgtc gcgacgacgg gcatgaaagg gacgcgggac aatatcgccg
ggaccctcgc 9900cgcgctggag caggccctgg cgaaaacacc gtggtcgatg
agcgatgtct ctcgcatcta 9960tcttaacgaa gccgcgccgg tgattggcga
tgtggcgatg gagaccatca ccgagaccat 10020tatcaccgaa tcgaccatga
tcggtcataa cccgcagacg ccgggcgggg tgggcgttgg 10080cgtggggacg
actatcgccc tcgggcggct ggcgacgctg ccggcggcgc agtatgccga
10140ggggtggatc gtactgattg acgacgccgt cgatttcctt gacgccgtgt
ggtggctcaa 10200tgaggcgctc gaccggggga tcaacgtggt ggcggcgatc
ctcaaaaagg acgacggcgt 10260gctggtgaac aaccgcctgc gtaaaaccct
gccggtggtg gatgaagtga cgctgctgga 10320gcaggtcccc gagggggtaa
tggcggcggt ggaagtggcc gcgccgggcc aggtggtgcg 10380gatcctgtcg
aatccctacg ggatcgccac cttcttcggg ctaagcccgg aagagaccca
10440ggccatcgtc cccatcgccc gcgccctgat tggcaaccgt tccgcggtgg
tgctcaagac 10500cccgcagggg gatgtgcagt cgcgggtgat cccggcgggc
aacctctaca ttagcggcga 10560aaagcgccgc ggagaggccg atgtcgccga
gggcgcggaa gccatcatgc aggcgatgag 10620cgcctgcgct ccggtacgcg
acatccgcgg cgaaccgggc acccacgccg gcggcatgct 10680tgagcgggtg
cgcaaggtaa tggcgtccct gaccggccat gagatgagcg cgatatacat
10740ccaggatctg ctggcggtgg atacgtttat tccgcgcaag gtgcagggcg
ggatggccgg 10800cgagtgcgcc atggagaatg ccgtcgggat ggcggcgatg
gtgaaagcgg atcgtctgca 10860aatgcaggtt atcgcccgcg aactgagcgc
ccgactgcag accgaggtgg tggtgggcgg 10920cgtggaggcc aacatggcca
tcgccggggc gttaaccact cccggctgtg cggcgccgct 10980ggcgatcctc
gacctcggcg ccggctcgac ggatgcggcg atcgtcaacg cggaggggca
11040gataacggcg gtccatctcg ccggggcggg gaatatggtc agcctgttga
ttaaaaccga 11100gctgggcctc gaggatcttt cgctggcgga agcgataaaa
aaatacccgc tggccaaagt 11160ggaaagcctg ttcagtattc gtcacgagaa
tggcgcggtg gagttctttc gggaagccct 11220cagcccggcg gtgttcgcca
aagtggtgta catcaaggag ggcgaactgg tgccgatcga 11280taacgccagc
ccgctggaaa aaattcgtct cgtgcgccgg caggcgaaag agaaagtgtt
11340tgtcaccaac tgcctgcgcg cgctgcgcca ggtctcaccc ggcggttcca
ttcgcgatat 11400cgcctttgtg gtgctggtgg gcggctcatc gctggacttt
gagatcccgc agcttatcac 11460ggaagccttg tcgcactatg gcgtggtcgc
cgggcagggc aatattcggg gaacagaagg 11520gccgcgcaat gcggtcgcca
ccgggctgct actggccggt caggcgaatt aaacgggcgc 11580tcgcgccagc
ctctctcttt aacgtgctat ttcaggatgc cgataatgaa ccagacttct
11640accttaaccg ggcagtgcgt ggccgagttt cttggcaccg gattgctcat
tttcttcggc 11700gcgggctgcg tcgctgcgct gcgggtcgcc ggggccagct
ttggtcagtg ggagatcagt 11760attatctggg gccttggcgt cgccatggcc
atctacctga cggccggtgt ctccggcgcg 11820cacctaaatc cggcggtgac
cattgccctg tggctgttcg cctgttttga acgccgcaag 11880gtgctgccgt
ttattgttgc ccagacggcc ggggccttct gcgccgccgc gctggtgtat
11940gggctctatc gccagctgtt tctcgatctt gaacagagtc agcatatcgt
gcgcggcact 12000gccgccagtc ttaacctggc cggggtcttt tccacgtacc
cgcatccaca tatcactttt 12060atacaagcgt ttgccgtgga gaccaccatc
acggcaatcc tgatggcgat gatcatggcc 12120ctgaccgacg acggcaacgg aattc
12145222DNAArtificial SequencePrimer 2gctttctgtg ctgcggcttt ag
22323DNAArtificial SequencePrimer 3tggtcgagga tccacttcac ttt
23451DNAArtificial SequencePrimer 4aaagtgaagt ggatcctcga ccaattggat
ggtggcgcag tagcaaacaa t 51523DNAArtificial SequencePrimer
5ggatcaccgc cgcagaaact acg 23625DNAArtificial SequencePrimer
6ctgtcagccg ttaagtgttc ctgtg 25723DNAArtificial SequencePrimer
7cagttcaacc tgttgatagt acg 23820DNAArtificial SequencePrimer
8atgagtcaaa catcaacctt 20920DNAArtificial SequencePrimer
9atggagaaaa aaatcactgg 201020DNAArtificial SequencePrimer
10ttacgccccg ccctgccact 201120DNAArtificial SequencePrimer
11tcagaggatg tgcacctgca 201226DNAArtificial SequencePrimer
12cgagcatgcc gcatttggca ctactc 261329DNAArtificial SequencePrimer
13gcgtctagag taggttattc ccactcttg 291426DNAArtificial
SequencePrimer 14gaagtcgacc gctgcgcctt atccgg 261528DNAArtificial
SequencePrimer 15cgcgtcgacg tttacaattt caggtggc 281623DNAArtificial
SequencePrimer 16gcagcatgct ggactggtag tag 231727DNAArtificial
SequencePrimer 17cagtctagag ttattggcaa acctacc 271825DNAArtificial
SequencePrimer 18gatgcatgcc cagggcggag acggc 251929DNAArtificial
SequencePrimer 19ctaacgattg ttctctagag aaaatgtcc
292030DNAArtificial SequencePrimer 20cacgcatgca gttcaacctg
ttgatagtac 302128DNAArtificial SequencePrimer 21gcgtctagat
ccttttaaat taaaaatg 282251DNAArtificial SequencePrimer 22gcgcggatcc
aggagtctag aattatggga ttgactacta aacctctatc t 512336DNAArtificial
SequencePrimer 23gatacgcccg ggttaccatt tcaacagatc gtcctt
362434DNAArtificial SequencePrimer 24ttgataatat aaccatggct
gctgctgctg atag 342539DNAArtificial SequencePrimer 25gtatgatatg
ttatcttgga tccaataaat ctaatcttc 392624DNAArtificial SequencePrimer
26catgactagt aaggaggaca attc 242724DNAArtificial SequencePrimer
27catggaattg tcctccttac tagt 242819DNAArtificial SequencePrimer
28ctagtaagga ggacaattc 192919DNAArtificial SequencePrimer
29catggaattg tcctcctta 193015DNAArtificial SequencePrimer
30gatccaggaa acaga 153115DNAArtificial SequencePrimer 31ctagtctgtt
tcctg 153294DNAArtificial SequenceTerminator sequence 32agcttaggag
tctagaatat tgagctcgaa ttcccgggca tgcggtaccg gatccagaaa 60aaagcccgca
cctgacagtg cgggcttttt tttt
943337DNAArtificial SequencePrimer 33ggaattcaga tctcagcaat
gagcgagaaa accatgc 373427DNAArtificial SequencePrimer 34gctctagatt
agcttccttt acgcagc 273533DNAArtificial SequencePrimer 35ggccaagctt
aaggaggtta attaaatgaa aag 333626DNAArtificial SequencePrimer
36gctctagatt attcaatggt gtcggg 263742DNAArtificial SequencePrimer
37gcgccgtcta gaattatgag ctatcgtatg tttgattatc tg
423836DNAArtificial SequencePrimer 38tctgatacgg gatcctcaga
atgcctggcg gaaaat 363918DNAArtificial SequenceLinker 39tcgacgaatt
caggagga 184018DNAArtificial SequenceLinker 40ctagtcctcc tgaattcg
18414549DNAArtificial Sequenceplasmid pCL1920 41agctcgtcag
cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag 60attgtactga
gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
120taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg
gcgatcggtg 180cgggcctctt cgctattacg ccagctggcg aaagggggat
gtgctgcaag gcgattaagt 240tgggtaacgc cagggttttc ccagtcacga
cgttgtaaaa cgacggccag tgaattcgag 300ctcggtaccc ggggatcctc
tagagtcgac ctgcaggcat gcaagcttgg cgtaatcatg 360gtcatagctg
tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
420cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
cattaattgc 480gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
tgccagctgc attaatgaat 540cggccaacgc gaattcccga cagtaagacg
ggtaagcctg ttgatgatac cgctgcctta 600ctgggtgcat tagccagtct
gaatgacctg tcacgggata atccgaagtg gtcagactgg 660aaaatcagag
ggcaggaact gctgaacagc aaaaagtcag atagcaccac atagcagacc
720cgccataaaa cgccctgaga agcccgtgac gggcttttct tgtattatgg
gtagtttcct 780tgcatgaatc cataaaaggc gcctgtagtg ccatttaccc
ccattcactg ccagagccgt 840gagcgcagcg aactgaatgt cacgaaaaag
acagcgactc aggtgcctga tggtcggaga 900caaaaggaat attcagcgat
ttgcccgagc ttgcgagggt gctacttaag cctttagggt 960tttaaggtct
gttttgtaga ggagcaaaca gcgtttgcga catccttttg taatactgcg
1020gaactgacta aagtagtgag ttatacacag ggctgggatc tattcttttt
atcttttttt 1080attctttctt tattctataa attataacca cttgaatata
aacaaaaaaa acacacaaag 1140gtctagcgga atttacagag ggtctagcag
aatttacaag ttttccagca aaggtctagc 1200agaatttaca gatacccaca
actcaaagga aaaggactag taattatcat tgactagccc 1260atctcaattg
gtatagtgat taaaatcacc tagaccaatt gagatgtatg tctgaattag
1320ttgttttcaa agcaaatgaa ctagcgatta gtcgctatga cttaacggag
catgaaacca 1380agctaatttt atgctgtgtg gcactactca accccacgat
tgaaaaccct acaaggaaag 1440aacggacggt atcgttcact tataaccaat
acgctcagat gatgaacatc agtagggaaa 1500atgcttatgg tgtattagct
aaagcaacca gagagctgat gacgagaact gtggaaatca 1560ggaatccttt
ggttaaaggc tttgagattt tccagtggac aaactatgcc aagttctcaa
1620gcgaaaaatt agaattagtt tttagtgaag agatattgcc ttatcttttc
cagttaaaaa 1680aattcataaa atataatctg gaacatgtta agtcttttga
aaacaaatac tctatgagga 1740tttatgagtg gttattaaaa gaactaacac
aaaagaaaac tcacaaggca aatatagaga 1800ttagccttga tgaatttaag
ttcatgttaa tgcttgaaaa taactaccat gagtttaaaa 1860ggcttaacca
atgggttttg aaaccaataa gtaaagattt aaacacttac agcaatatga
1920aattggtggt tgataagcga ggccgcccga ctgatacgtt gattttccaa
gttgaactag 1980atagacaaat ggatctcgta accgaacttg agaacaacca
gataaaaatg aatggtgaca 2040aaataccaac aaccattaca tcagattcct
acctacataa cggactaaga aaaacactac 2100acgatgcttt aactgcaaaa
attcagctca ccagttttga ggcaaaattt ttgagtgaca 2160tgcaaagtaa
gtatgatctc aatggttcgt tctcatggct cacgcaaaaa caacgaacca
2220cactagagaa catactggct aaatacggaa ggatctgagg ttcttatggc
tcttgtatct 2280atcagtgaag catcaagact aacaaacaaa agtagaacaa
ctgttcaccg ttacatatca 2340aagggaaaac tgtccatatg cacagatgaa
aacggtgtaa aaaagataga tacatcagag 2400cttttacgag tttttggtgc
attcaaagct gttcaccatg aacagatcga caatgtaaca 2460gatgaacagc
atgtaacacc taatagaaca ggtgaaacca gtaaaacaaa gcaactagaa
2520catgaaattg aacacctgag acaacttgtt acagctcaac agtcacacat
agacagcctg 2580aaacaggcga tgctgcttat cgaatcaaag ctgccgacaa
cacgggagcc agtgacgcct 2640cccgtgggga aaaaatcatg gcaattctgg
aagaaatagc gctttcagcc ggcaaaccgg 2700ctgaagccgg atctgcgatt
ctgataacaa actagcaaca ccagaacagc ccgtttgcgg 2760gcagcaaaac
ccgtgggaat taattcccct gctcgcgcag gctgggtgcc aagctctcgg
2820gtaacatcaa ggcccgatcc ttggagccct tgccctcccg cacgatgatc
gtgccgtgat 2880cgaaatccag atccttgacc cgcagttgca aaccctcact
gatccgcatg cccgttccat 2940acagaagctg ggcgaacaaa cgatgctcgc
cttccagaaa accgaggatg cgaaccactt 3000catccggggt cagcaccacc
ggcaagcgcc gcgacggccg aggtcttccg atctcctgaa 3060gccagggcag
atccgtgcac agcaccttgc cgtagaagaa cagcaaggcc gccaatgcct
3120gacgatgcgt ggagaccgaa accttgcgct cgttcgccag ccaggacaga
aatgcctcga 3180cttcgctgct gcccaaggtt gccgggtgac gcacaccgtg
gaaacggatg aaggcacgaa 3240cccagtggac ataagcctgt tcggttcgta
agctgtaatg caagtagcgt atgcgctcac 3300gcaactggtc cagaaccttg
accgaacgca gcggtggtaa cggcgcagtg gcggttttca 3360tggcttgtta
tgactgtttt tttggggtac agtctatgcc tcgggcatcc aagcagcaag
3420cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag caacgatgtt
acgcagcagg 3480gcagtcgccc taaaacaaag ttaaacatca tgagggaagc
ggtgatcgcc gaagtatcga 3540ctcaactatc agaggtagtt ggcgtcatcg
agcgccatct cgaaccgacg ttgctggccg 3600tacatttgta cggctccgca
gtggatggcg gcctgaagcc acacagtgat attgatttgc 3660tggttacggt
gaccgtaagg cttgatgaaa caacgcggcg agctttgatc aacgaccttt
3720tggaaacttc ggcttcccct ggagagagcg agattctccg cgctgtagaa
gtcaccattg 3780ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa
gcgcgaactg caatttggag 3840aatggcagcg caatgacatt cttgcaggta
tcttcgagcc agccacgatc gacattgatc 3900tggctatctt gctgacaaaa
gcaagagaac atagcgttgc cttggtaggt ccagcggcgg 3960aggaactctt
tgatccggtt cctgaacagg atctatttga ggcgctaaat gaaaccttaa
4020cgctatggaa ctcgccgccc gactgggctg gcgatgagcg aaatgtagtg
cttacgttgt 4080cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc
gaaggatgtc gctgccgact 4140gggcaatgga gcgcctgccg gcccagtatc
agcccgtcat acttgaagct agacaggctt 4200atcttggaca agaagaagat
cgcttggcct cgcgcgcaga tcagttggaa gaatttgtcc 4260actacgtgaa
aggcgagatc accaaggtag tcggcaaata atgtctaaca attcgttcaa
4320gccgacgccg cttcgcggcg cggcttaact caagcgttag atgcactaag
cacataattg 4380ctcacagcca aactatcagg tcaagtctgc ttttattatt
tttaagcgtg cataataagc 4440cctacacaaa ttgggagata tatcatgaaa
ggctggcttt ttcttgttat cgcaatagtt 4500ggcgaagtaa tcgcaacatc
cgcattaaaa tctagcgagg gctttacta 454942199DNAArtificial
Sequenceglucose isomerase promoter 42gaattcacta gtcgatctgt
gctgtttgcc acggtatgca gcaccagcgc gagattatgg 60gctcgcacgc tcgactgtcg
gacgggggca ctggaacgag aagtcaggcg agccgtcacg 120cccttgacaa
tgccacatcc tgagcaaata attcaaccac taaacaaatc aaccgcgttt
180cccggaggta accaagctt 1994321DNAArtificial SequencePrimer
43gacgcaacag tattccgtcg c 214442DNAArtificial SequencePrimer
44atgagctatc gtatgttccg ccaggcattc tgagtgttaa cg
424533DNAArtificial SequencePrimer 45gcctggcgga acatacgata
gctcataata tac 334621DNAArtificial SequencePrimer 46cggggcgctg
ggccagtact g 214728DNAArtificial SequencePrimer 47tcaaacccgg
tggtttctcg cgaccggg 284828DNAArtificial SequencePrimer 48ctcagccgga
tatcgacggc gcgctggt 284960DNAArtificial SequencePrimer 49accagcgcgc
cgtcgatatc cggctgagta ctcaacacct gccagctctt tacgcaggtt
605028DNAArtificial SequencePrimer 50cagcatgcct gcgaaccaca ggcctatc
285128DNAArtificial SequencePrimer 51atgaacaagt ggggcgtagg gttaacat
285228DNAArtificial SequencePrimer 52ttaattactt gatttattgt cggcttta
28531380DNASaccharomyces cerevisiae 53ctttaatttt cttttatctt
actctcctac ataagacatc aagaaacaat tgtatattgt 60acaccccccc cctccacaaa
cacaaatatt gataatataa agatgtctgc tgctgctgat 120agattaaact
taacttccgg ccacttgaat gctggtagaa agagaagttc ctcttctgtt
180tctttgaagg ctgccgaaaa gcctttcaag gttactgtga ttggatctgg
taactggggt 240actactattg ccaaggtggt tgccgaaaat tgtaagggat
acccagaagt tttcgctcca 300atagtacaaa tgtgggtgtt cgaagaagag
atcaatggtg aaaaattgac tgaaatcata 360aatactagac atcaaaacgt
gaaatacttg cctggcatca ctctacccga caatttggtt 420gctaatccag
acttgattga ttcagtcaag gatgtcgaca tcatcgtttt caacattcca
480catcaatttt tgccccgtat ctgtagccaa ttgaaaggtc atgttgattc
acacgtcaga 540gctatctcct gtctaaaggg ttttgaagtt ggtgctaaag
gtgtccaatt gctatcctct 600tacatcactg aggaactagg tattcaatgt
ggtgctctat ctggtgctaa cattgccacc 660gaagtcgctc aagaacactg
gtctgaaaca acagttgctt accacattcc aaaggatttc 720agaggcgagg
gcaaggacgt cgaccataag gttctaaagg ccttgttcca cagaccttac
780ttccacgtta gtgtcatcga agatgttgct ggtatctcca tctgtggtgc
tttgaagaac 840gttgttgcct taggttgtgg tttcgtcgaa ggtctaggct
ggggtaacaa cgcttctgct 900gccatccaaa gagtcggttt gggtgagatc
atcagattcg gtcaaatgtt tttcccagaa 960tctagagaag aaacatacta
ccaagagtct gctggtgttg ctgatttgat caccacctgc 1020gctggtggta
gaaacgtcaa ggttgctagg ctaatggcta cttctggtaa ggacgcctgg
1080gaatgtgaaa aggagttgtt gaatggccaa tccgctcaag gtttaattac
ctgcaaagaa 1140gttcacgaat ggttggaaac atgtggctct gtcgaagact
tcccattatt tgaagccgta 1200taccaaatcg tttacaacaa ctacccaatg
aagaacctgc cggacatgat tgaagaatta 1260gatctacatg aagattagat
ttattggaga aagataacat atcatacttc ccccactttt 1320ttcgaggctc
ttctatatca tattcataaa ttagcattat gtcatttctc ataactactt
138054391PRTSaccharomyces cerevisiae 54Met Ser Ala Ala Ala Asp Arg
Leu Asn Leu Thr Ser Gly His Leu Asn1 5 10 15Ala Gly Arg Lys Arg Ser
Ser Ser Ser Val Ser Leu Lys Ala Ala Glu 20 25 30Lys Pro Phe Lys Val
Thr Val Ile Gly Ser Gly Asn Trp Gly Thr Thr 35 40 45Ile Ala Lys Val
Val Ala Glu Asn Cys Lys Gly Tyr Pro Glu Val Phe 50 55 60Ala Pro Ile
Val Gln Met Trp Val Phe Glu Glu Glu Ile Asn Gly Glu65 70 75 80Lys
Leu Thr Glu Ile Ile Asn Thr Arg His Gln Asn Val Lys Tyr Leu 85 90
95Pro Gly Ile Thr Leu Pro Asp Asn Leu Val Ala Asn Pro Asp Leu Ile
100 105 110Asp Ser Val Lys Asp Val Asp Ile Ile Val Phe Asn Ile Pro
His Gln 115 120 125Phe Leu Pro Arg Ile Cys Ser Gln Leu Lys Gly His
Val Asp Ser His 130 135 140Val Arg Ala Ile Ser Cys Leu Lys Gly Phe
Glu Val Gly Ala Lys Gly145 150 155 160Val Gln Leu Leu Ser Ser Tyr
Ile Thr Glu Glu Leu Gly Ile Gln Cys 165 170 175Gly Ala Leu Ser Gly
Ala Asn Ile Ala Thr Glu Val Ala Gln Glu His 180 185 190Trp Ser Glu
Thr Thr Val Ala Tyr His Ile Pro Lys Asp Phe Arg Gly 195 200 205Glu
Gly Lys Asp Val Asp His Lys Val Leu Lys Ala Leu Phe His Arg 210 215
220Pro Tyr Phe His Val Ser Val Ile Glu Asp Val Ala Gly Ile Ser
Ile225 230 235 240Cys Gly Ala Leu Lys Asn Val Val Ala Leu Gly Cys
Gly Phe Val Glu 245 250 255Gly Leu Gly Trp Gly Asn Asn Ala Ser Ala
Ala Ile Gln Arg Val Gly 260 265 270Leu Gly Glu Ile Ile Arg Phe Gly
Gln Met Phe Phe Pro Glu Ser Arg 275 280 285Glu Glu Thr Tyr Tyr Gln
Glu Ser Ala Gly Val Ala Asp Leu Ile Thr 290 295 300Thr Cys Ala Gly
Gly Arg Asn Val Lys Val Ala Arg Leu Met Ala Thr305 310 315 320Ser
Gly Lys Asp Ala Trp Glu Cys Glu Lys Glu Leu Leu Asn Gly Gln 325 330
335Ser Ala Gln Gly Leu Ile Thr Cys Lys Glu Val His Glu Trp Leu Glu
340 345 350Thr Cys Gly Ser Val Glu Asp Phe Pro Leu Phe Glu Ala Val
Tyr Gln 355 360 365Ile Val Tyr Asn Asn Tyr Pro Met Lys Asn Leu Pro
Asp Met Ile Glu 370 375 380Glu Leu Asp Leu His Glu Asp385
39055753DNASaccharomyces cerevisiae 55atgggattga ctactaaacc
tctatctttg aaagttaacg ccgctttgtt cgacgtcgac 60ggtaccatta tcatctctca
accagccatt gctgcattct ggagggattt cggtaaggac 120aaaccttatt
tcgatgctga acacgttatc caagtctcgc atggttggag aacgtttgat
180gccattgcta agttcgctcc agactttgcc aatgaagagt atgttaacaa
attagaagct 240gaaattccgg tcaagtacgg tgaaaaatcc attgaagtcc
caggtgcagt taagctgtgc 300aacgctttga acgctctacc aaaagagaaa
tgggctgtgg caacttccgg tacccgtgat 360atggcacaaa aatggttcga
gcatctggga atcaggagac caaagtactt cattaccgct 420aatgatgtca
aacagggtaa gcctcatcca gaaccatatc tgaagggcag gaatggctta
480ggatatccga tcaatgagca agacccttcc aaatctaagg tagtagtatt
tgaagacgct 540ccagcaggta ttgccgccgg aaaagccgcc ggttgtaaga
tcattggtat tgccactact 600ttcgacttgg acttcctaaa ggaaaaaggc
tgtgacatca ttgtcaaaaa ccacgaatcc 660atcagagttg gcggctacaa
tgccgaaaca gacgaagttg aattcatttt tgacgactac 720ttatatgcta
aggacgatct gttgaaatgg taa 75356250PRTSaccharomyces cerevisiae 56Met
Gly Leu Thr Thr Lys Pro Leu Ser Leu Lys Val Asn Ala Ala Leu1 5 10
15Phe Asp Val Asp Gly Thr Ile Ile Ile Ser Gln Pro Ala Ile Ala Ala
20 25 30Phe Trp Arg Asp Phe Gly Lys Asp Lys Pro Tyr Phe Asp Ala Glu
His 35 40 45Val Ile Gln Val Ser His Gly Trp Arg Thr Phe Asp Ala Ile
Ala Lys 50 55 60Phe Ala Pro Asp Phe Ala Asn Glu Glu Tyr Val Asn Lys
Leu Glu Ala65 70 75 80Glu Ile Pro Val Lys Tyr Gly Glu Lys Ser Ile
Glu Val Pro Gly Ala 85 90 95Val Lys Leu Cys Asn Ala Leu Asn Ala Leu
Pro Lys Glu Lys Trp Ala 100 105 110Val Ala Thr Ser Gly Thr Arg Asp
Met Ala Gln Lys Trp Phe Glu His 115 120 125Leu Gly Ile Arg Arg Pro
Lys Tyr Phe Ile Thr Ala Asn Asp Val Lys 130 135 140Gln Gly Lys Pro
His Pro Glu Pro Tyr Leu Lys Gly Arg Asn Gly Leu145 150 155 160Gly
Tyr Pro Ile Asn Glu Gln Asp Pro Ser Lys Ser Lys Val Val Val 165 170
175Phe Glu Asp Ala Pro Ala Gly Ile Ala Ala Gly Lys Ala Ala Gly Cys
180 185 190Lys Ile Ile Gly Ile Ala Thr Thr Phe Asp Leu Asp Phe Leu
Lys Glu 195 200 205Lys Gly Cys Asp Ile Ile Val Lys Asn His Glu Ser
Ile Arg Val Gly 210 215 220Gly Tyr Asn Ala Glu Thr Asp Glu Val Glu
Phe Ile Phe Asp Asp Tyr225 230 235 240Leu Tyr Ala Lys Asp Asp Leu
Leu Lys Trp 245 25057387PRTE. coli 57Met Asn Asn Phe Asn Leu His
Thr Pro Thr Arg Ile Leu Phe Gly Lys1 5 10 15Gly Ala Ile Ala Gly Leu
Arg Glu Gln Ile Pro His Asp Ala Arg Val 20 25 30Leu Ile Thr Tyr Gly
Gly Gly Ser Val Lys Lys Thr Gly Val Leu Asp 35 40 45Gln Val Leu Asp
Ala Leu Lys Gly Met Asp Val Leu Glu Phe Gly Gly 50 55 60Ile Glu Pro
Asn Pro Ala Tyr Glu Thr Leu Met Asn Ala Val Lys Leu65 70 75 80Val
Arg Glu Gln Lys Val Thr Phe Leu Leu Ala Val Gly Gly Gly Ser 85 90
95Val Leu Asp Gly Thr Lys Phe Ile Ala Ala Ala Ala Asn Tyr Pro Glu
100 105 110Asn Ile Asp Pro Trp His Ile Leu Gln Thr Gly Gly Lys Glu
Ile Lys 115 120 125Ser Ala Ile Pro Met Gly Cys Val Leu Thr Leu Pro
Ala Thr Gly Ser 130 135 140Glu Ser Asn Ala Gly Ala Val Ile Ser Arg
Lys Thr Thr Gly Asp Lys145 150 155 160Gln Ala Phe His Ser Ala His
Val Gln Pro Val Phe Ala Val Leu Asp 165 170 175Pro Val Tyr Thr Tyr
Thr Leu Pro Pro Arg Gln Val Ala Asn Gly Val 180 185 190Val Asp Ala
Phe Val His Thr Val Glu Gln Tyr Val Thr Lys Pro Val 195 200 205Asp
Ala Lys Ile Gln Asp Arg Phe Ala Glu Gly Ile Leu Leu Thr Leu 210 215
220Ile Glu Asp Gly Pro Lys Ala Leu Lys Glu Pro Glu Asn Tyr Asp
Val225 230 235 240Arg Ala Asn Val Met Trp Ala Ala Thr Gln Ala Leu
Asn Gly Leu Ile 245 250 255Gly Ala Gly Val Pro Gln Asp Trp Ala Thr
His Met Leu Gly His Glu 260 265 270Leu Thr Ala Met His Gly Leu Asp
His Ala Gln Thr Leu Ala Ile Val 275 280 285Leu Pro Ala Leu Trp Asn
Glu Lys Arg Asp Thr Lys Arg Ala Lys Leu 290 295 300Leu Gln Tyr Ala
Glu Arg Val Trp Asn Ile Thr Glu Gly Ser Asp Asp305 310 315 320Glu
Arg Ile Asp Ala Ala Ile Ala Ala Thr Arg Asn Phe Phe Glu Gln 325 330
335Leu Gly Val Pro Thr His Leu Ser Asp Tyr Gly Leu Asp Gly Ser Ser
340 345 350Ile Pro Ala Leu Leu Lys Lys Leu Glu Glu His Gly Met Thr
Gln Leu 355 360 365Gly Glu Asn His Asp Ile Thr Leu Asp Val Ser Arg
Arg Ile Tyr Glu 370 375 380Ala Ala
Arg385581164DNAE. coli 58atgaacaact ttaatctgca caccccaacc
cgcattctgt ttggtaaagg cgcaatcgct 60ggtttacgcg aacaaattcc tcacgatgct
cgcgtattga ttacctacgg cggcggcagc 120gtgaaaaaaa ccggcgttct
cgatcaagtt ctggatgccc tgaaaggcat ggacgtgctg 180gaatttggcg
gtattgagcc aaacccggct tatgaaacgc tgatgaacgc cgtgaaactg
240gttcgcgaac agaaagtgac tttcctgctg gcggttggcg gcggttctgt
actggacggc 300accaaattta tcgccgcagc ggctaactat ccggaaaata
tcgatccgtg gcacattctg 360caaacgggcg gtaaagagat taaaagcgcc
atcccgatgg gctgtgtgct gacgctgcca 420gcaaccggtt cagaatccaa
cgcaggcgcg gtgatctccc gtaaaaccac aggcgacaag 480caggcgttcc
attctgccca tgttcagccg gtatttgccg tgctcgatcc ggtttatacc
540tacaccctgc cgccgcgtca ggtggctaac ggcgtagtgg acgcctttgt
acacaccgtg 600gaacagtatg ttaccaaacc ggttgatgcc aaaattcagg
accgtttcgc agaaggcatt 660ttgctgacgc taatcgaaga tggtccgaaa
gccctgaaag agccagaaaa ctacgatgtg 720cgcgccaacg tcatgtgggc
ggcgactcag gcgctgaacg gtttgattgg cgctggcgta 780ccgcaggact
gggcaacgca tatgctgggc cacgaactga ctgcgatgca cggtctggat
840cacgcgcaaa cactggctat cgtcctgcct gcactgtgga atgaaaaacg
cgataccaag 900cgcgctaagc tgctgcaata tgctgaacgc gtctggaaca
tcactgaagg ttccgatgat 960gagcgtattg acgccgcgat tgccgcaacc
cgcaatttct ttgagcaatt aggcgtgccg 1020acccacctct ccgactacgg
tctggacggc agctccatcc cggctttgct gaaaaaactg 1080gaagagcacg
gcatgaccca actgggcgaa aatcatgaca ttacgttgga tgtcagccgc
1140cgtatatacg aagccgcccg ctaa 11645932DNAArtificial SequencePrimer
59gcggtaccgt tgctcgacgc tcaggttttc gg 326033DNAArtificial
SequencePrimer 60gcgagctcga cgcttgccct gatcgagttt tgc
336133DNAArtificial SequencePrimer 61gcgagctcga cgcttgccct
gatcgagttt tgc 336220DNAArtificial SequencePrimer 62cagctggcaa
ttccggttcg 206330DNAArtificial SequencePrimer 63cccagctggc
aattccggtt cgcttgctgt 306430DNAArtificial SequencePrimer
64ggcgacccga cgctccagac ggaagctggt 306530DNAArtificial
SequencePrimer 65ccgcaagatt cacggatgca tcgtgaaggg
306627DNAArtificial SequencePrimer 66cgccttcttg acgagttctg agcggga
276730DNAArtificial SequencePrimer 67ggaattcatg aacaacttta
atctgcacac 306830DNAArtificial SequencePrimer 68gtttgaggcg
taaaaagctt agcgggcggc 30
* * * * *
References