U.S. patent application number 11/282498 was filed with the patent office on 2006-08-10 for method for the production of glycerol by recombinant organisms.
Invention is credited to Ramesh V. Nair, Mark S. Payne, Donald E. Trimbur, Fernando Valle.
Application Number | 20060177915 11/282498 |
Document ID | / |
Family ID | 21855002 |
Filed Date | 2006-08-10 |
United States Patent
Application |
20060177915 |
Kind Code |
A1 |
Nair; Ramesh V. ; et
al. |
August 10, 2006 |
Method for the production of glycerol by recombinant organisms
Abstract
Recombinant organisms are provided comprising genes encoding a
glycerol-3-phosphate dehydrogenase and/or a glycerol-3-phosphatase
activity useful for the production of glycerol from a variety of
carbon substrates. The organisms further contain disruptions in the
endogenous genes encoding proteins having glycerol kinase and
glycerol dehydrogenase activities.
Inventors: |
Nair; Ramesh V.;
(Wilmington, DE) ; Payne; Mark S.; (Wilmington,
DE) ; Trimbur; Donald E.; (Redwood City, CA) ;
Valle; Fernando; (Burlingame, CA) |
Correspondence
Address: |
E I DU PONT DE NEMOURS AND COMPANY;LEGAL PATENT RECORDS CENTER
BARLEY MILL PLAZA 25/1128
4417 LANCASTER PIKE
WILMINGTON
DE
19805
US
|
Family ID: |
21855002 |
Appl. No.: |
11/282498 |
Filed: |
November 18, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09695786 |
Oct 25, 2000 |
7005291 |
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11282498 |
Nov 18, 2005 |
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08982783 |
Dec 2, 1997 |
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11282498 |
Nov 18, 2005 |
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08968418 |
Nov 12, 1997 |
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11282498 |
Nov 18, 2005 |
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60030602 |
Nov 13, 1996 |
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Current U.S.
Class: |
435/159 ;
435/190; 435/196 |
Current CPC
Class: |
C12P 7/20 20130101 |
Class at
Publication: |
435/159 ;
435/196; 435/190 |
International
Class: |
C12P 7/20 20060101
C12P007/20; C12N 9/04 20060101 C12N009/04; C12N 9/16 20060101
C12N009/16 |
Claims
1-16. (canceled)
17. A method for the production of 1,3-propanediol from a
recombinant organism comprising: (i) transforming a suitable host
cell with an expression cassette comprising either one or both of
(a) a gene encoding a protein having glycerol-3-phosphate
dehydrogenase activity, and (b) a gene encoding a protein having
glycerol-3-phosphate phosphatase activity, the suitable host cell
having at least one gene encoding a protein having a dehydratase
activity and having a disruption in either one or both of: (a) an
endogenous gene encoding a polypeptide having glycerol kinase
activity, and (b) an endogenous gene encoding a polypeptide having
glycerol dehydrogenase activity, wherein the disruption in the
genes of (a) or (b) prevents the expression of active gene product;
(ii) culturing the transformed host cell of (i) in the presence of
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
(iii) recovering the 1,3-propanediol produced in (ii).
18. The method of claim 17 wherein the protein having a dehydratase
activity is selected from the group consisting of a glycerol
dehydratase enzyme and a diol dehydratase enzyme.
19. The method of claim 18 wherein the glycerol dehydratase enzyme
is encoded by a gene, the gene isolated from a microorganism, the
microorganism selected from the group consisting of Klebsiella,
Lactobacillus, Enterobacter, Citrobacter, Pelobacter, Ilyobacter,
and Clostridium.
20. The method of claim 18 wherein the diol dehydratase enzyme is
encoded by a gene, the gene isolated from a microorganism, the
microorganism selected from the group consisting of Klebsiella and
Salmonella.
21. (canceled)
Description
FIELD OF INVENTION
[0001] This application is a divisional of U.S. application Ser.
No. 09/695,786, which is a divisional of U.S. application Ser. No.
08/982,783 filed 3 Dec. 1997 (now abandoned), which is a
continuation-in-part of U.S. application Ser. No. 08/968,418 filed
12 Nov. 1997, (now abandoned), claiming benefit to U.S. Provisional
Application No. 60/030,602, filed 13 Nov. 1996.
[0002] The present invention relates to the field of molecular
biology and the use of recombinant organisms for the production of
glycerol and compounds derived from the glycerol biosynthetic
pathway. More specifically the invention describes the construction
of a recombinant cell for the production of glycerol and derived
compounds from a carbon substrate, the cell containing foreign
genes encoding proteins having glycerol-3-phosphate dehydrogenase
(G3PDH) and glycerol-3-phosphatase (G3P phosphatase) activities
where the endogenous genes encoding the glycerol-converting
glycerol kinase and glycerol dehydrogenase activities have been
deleted.
BACKGROUND
[0003] Glycerol is a compound in great demand by industry for use
in cosmetics, liquid soaps, food, pharmaceuticals, lubricants,
anti-freeze solutions, and in numerous other applications. The
esters of glycerol are important in the fat and oil industry.
Historically, glycerol has been isolated from animal fat and
similar sources; however, the process is laborious and inefficient.
Microbial production of glycerol is preferred.
[0004] Not all organisms have a natural capacity to synthesize
glycerol. However, the biological production of glycerol is known
for some species of bacteria, algae, and yeast. The bacteria
Bacillus licheniformis and Lactobacillus lycopersica synthesize
glycerol. Glycerol production is found in the halotolerant algae
Dunaliella sp. and Asteromonas gracilis for protection against high
external salt concentrations (Ben-Amotz et al., (1982) Experientia
38:49-52). Similarly, various osmotolerant yeast synthesize
glycerol as a protective measure. Most strains of Saccharomyces
produce some glycerol during alcoholic fermentation and this
production can be increased by the application of osmotic stress
(Albertyn et al., (1994) Mol. Cell. Biol. 14, 4135-4144). Earlier
this century glycerol was produced commercially with 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 dihydroxyacetone phosphate (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 which
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.
Thus, although production of glycerol is possible from naturally
occurring organisms, production is often subject to the need to
control osmotic stress of the cultures and the production of
sulfites. A method free from these limitations is desirable.
Production of glycerol from recombinant organisms containing
foreign genes encoding key steps in the glycerol biosynthetic
pathway is one possible route to such a method.
[0005] A number of the genes involved in the glycerol biosynthetic
pathway have been isolated. For example, the gene encoding
glycerol-3-phosphate dehydrogenase (DAR1, GPD1) has been cloned and
sequenced from Saccharomyces diastaticus (Wang et al., (1994), J.
Bact. 176:7091-7095). The DAR1 gene was cloned into a shuttle
vector and used to transform E. coli where expression produced
active enzyme. Wang et al., supra, recognizes that DAR1 is
regulated by the cellular osmotic environment but does not suggest
how the gene might be used to enhance glycerol production in a
recombinant organism.
[0006] Other glycerol-3-phosphate dehydrogenase enzymes have been
isolated. For example, sn-glycerol-3-phosphate dehydrogenase has
been cloned and sequenced from S. cerevisiae (Larason et al.,
(1993) Mol. Microbiol., 10:1101). Albertyn et al., (1994) Mol.
Cell. Biol., 14:4135) teach the cloning of GPD1 encoding a
glycerol-3-phosphate dehydrogenase from S. cerevisiae. Like Wang et
al., both Albertyn et al. and Larason et al. recognize the
osmo-sensitvity of the regulation of this gene but do not suggest
how the gene might be used in the production of glycerol in a
recombinant organism.
[0007] 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., (1996) J. Biol.
Chem., 271:13875). Like the genes encoding G3PDH, it appears that
GPP2 is osmotically-induced.
[0008] Although the genes encoding G3PDH and G3P phosphatase have
been isolated, there is no teaching in the art that demonstrates
glycerol production from recombinant organisms with G3PDH/G3P
phosphatase expressed together or separately. Further, there is no
teaching to suggest that efficient glycerol production from any
wild-type organism is possible using these two enzyme activities
that does not require applying some stress (salt or an osmolyte) to
the cell. In fact, the art suggests that G3PDH activities may not
affect glycerol production. For example, Eustace ((1987), Can. J.
Microbiol., 33:112-117)) teaches hybridized yeast strains that
produced glycerol at greater levels than the parent strains.
However, Eustace also demonstrates that G3PDH activity remained
constant or slightly lower in the hybridized strains as opposed to
the wild type.
[0009] Glycerol is an industrially useful material. However, other
compounds may be derived from the glycerol biosynthetic pathway
that also have commercial significance. For example,
glycerol-producing organisms may be engineered to produce
1,3-propanediol (U.S. Pat. No. 5,686,276), a monomer having
potential utility in the production of polyester fibers and the
manufacture of polyurethanes and cyclic compounds. It is known for
example that in some organisms, glycerol is converted to
3-hydroxypropionaldehyde and then to 1,3-propanediol through the
actions of a dehydratase enzyme and an oxidoreductase enzyme,
respectively. Bacterial strains able to produce 1,3-propanediol
have been found, for example, in the groups Citrobacter,
Clostridium, Enterobacter, Ilyobacter, Klebsiella, Lactobacillus,
and Pelobacter. Glycerol dehydratase and diol dehydratase systems
are described by Seyfried et al. (1996) J. Bacteriol. 178:5793-5796
and Tobimatsu et al. (1995) J. Biol. Chem. 270:7142-7148,
respectively. Recombinant organisms, containing exogenous
dehydratase enzyme, that are able to produce 1,3-propanediol have
been described (U.S. Pat. No. 5,686,276). Although these organisms
produce 1,3-propanediol, it is clear that they would benefit from a
system that would minimize glycerol conversion.
[0010] There are a number of advantages in engineering a
glycerol-producing organism for the production of 1,3-propanediol
where conversion of glycerol is minimized. A microorganism capable
of efficiently producing glycerol under physiological conditions is
industrially desirable, especially when the glycerol itself will be
used as a substrate in vivo as part of a more complex catabolic or
biosynthetic pathway that could be perturbed by osmotic stress or
the addition of steering agents (e.g., the production of
1,3-propanediol). Some attempts at creating glycerol kinase and
glycerol dehydrogenase mutants have been made. For example, De
Koning et al. (1990) Appl. Microbiol Biotechnol. 32:693-698 report
the methanol-dependent production of dihydroxyacetone and glycerol
by mutants of the methylotrophic yeast Hansenula polymorpha blocked
in dihydroxyacetone kinase and glycerol kinase. Methanol and an
additional substrate, required to replenish the xyulose-5-phosphate
co-substrate of the assimilation reaction, were used to produce
glycerol; however, a dihydroxyacetone reductase (glycerol
dehydrogenase) is also required. Similarly, Shaw and Cameron, Book
of Abstracts, 211th ACS National Meeting, New Orleans, La., Mar.
24-28 (1996), BIOT-154 Publisher: American Chemical Society,
Washington, D.C., investigate the deletion of of ldhA (lactate
dehydrogenase), glpK (glycerol kinase), and tpiA (triosephosphate
isomerase) for the optimization of 1,3-propanediol production. They
do not suggest the expression of cloned genes for G3PDH or G3P
phosphatase for the production of glycerol or 1,3-propanediol and
they do not discuss the impact of glycerol dehydrogenase.
[0011] The problem to be solved, therefore, is the lack of a
process to direct carbon flux towards glycerol production by the
addition or enhancement of certain enzyme activities, especially
G3PDH and G3P phosphatase which respectively catalyze the
conversion of dihydroxyacetone phosphate (DHAP) to
glycerol-3-phosphate (G3P) and then to glycerol. The problem is
complicated by the need to control the carbon flux away from
glycerol by deletion or decrease of certain enzyme activities,
especially glycerol kinase and glycerol dehydrogenase which
respectively catalyze the conversion of glycerol plus ATP to G3P
and glycerol to dihydroxyacetone (or glyceraldehyde).
SUMMARY OF THE INVENTION
[0012] The present invention provides a method for the production
of glycerol from a recombinant organism comprising: transforming a
suitable host cell with an expression cassette comprising either
one or both of (a) a gene encoding a protein having
glycerol-3-phosphate dehydrogenase activity and (b) a gene encoding
a protein having glycerol-3-phosphate phosphatase activity, where
the suitable host cell contains a disruption in either one or both
of (a) a gene encoding an endogenous glycerol kinase and (b) a gene
encoding an endogenous glycerol dehydrogenase, wherein the
disruption prevents the expression of active gene product;
culturing the transformed host cell in the presence of at least one
carbon source selected from the group consisting of
monosaccharides, oligosaccharides, polysaccharides, and
single-carbon substrates, whereby glycerol is produced; and
recovering the glycerol produced.
[0013] The present invention further provides a process for the
production of 1,3-propanediol from a recombinant organism
comprising: transforming a suitable host cell with an expression
cassette comprising either one or both of (a) a gene encoding a
protein having glycerol-3-phosphate dehydrogenase activity and (b)
a gene encoding a protein having glycerol-3-phosphate phosphatase
activity, the suitable host cell having at least one gene encoding
a protein having a dehydratase activity and having a disruption in
either one or both of (a) a gene encoding an endogenous glycerol
kinase and (b) a gene encoding an endogenous glycerol
dehydrogenase, wherein the disruption in the genes of (a) or (b)
prevents the expression of active gene product; culturing the
transformed host cell in the presence of 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 recovering the
1,3-propanediol produced.
[0014] Additionally, the invention provides for a process for the
production of 1,3-propanediol from a recombinant organism where
multiple copies of endogeneous genes are introduced.
[0015] Further embodiments of the invention include host cells
transformed with heterologous genes for the glycerol pathway as
well as host cells which contain endogeneous genes for the glycerol
pathway.
[0016] Additionally, the invention provides recombinant cells
suitable for the production either glycerol or 1,3-propanediol, the
host cells having genes expressing either one or both of a
glycerol-3-phosphate dehydrogenase activity and a
glycerol-3-phosphate phosphatase activity wherein the cell also has
disruptions in either one or both of a gene encoding an endogenous
glycerol kinase and a gene encoding an endogenous glycerol
dehydrogenase, wherein the disruption in the genes prevents the
expression of active gene product.
BRIEF DESCRIPTION OF THE FIGURES, BIOLOGICAL DEPOSITS AND SEQUENCE
LISTING
[0017] FIG. 1 illustrates the representative enzymatic pathways
involving glycerol metabolism.
[0018] 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 Depositor Identification Int'l.
Depository Reference Designation Date of Deposit Escherichia coli
pAH21/DH5.alpha. ATCC 98187 26 Sep. 1996 (containing the GPP2 gene)
Escherichia coli (pDAR1A/AA200) ATCC 98248 6 Nov. 1996 (containing
the DAR1 gene) FM5 Escherichia coli RJF10m ATCC 98597 25 Nov. 1997
(containing a glpK disruption) FM5 Escherichia coli MSP33.6 ATCC
98598 25 Nov. 1997 (containing a gldA disruption)
[0019] "ATCC" refers to the American Type Culture Collection
international depository located at 10801 University Blvd,
Manassas, Va. 20110-2209 U.S.A. The designation is the accession
number of the deposited material.
[0020] Applicants have provided 43 sequences in conformity with the
Rules for the Standard Representation of Nucleotide and Amino Acid
Sequences in Patent Applications (Annexes I and II to the Decision
of the President of the EPO, published in Supplement No. 2 to OJ
EPO, 12/1992) and with 37 C.F.R. 1.821-1.825 and Appendices A and B
(Requirements for Application Disclosures Containing Nucleotides
and/or Amino Acid Sequences).
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention solves the problem stated above by
providing a method for the biological production of glycerol from a
fermentable carbon source in a recombinant organism. The method
provides a rapid, inexpensive and environmentally-responsible
source of glycerol useful in the cosmetics and pharmaceutical
industries. The method uses a microorganism containing cloned
homologous or heterologous genes encoding glycerol-3-phosphate
dehydrogenase (G3PDH) and/or glycerol-3-phosphatase (G3P
phosphatase). These genes are expressed in a recombinant host
having disruptions in genes encoding endogenous glycerol kinase
and/or glycerol dehydrogenase enzymes. The method is useful for the
production of glycerol, as well as any end products for which
glycerol is an intermediate. The recombinant microorganism is
contacted with a carbon source and cultured and then glycerol or
any end products derived therefrom are isolated from the
conditioned media. The genes may be incorporated into the host
microorganism separately or together for the production of
glycerol.
[0022] Applicants' process has not previously been described for a
recombinant organism and required the isolation of genes encoding
the two enzymes and their subsequent expression in a host cell
having disruptions in the endogenous kinase and dehydrogenase
genes. It will be appreciated by those familiar with this art that
Applicants' process may be generally applied to the production
compounds where glycerol is a key intermediate, e.g.,
1,3-propanediol.
[0023] As used herein the following terms may be used for
interpretation of the claims and specification.
[0024] 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. 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 Z35169x1), or GPD3 (GenBank
G984182), or DAR1 (GenBank Z74071x2). 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 Z47047x23), or glpD (GenBank
G147838), or glpABC (GenBank M20938).
[0025] The terms "glycerol-3-phosphatase",
"sn-glycerol-3-phosphatase", or "d,l-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 U18813x11).
[0026] 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).
[0027] 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. 1.1.1.12), or other cofactors (e.g., E.C.
1.1.99.22). A NADH-dependent glycerol dehydrogenase is encoded, for
example, by gldA (GenBank U00006).
[0028] The term "dehydratase enzyme" will refer to any enzyme that
is capable of isomerizing or converting a glycerol molecule to the
product 3-hydroxypropion-aldehyde. 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.
In Citrobacter freundii, for example, glycerol dehydratase is
encoded by three polypeptides whose gene sequences are represented
by dhaB, dhaC and dhaE (GenBank U09771: base pairs 8556-10223,
10235-10819, and 10822-11250, respectively). In Klebsiella oxytoca,
for example, diol dehydratase is encoded by three polypeptides
whose gene sequences are represented by pddA, pddB, and pddC
(GenBank D45071: base pairs 121-1785, 1796-2470, and 2485-3006,
respectively).
[0029] The terms "GPD1", "DAR1", "OSG1", "D2830", and "YDL022W"
will be used interchangeably and refer to a gene that encodes a
cytosolic glycerol-3-phosphate dehydrogenase and is characterized
by the base sequence given as SEQ ID NO: 1.
[0030] The term "GPD2" refers to a gene that encodes a cytosolic
glycerol-3-phosphate dehydrogenase and is characterized by the base
sequence given in SEQ ID NO: 2.
[0031] The terms "GUT2" and "YIL155C" are used interchangeably and
refer to a gene that encodes a mitochondrial glycerol-3-phosphate
dehydrogenase and is characterized by the base sequence given in
SEQ ID NO: 3.
[0032] The terms "GPP1", "RHR2" and "YIL053W" are used
interchangeably and refer to a gene that encodes a cytosolic
glycerol-3-phosphatase and is characterized by the base sequence
given in SEQ ID NO: 4.
[0033] The terms "GPP2", "HOR2" and "YER062C" are used
interchangeably and refer to a gene that encodes a cytosolic
glycerol-3-phosphatase and is characterized by the base sequence
given as SEQ ID NO: 5.
[0034] The term "GUT1" refers to a gene that encodes a cytosolic
glycerol kinase and is characterized by the base sequence given as
SEQ ID NO: 6. The term "glpK" refers to another gene that encodes a
glycerol kinase and is characterized by the base sequence given in
GeneBank L19201, base pairs 77347-78855.
[0035] The term "gldA" refers to a gene that encodes a glycerol
dehydrogenase and is characterized by the base sequence given in
GeneBank U00006, base pairs 31744316. The term "dhaD" refers to
another gene that encodes a glycerol dehydrogenase and is
characterized by the base sequence given in GeneBank U09771, base
pairs 2557-3654.
[0036] As used herein, the terms "function" and "enzyme function"
refer to the catalytic activity of an enzyme in altering the energy
required to perform a specific chemical reaction. Such an activity
may apply to a reaction in equilibrium where the production of both
product and substrate may be accomplished under suitable
conditions.
[0037] The terms "polypeptide" and "protein" are used
interchangeably.
[0038] The terms "carbon substrate" and "carbon source" refer to a
carbon source capable of being metabolized by host organisms of the
present invention and particularly mean carbon sources selected
from the group consisting of monosaccharides, oligosaccharides,
polysaccharides, and one-carbon substrates or mixtures thereof.
[0039] "Conversion" refers to the metabolic processes of an
organism or cell that by means of a chemical reaction degrades or
alters the complexity of a chemical compound or substrate.
[0040] The terms "host cell" and "host organism" refer to a
microorganism capable of receiving foreign or heterologous genes
and additional copies of endogeneous genes and expressing those
genes to produce an active gene product.
[0041] The terms "production cell" and "production organism" refer
to a cell engineered for the production of glycerol or compounds
that may be derived from the glycerol biosynthetic pathway. The
production cell will be recombinant and contain either one or both
of a gene that encodes a protein having a glycerol-3-phosphate
dehydrogenase activity and a gene encoding a protein having
glycerol-3-phosphatase activity. In aon to the G3PDH and G3P
phosphatase genes, the host cell will contain disruptions in one or
both of a gene encoding an endogenous glycerol kinase and a gene
encoding an endogenous glycerol dehydrogenase. Where the production
cell is designed to produce 1,3-propanediol, it will additionally
contain a gene encoding a protein having a dehydratase
activity.
[0042] The terms "foreign gene", "foreign DNA", "heterologous
gene", and "heterologous DNA" all refer to genetic material native
to one organism that has been placed within a different host
organism.
[0043] The term "endogenous" as used herein with reference to genes
or polypeptides expressed by genes, refers to genes or polypeptides
that are native to a production cell and are not derived from
another organism. Thus an "endogenous glycerol kinase" and an
"endogenous glycerol dehydrogenase" are terms referring to
polypeptides encoded by genes native to the production cell.
[0044] The terms "recombinant organism" and "transformed host"
refer to any organism transformed with heterologous or foreign
genes. The recombinant organisms of the present invention express
foreign genes encoding G3PDH and G3P phosphatase for the production
of glycerol from suitable carbon substrates. Additionally, the
terms "recombinant organism" and "transformed host" refer to any
organism transformed with endogenous (or homologous) genes so as to
increase the copy number of the genes.
[0045] "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" gene refer to the gene as found
in nature with its own regulatory sequences.
[0046] 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. The process of
encoding a specific amino acid sequence is meant to include 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. Therefore,
the invention encompasses more than the specific exemplary
sequences. 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, alterations 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.
[0047] The term "expression" refers to the transcription and
translation to gene product from a gene coding for the sequence of
the gene product.
[0048] The terms "plasmid", "vector", and "cassette" as used herein
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 facilitate 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.
[0049] 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 cell resulting from a
transformation.
[0050] The term "genetically altered" refers to the process of
changing hereditary material by transformation or mutation. The
terms "disruption" and "gene interrupt" as applied to genes refer
to a method of genetically altering an organism by adding to or
deleting from a gene a significant portion of that gene such that
the protein encoded by that gene is either not expressed or not
expressed in active form.
Glycerol Biosynthetic Pathway
[0051] It is contemplated that glycerol may be produced in
recombinant organisms by the manipulation of the glycerol
biosynthetic pathway found in most microorganisms. Typically, a
carbon substrate such as glucose is converted to
glucose-6-phosphate via hexokinase in the presence of ATP.
Glucose-phosphate isomerase catalyzes the conversion of
glucose-6-phosphate to fructose-6-phosphate and then to
fructose-1,6-diphosphate through the action of
6-phosphofructokinase. The diphosphate is then taken to
dihydroxyacetone phosphate (DHAP) via aldolase. Finally
NADH-dependent G3PDH converts DHAP to glycerol-3-phosphate which is
then dephosphorylated to glycerol by G3P phosphatase. (Agarwal
(1990), Adv. Biochem. Engrg. 41:114).
Genes Encoding G3PDH, Glycerol Dehydrogenase, G3P Phosphatase and
Glycerol Kinase
[0052] The present invention provides genes suitable for the
expression of G3PDH and G3P phosphatase activities in a host
cell.
[0053] Genes encoding G3PDH are known. For example, GPD1 has been
isolated from Saccharomyces and has the base sequence given by SEQ
ID NO: 1, encoding the amino acid sequence given in SEQ ID NO: 7
(Wang et al., supra). Similarly, G3PDH activity has also been
isolated from Saccharomyces encoded by GPD2 having the base
sequence given in SEQ ID NO: 2 encoding the amino acid sequence
given in SEQ ID NO: 8 (Eriksson et al., (1995) Mol. Microbiol.,
17:95).
[0054] For the purposes of the present invention it is contemplated
that any gene encoding a polypeptide responsible for 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 G3PDH as given by SEQ ID
NOS: 7, 8, 9, 10, 11 and 12 corresponding to the genes GPD1, GPD2,
GUT2, gpsA, glpD, and the .alpha. subunit of glpABC respectively,
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. For example, genes isolated from prokaryotes include
GenBank accessions M34393, M20938, L06231, U12567, L45246, L45323,
L45324, L45325, U32164, U32689, and U39682. Genes isolated from
fungi include GenBank accessions U30625, U30876 and X56162; genes
isolated from insects include GenBank accessions X61223 and X14179;
and genes isolated from mammalian sources include GenBank
accessions U12424, M25558 and X78593.
[0055] 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: 5, which encodes the amino acid
sequence given in SEQ ID NO: 13 (Norbeck et al., (1996), J. Biol.
Chem., 271:13875).
[0056] 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 and water to glycerol and inorganic phosphate.
Further, any gene encoding the amino acid sequence of G3P
phosphatase as given by SEQ ID NOS: 13 and 14 corresponding to the
genes GPP2 and GPP1 respectively, 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. For
example, the dephosphorylation of glycerol-3-phosphate to yield
glycerol may be achieved with one or more of the following general
or specific phosphatases: alkaline phosphatase (EC 3.1.3.1)
[GenBank M19159, M29663, U02550 or M33965]; acid phosphatase (EC
3.1.3.2) [GenBank U51210, U19789, U28658 or L20566];
glycerol-3-phosphatase (EC 3.1.3.21) [GenBank Z38060 or U18813x11];
glucose-1-phosphatase (EC 3.1.3.10) [GenBank M33807];
glucose-6-phosphatase (EC 3.1.3.9) [GenBank U00445];
fructose-1,6-bisphosphatase (EC 3.1.3.11) [GenBank XI 2545 or
J03207] or phosphotidyl glycero phosphate phosphatase (EC 3.1.3.27)
[GenBank M23546 and M23628].
[0057] Genes encoding glycerol kinase are known. For example, GUT1
encoding the glycerol kinase from Saccharomyces has been isolated
and sequenced (Pavlik et al. (1993), Curr. Genet., 24:21) and the
base sequence is given by SEQ ID NO: 6, which encodes the amino
acid sequence given in SEQ ID NO: 15. Alternatively, glpK encodes a
glycerol kinase from E. coli and is characterized by the base
sequence given in GeneBank L19201, base pairs 77347-78855.
[0058] Genes encoding glycerol dehydrogenase are known. For
example, gldA encodes a glycerol dehydrogenase from E. coli and is
characterized by the base sequence given in GeneBank U00006, base
pairs 3174-4316. Alternatively, dhaD refers to another gene that
encodes a glycerol dehydrogenase from Citrobacter freundii and is
characterized by the base sequence given in GeneBank U09771, base
pairs 2557-3654.
Host Cells
[0059] Suitable host cells for the recombinant production of
glycerol by the expression of G3PDH and G3P phosphatase may be
either prokaryotic or eukaryotic and will be limited only by their
ability to express active enzymes. Preferred host cells will be
those bacteria, yeasts, and filamentous fungi typically useful for
the production of glycerol 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 and
Saccharomyces.
[0060] Where glycerol is a key intermediate in the production of
1,3-propane-diol the host cell will either have an endogenous gene
encoding a protein having a dehydratase activity or will acquire
such a gene through transformation. Host cells particularly suited
for production of 1,3-propanediol are Citrobacter, 796
Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus,
and Salmonella, which have endogenous genes encoding dehydratase
enzymes. Additionally, host cells that lack such an endogeneous
gene include E. coli.
Vectors and Expression Cassettes
[0061] The present invention provides a variety of vectors and
transformation and expression cassettes suitable for the cloning,
transformation and expression of G3PDH and G3P phosphatase into a
suitable host cell. Suitable vectors will be those which are
compatible with the bacterium 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)).
[0062] 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.
[0063] Initiation control regions, or promoters, which are useful
to drive expression of the G3PDH and G3P phosphatase genes 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).
[0064] 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.
[0065] 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.
Transformation of Suitable Hosts and Expression of G3PDH and G3P
Phosphatase for the Production of Glycerol
[0066] Once suitable cassettes are constructed they are used to
transform appropriate host cells. Introduction of the cassette
containing the genes encoding G3PDH and/or G3P phosphatase 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).
[0067] In the present invention AH21 and DAR1 cassettes were used
to transform the E. coli DH5.alpha. and FM5 as fully described in
the GENERAL METHODS and EXAMPLES.
Random and Site Specific Mutagenisis for Disrupting Enzyme
Activities:
[0068] Enzyme pathways by which organisms metabolize glycerol are
known in the art, FIG. 1. Glycerol is converted to
glycerol-3-phosphate (G3P) by an ATP-dependent glycerol kinase; the
G3P may then be oxidized to DHAP by G3PDH. In a second pathway,
glycerol is oxidized to dihydroxyacetone (DHA) by a glycerol
dehydrogenase; the DHA may then be converted to DHAP by an
ATP-dependent DHA kinase. In a third pathway, glycerol is oxidized
to glyceraldehyde by a glycerol dehydrogenase; the glyceraldehyde
may be phosphorylated to glyceraldehyde-3-phosphate by an
ATP-dependent kinase. DHAP and glyceraldehyde-3-phosphate,
interconverted by the action of triosephosphate isomerase, may be
further metabolized via central metabolism pathways. These
pathways, by introducing by-products, are deleterious to glycerol
production.
[0069] One aspect of the present invention is the ability to
provide a production organism for the production of glycerol where
the glycerol-converting activities of glycerol kinase and glycerol
dehydrogenase have been deleted. Methods of creating deletion
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.
[0070] 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.
[0071] 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 Brock, Supra., DeMancilha et al., Food Chem., 14,
313, (1984).
[0072] Biological mutagenic agents which target genes randomly are
well known in the art. See for example De Bruijn and Rossbach in
Methods for General and Molecular Bacteriology (1994) American
Society for Microbiology, Washington, D.C. Alternatively, provided
that gene sequence is known, chromosomal gene disruption with
specific deletion or replacement is achieved by homologous
recombination with an appropriate plasmid. See for example Hamilton
et al. (1989) J. Bacteriol. 171:4617-4622, Balbas et al. (1993)
Gene 136: 211-213, Gueldener et al. (1996) Nucleic Acids Res. 24:
2519-2524, and Smith et al. (1996) Methods Mol. Cell. Biol. 5:
270-277.
[0073] It is contemplated that any of the above cited methods may
be used for the deletion or inactivation of glycerol kinase and
glycerol dehydrogenase activities in the preferred production
organism.
Media and Carbon Substrates
[0074] Fermentation media in the present invention must contain
suitable carbon substrates. Suitable substrates may include but are
not limited to mono-saccharides 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.
[0075] Glycerol production from single carbon sources (e.g.,
methanol, formaldehyde or formate) has been reported in
methylotrophic yeasts (Yamada et al. (1989), Agric. Biol. Chem.,
53(2):541-543) and in bacteria (Hunter et al. (1985), Biochemistry,
24:4148-4155). These organisms 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-monophosphate (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.
[0076] In addition to one and two carbon substrates, methylotrophic
organisms 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. (1993),
Microb. Growth C1 Compd., [Int. Symp.], 7th, 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. (1990), Arch. Microbiol.,
153(5):485-9). Hence, 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 organism.
[0077] Although all of the above mentioned carbon substrates and
mixtures thereof are suitable in the present invention, preferred
carbon substrates are monosaccharides, oligosaccharides,
polysaccharides, single-carbon substrates or mixtures thereof. More
preferred are sugars such as glucose, fructose, sucrose, maltose,
lactose and single carbon substrates such as methanol and carbon
dioxide. Most preferred as a carbon substrate is glucose.
[0078] 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 glycerol production.
Culture Conditions
[0079] Typically cells are grown at 30.degree. C. in appropriate
media. Preferred growth media 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 one 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 3':5'-monophosphate, may also be
incorporated into the reaction media. Similarly, the use of agents
known to modulate enzymatic activities (e.g., sulfites, bisulfites,
and alkalis) that lead to enhancement of glycerol production may be
used in conjunction with or as an alternative to genetic
manipulations.
[0080] Suitable pH ranges for the fermentation are between pH 5.0
to pH 9.0 where the range of pH 6.0 to pH 8.0 is preferred for the
initial condition.
[0081] Reactions may be performed under aerobic or anaerobic
conditions where anaerobic or microaerobic conditions are
preferred.
Identification of G3PDH, Glycerol Dehydrogenase, G3P Phosphatase,
and Glycerol Kinase Activities
[0082] The levels of expression of the proteins G3PDH, G3P
phosphatase glycerol dehydrogenase, and glycerol kinase are
measured by enzyme assays. Generally, G3PDH activity and glycerol
dehydrogenase activity assays rely on the spectral properties of
the cosubstrate, NADH, in the DHAP conversion to G-3-P and the DHA
conversion to glycerol, respectively. NADH has intrinsic UV/vis
absorption and its consumption can be monitored
spectrophotometrically at 340 nm. G3P phosphatase activity can be
measured by any method of measuring the inorganic phosphate
liberated in the reaction. The most commonly used detection method
uses the visible spectroscopic determination of a blue-colored
phosphomolybdate ammonium complex. Glycerol kinase activity can be
measured by the detection of G3P from glycerol and ATP, for
example, by NMR. Assays can be directed toward more specific
characteristics of individual enzymes if necessary, for example, by
the use of alternate cofactors.
Identification and Recovery of Glycerol and Other Products (e.g.
1,3-propanediol)
[0083] Glycerol and other products (e.g. 1,3-propanediol) may be
identified and quantified by high performance liquid chromatography
(HPLC) and gas chromatography/mass spectroscopy (GC/MS) analyses on
the cell-free extracts. Preferred is a HPLC method where the
fermentation media are analyzed on an analytical ion exchange
column using a mobile phase of 0.01N sulfuric acid in an isocratic
fashion.
[0084] Methods for the recovery of glycerol from fermentation media
are known in the art. For example, glycerol can be obtained from
cell media by subjecting the reaction mixture to the following
sequence of steps: filtration; water removal; organic solvent
extraction; and fractional distillation (U.S. Pat. No.
2,986,495).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0085] Production of Glycerol
[0086] The present invention describes a method for the production
of glycerol from a suitable carbon source utilizing a recombinant
organism. Particularly suitable in the invention is a bacterial
host cell, transformed with an expression cassette carrying either
or both of a gene that encodes a protein having a
glycerol-3-phosphate dehydrogenase activity and a gene encoding a
protein having a glycerol-3-phosphatase activity. In addition to
the G3PDH and G3P phosphatase genes, the host cell will contain
disruptions in either or both of genes encoding endogenous glycerol
kinase and glycerol dehydrogenase enzymes. The combined effect of
the foreign G3PDH and G3P phosphatase genes (providing a pathway
from the carbon source to glycerol) with the gene disruptions
(blocking the conversion of glycerol) results in an organism that
is capable of efficient and reliable glycerol production.
[0087] Although the optimal organism for glycerol production
contains the above mentioned gene disruptions, glycerol production
is possible with a host cell containing either one or both of the
foreign G3PDH and G3P phosphatase genes in the absence of such
disruptions. For example, the recombinant E. coli strain AA200
carrying the DAR1 gene (Example 1) was capable of producing between
0.38 g/L and 0.48 g/L of glycerol depending on fermentation
parameters. Similarly, the E. coli DH5.alpha., carrying and
expressible GPP2 gene (Example 2), was capable of 0.2 g/L of
glycerol production. Where both genes are present, (Example 3 and
4), glycerol production attained about 40 g/L. Where both genes are
present in conjunction with an elimination of the endogenous
glycerol kinase activity, a reduction in the conversion of glycerol
may be seen (Example 8). Furthermore, the presence of glycerol
dehydrogenase activity is linked to the conversion of glycerol
under glucose-limited conditions; thus, it is anticipated that the
elimination of glycerol dehydrogenase activity will result in the
reduction of glycerol conversion (Example 8).
[0088] Production of 1.3-propanediol
[0089] The present invention may also be adapted for the production
of 1,3-propanediol by utilizing recombinant organisms expressing
the foreign G3PDH and/or G3P phosphatase genes and containing
disruptions in the endogenous glycerol kinase and/or glycerol
dehydrogenase activities. Additionally, the invention provides for
the process for the production of 1,3-propanediol from a
recombinant organism where multiple copies of endogeneous genes are
introduced. In addition to these genetic alterations, the
production cell will require the presence of a gene encoding an
active dehydratase enzyme. The dehydratase enzyme activity may
either be a glycerol dehydratase or a diol dehydratase. The
dehydratase enzyme activity may result from either the expression
of an endogenous gene or from the expression of a foreign gene
transfected into the host organism. Isolation and expression of
genes encoding suitable dehydratase enzymes are well known in the
art and are taught by applicants in PCT/US96/06705, filed 5 Nov.
1996 and U.S. Pat. No. 5,686,276 and U.S. Pat. No. 5,633,362,
hereby incorporated by reference. It will be appreciated that, as
glycerol is a key intermediate in the production of
1,3-propanediol, where the host cell contains a dehydratase
activity in conjunction with expressed foreign G3PDH and/or G3P
phosphatase genes and in the absence of the glycerol-converting
glycerol kinase or glycerol dehydrogenase activities, the cell will
be particularly suited for the production of 1,3-propanediol.
[0090] The present invention is further defined in the following
Examples. It should be understood that these Examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only. From the above discussion and these Examples,
one skilled in the art can ascertain the essential characteristics
of this invention, and without departing from the spirit and scope
thereof, can make various changes and modifications of the
invention to adapt it to various usages and conditions.
EXAMPLES
General Methods
[0091] 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 et al.,
Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring
Harbor Laboratory Press (1989).
[0092] 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 in Biotechnology: A
Textbook of Industrial Microbiology (Thomas D. Brock, 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.
[0093] The meaning of abbreviations is as follows: "h" means
hour(s), "min" means minute(s), "sec" means second(s), "d" means
day(s), "1 nL" means milliliters, "L" means liters.
Cell Strains
[0094] The following Escherichia coli strains were used for
transformation and expression of G3PDH and G3P phosphatase. Strains
were obtained from the E. coli Genetic Stock Center, ATCC, or Life
Technologies (Gaithersburg, Md.). [0095] AA200 (garB10 fhuA22
ompF627 fadL701 relA1 pit-10 spoT1 tpi-1 phoM510 mcrB1) (Anderson
et al., (1970), J. Gen. Microbiol., 62:329). [0096] BB20 (tonA22
.DELTA.phoA8 fadL701 relA1 glpR2 glpD3 pit-10 gpsA20 spot1 T2R)
(Cronan et al., J. Bact., 118:598). [0097] DH5.alpha. (deoR endA1
gyrA96 hsdR17 recA1 relA1 supE44 thi-1 .DELTA.(lacZYA-argFV169)
phi80lacZ.DELTA.M15 F.sup.-) (Woodcock et al., (1989), Nucl. Acids
Res., 17:3469). [0098] FM5 Escherichia coli (ATCC 53911)
Identification of Glycerol
[0099] The conversion of glucose to glycerol was monitored by HPLC
and/or GC. 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 R1 detection. Samples were injected onto a Shodex
SH-1011 column (8 mm.times.300 mm; Waters, Milford, Mass.) equipped
with a Shodex SH-1011P precolumn (6 mm.times.50 mm),
temperature-controlled at 50.degree. C., using 0.01 N
H.sub.2SO.sub.4 as mobile phase at a flow rate of 0.69 mL/min. When
quantitative analysis was desired, samples were prepared with a
known amount of trimethylacetic acid as an external standard.
Typically, the retention times of 1,3-propanediol (R1 detection),
glycerol (R1 detection) and glucose (R1 detection) were 21.39 min,
17.03 min and 12.66 min, respectively.
[0100] Glycerol was also analyzed by GC/MS. Gas chromatography with
mass spectrometry detection for separation and quantitation of
glycerol was performed using a DB-WAX column (30 m, 0.32 mm I.D.,
0.25 um film thickness, J & W Scientific, Folsom, Calif.) at
the following conditions: injector: split, 1:15; sample volume: 1
uL; temperature profile: 150.degree. C. intitial temperature with
30 sec hold, 40.degree. C./min to 180.degree. C., 20.degree. C./min
to 240.degree. C., hold for 2.5 min. Detection: EI Mass
Spectrometry (Hewlett Packard 5971, San Fernando, Calif.),
quantitative SIM using ions 61 m/z and 64 m/z as target ions for
glycerol and glycerol-d8, and ion 43 m/z as qualifier ion for
glycerol. Glycerol-d8 was used as an internal standard.
Assay for Glycerol-3-phosphatase, G3P Phosphatase
[0101] 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
l-.alpha.-glycerol phosphate, or d,l-.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.
Spectrophotometric Assay for Glycerol 3-Phosphate Dehydrogenase
(G3PDH) Activity
[0102] The following procedure was used as modified below from a
method published by Bell et al. (1975), J. Biol. Chem., 250:7153-8.
This method involved incubating an enzyme 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. The spectrophotometer was
set to monitor absorbance changes at the fixed wavelength of 340
nm. The instrument was blanked on a cuvette containing buffer only.
After the enzyme was added to the cuvette, an absorbance reading
was taken. The first substrate, NADH (50 uL 4 mM NADH; absorbance
should increase approx 1.25 AU), was added to determine the
background rate. The rate should be followed for at least 3 min.
The second substrate, DHAP (50 .mu.L 40 mM DHAP), was then added
and the absorbance change over time was monitored for at least 3
min to determine to determine the gross rate. G3PDH activity was
defined by subtracting the background rate from the gross rate.
.sub.13C-NMR Assay for Glycerol Kinase Activity
[0103] 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 1.sup.3C 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, d, J=41 Hz and 72.66 ppm, t, J=41 Hz); glycerol 3-phosphate
(62.93 ppm, d, 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
[0104] NADH-linked glycerol dehydrogenase activity in E. coli
strains (gldA) 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 140:182).
[0105] 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 (approx. 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.
[0106] 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).
Plasmid Construction and Strain Construction
Cloning and Expression of Glycerol 3-Phosphatase for Increase of
Glycerol Production in E. coli DH5.alpha. and FM5
[0107] The Saccharomyces cerevisiae chromosomeV lamda clone 6592
(Gene Bank, accession # U18813x11) was obtained from ATCC. The
glycerol 3-phosphate phosphatase (GPP2) gene was cloned by cloning
from the lamda clone as target DNA using synthetic primers (SEQ ID
NO: 16 with SEQ ID NO: 17) 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 plasmids 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.
Plasmids for the Over-Expression of DAR1 in E. coli
[0108] DAR1 was Isolated by PCR Cloning from Genomic S. cerevisiae
DNA using synthetic primers (SEQ ID NO: 18 with SEQ ID NO: 19).
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.
[0109] 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: 20 with SEQ ID NO: 21) 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 fragement from pDAR1A and an second set of
SpeI-RBS-NcoI linker obtained by annealing synthetic primers (SEQ
ID NO: 22 with SEQ ID NO: 23) 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
[0110] 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.
[0111] 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: 24) with
CTAGTCTGTTTCCTG (SEQ ID NO: 25) 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
[0112] All the plasmids described here were transformed into E.
coli DH5.alpha. or FM5 using standard molecular biology techniques.
The transformants were verified by its DNA RPLP pattern.
Example 1
Production of Glycerol from E. coli Transformed with G3PDH Gene
Media
[0113] Synthetic media was used for anaerobic or aerobic production
of glycerol using E. coli cells transformed with pDAR1A. The media
contained per liter 6.0 g Na.sub.2HPO.sub.4, 3.0 g
KH.sub.2PO.sub.4, 1.0 g NH.sub.4Cl, 0.5 g NaCl, 1 mL 20%
MgSO.sub.40.7H.sub.2O, 8.0 g glucose, 40 mg casamino acids, 0.5 ml
1% thiamine hydrochloride, 100 mg ampicillin.
Growth Conditions
[0114] Strain AA200 harboring pDAR1A or the pTrc99A vector was
grown in aerobic conditions in 50 mL of media shaking at 250 rpm in
250 mL flasks at 37.degree. C. At A.sub.600 0.2-0.3
isopropylthio-.beta.-D-galactoside was added to a final
concentration of 1 mM and incubation continued for 48 h. For
anaerobic growth samples of induced cells were used to fill Falcon
#2054 tubes which were capped and gently mixed by rotation at
37.degree. C. for 48 h. Glycerol production was determined by HPLC
analysis of the culture supernatants. Strain pDAR1A/AA200 produced
0.38 g/L glycerol after 48 h under anaerobic conditions, and 0.48
g/L under aerobic conditions.
Example 2
Production of Glycerol from E. coli Transformed with G3P
Phosphatase Gene (GPP2)
Media
[0115] Synthetic phoA media was used in shake flasks to demonstrate
the increase of glycerol by GPP2 expression in E. coli. The phoA
medium contained per liter: Amisoy, 12 g; ammonium sulfate, 0.62 g;
MOPS, 10.5 g; Na-citrate, 1.2 g; NaOH (1 M), 10 mL; 1 M MgSO.sub.4,
12 mL; 100.times. trace elements, 12 mL; 50% glucose, 10 mL; 1%
thiamine, 10 mL; 100 mg/mL L-proline, 10 mL; 2.5 mM FeCl.sub.3, 5
mL; mixed phosphates buffer, 2 mL (5 mL 0.2 M NaH.sub.2PO.sub.4+9
mL 0.2 M K.sub.2HPO.sub.4), and pH to 7.0. The 100.times. traces
elements for phoA medium/L contained: ZnSO.sub.4.7H.sub.2O, 0.58 g;
MnSO.sub.4.H.sub.2O, 0.34 g; CuSO.sub.4.5H.sub.2O, 0.49 g;
CoCl.sub.2.6H.sub.2O, 0.47 g; H.sub.3BO.sub.3, 0.12 g,
NaMoO.sub.4.2H.sub.2O, 0.48 g.
Shake Flasks Experiments
[0116] The strains pAH21/DH5.alpha. (containing GPP2 gene) and
pPHOX2/DH5.alpha. (control) were grown in 45 mL of media (phoA
media, 50 ug/mL carbenicillin, and 1 ug/mL vitamin B.sub.12) in a
250 mL shake flask at 37.degree. C. The cultures were grown under
aerobic condition (250 rpm shaking) for 24 h. Glycerol production
was determined by HPLC analysis of the culture supernatant.
pAH21/DH5.alpha. produced 0.2 g/L glycerol after 24 h.
Example 3
Production of Glycerol from D-Glucose Using Recombinant E. coli
Containing Both GPP2 and DAR1
[0117] Growth for demonstration of increased glycerol production by
E. coli DH5.alpha.-containing pAH43 proceeds aerobically at
37.degree. C. in shake-flask cultures (erlenmeyer flasks, liquid
volume 1/5th of total volume).
[0118] Cultures in minimal media/1% glucose shake-flasks are
started by inoculation from overnight LB/1% glucose culture with
antibiotic selection. Minimal media are: filter-sterilized defined
media, final pH 6.8 (HCl), contained per liter: 12.6 g
(NH.sub.4).sub.2SO.sub.4, 13.7 g K.sub.2HPO.sub.4, 0.2 g yeast
extract (Difco), 1 g NaHCO.sub.3, 5 mg vitamin B.sub.12, 5 mL
Modified Balch's Trace-Element Solution (the composition of which
can be found in Methods for General and Molecular Bacteriology (P.
Gerhardt et al., eds, p. 158, American Society for Microbiology,
Washington, D.C. (1994)). The shake-flasks are incubated at
37.degree. C. with vigorous shaking for overnight, after which they
are sampled for GC analysis of the supernatant. The
pAH43/DH5.alpha. showed glycerol production of 3.8 g/L after 24
h.
Example 4
Production of Glycerol from D-Glucose Using Recombinant E. coli
Containing Both GPP2 and DAR1
[0119] Example 4 illustrates the production of glucose from the
recombinant E. coli DH5.alpha./pAH48, containing both the GPP2 and
DAR1 genes.
[0120] The strain DH5.alpha./pAH48 was constructed as described
above in the GENERAL METHODS.
Pre-Culture
[0121] DH5.alpha./pAH48 were pre-cultured for seeding into a
fermentation run. Components and protocols for the pre-culture are
listed below. TABLE-US-00002 Pre-Culture Media KH.sub.2PO.sub.4
30.0 g/L Citric acid 2.0 g/L MgSO.sub.4.7H.sub.2O 2.0 g/L 98%
H.sub.2SO.sub.4 2.0 mL/L Ferric ammonium citrate 0.3 g/L
CaCl.sub.2.2H.sub.2O 0.2 g/L Yeast extract 5.0 g/L Trace metals 5.0
mL/L Glucose 10.0 g/L Carbenicillin 100.0 mg/L
[0122] The above media components were mixed together and the pH
adjusted to 6.8 with NH.sub.4OH. The media was then filter
sterilized.
[0123] Trace metals were used according to the following recipe:
TABLE-US-00003 Citric acid, monohydrate 4.0 g/L
MgSO.sub.4.7H.sub.2O 3.0 g/L MnSO4.H.sub.2O 0.5 g/L NaCl 1.0 g/L
FeSO4.7H.sub.2O 0.1 g/L CoCl2.6H.sub.2O 0.1 g/L CaCl.sub.2 0.1 g/L
ZnSO.sub.4.7H.sub.2O 0.1 g/L CuSO.sub.4.5 H.sub.2O 10 mg/L
AlK(SO.sub.4).sub.2.12H.sub.2O 10 mg/L H.sub.3BO.sub.3 10 mg/L
Na.sub.2MoO.sub.4.2H.sub.2O 10 mg/L NiSO4.6H.sub.2O 10 mg/L
Na.sub.2SeO.sub.3 10 mg/L Na.sub.2WO.sub.4.2H.sub.2O 10 mg/L
[0124] Cultures were started from seed culture inoculated from 50
.mu.L frozen stock (15% glycerol as cryoprotectant) to 600 mL
medium in a 2-L Erlenmeyer flask. Cultures were grown at 30.degree.
C. in a shaker at 250 rpm for approximately 12 h and then used to
seed the fermenter. TABLE-US-00004 Fermentation growth Vessel 15-L
stirred tank fermenter Medium KH.sub.2PO.sub.4 6.8 g/L Citric acid
2.0 g/L MgSO.sub.4.7H.sub.2O 2.0 g/L 98% H.sub.2SO.sub.4 2.0 mL/L
Ferric ammonium citrate 0.3 g/L CaCl.sub.2.2H.sub.2O 0.2 g/L Mazu
DF204 antifoam 1.0 mL/L
[0125] The above components were sterilized together in the
fermenter vessel.
[0126] The pH was raised to 6.7 with NH.sub.4OH. Yeast extract (5
g/L) and trace metals solution (5 mL/L) were added aseptically from
filter sterilized stock solutions. Glucose was added from 60% feed
to give final concentration of 10 g/L. Carbenicillin was added at
100 mg/L. Volume after inoculation was 6 L.
Environmental Conditions for Fermentation
[0127] The temperature was controlled at 36.degree. C. and the air
flow rate was controlled at 6 standard liters per minute. Back
pressure was controlled at 0.5 bar. The agitator was set at 350
rpm. Aqueous ammonia was used to control pH at 6.7. The glucose
feed (60% glucose monohydrate) rate was controlled to maintain
excess glucose.
Results
[0128] The results of the fermentation run are given in Table 1.
TABLE-US-00005 TABLE 1 EFT OD550 [Glucose] [Glycerol] Total Glucose
Total Glycerol (hr) (AU) (g/L) (g/L) Fed (g) Produced (g) 0 0.8 9.3
25 6 4.7 4.0 2.0 49 14 8 5.4 0 3.6 71 25 10 6.7 0.0 4.7 116 33 12
7.4 2.1 7.0 157 49 14.2 10.4 0.3 10.0 230 70 16.2 18.1 9.7 15.5 259
106 18.2 12.4 14.5 305 20.2 11.8 17.4 17.7 353 119 22.2 11.0 12.6
382 24.2 10.8 6.5 26.6 404 178 26.2 10.9 6.8 442 28.2 10.4 10.3
31.5 463 216 30.2 10.2 13.1 30.4 493 213 32.2 10.1 8.1 28.2 512 196
34.2 10.2 3.5 33.4 530 223 36.2 10.1 5.8 548 38.2 9.8 5.1 36.1 512
233
Example 5
Engineering of Glycerol Kinase Mutants of E. coli FM5 for
Production of Glycerol from Glucose
Construction of Integration Plasmid for Glycerol Kinase Gene
Replacement in E. coli FM5
[0129] E. coli FM5 genomic DNA was prepared using the Puregene DNA
Isolation Kit (Gentra Systems, Minreapolis, Minn.). A 1.0 kb DNA
fragment containing partial glpF and glycerol kinase (glpK) genes
was amplified by PCR (Mullis and Faloona, Methods Enzymol.,
155:335-350, 1987) from FM5 genomic DNA using primers SEQ ID NO: 26
and SEQ ID NO: 27. 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: 28 and SEQ ID NO: 29. A MunI site was
incorporated into primer SEQ ID NO: 28. The 5' end of primer SEQ ID
NO: 28 was the reverse complement of primer SEQ ID NO: 27 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 2.1 kb fragment by PCR using the above two
PCR fragments as templates and primers SEQ ID NO: 26 and SEQ ID NO:
29. 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.
[0130] 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, 166:173-174,
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: 30 and SEQ ID NO: 31, 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
[0131] 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: 32 and SEQ ID NO:
33, SEQ ID NO: 34 and SEQ ID NO: 35, and SEQ ID NO: 32 and SEQ ID
NO: 35 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: 32 and SEQ ID NO: 35. 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
[0132] 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, 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: 32 and SEQ ID NO: 35 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 6
Construction of E. coli Strain with GLDA Gene Knockout
[0133] The gldA gene was isolated from E. coli by PCR (K. B. Mullis
and F. A. Faloona (1987) Meth. Enzymol. 155:335-350) using primers
SEQ ID NO: 36 and SEQ ID NO: 37, 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: 38 and SEQ ID NO:
39, 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: 40 and SEQ ID NO: 41, 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 ug/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 ug/mL kan. Approximately 32,000 cells were plated on 20
ug/mL kan and incubated for 16 hrs at 44.degree. C., which is the
restrictive temperature for pMAK705 replication. Transformants
growing at 44.degree. C. have plasmid integrated into the
chromosome, occuring at a frequency of approximately 0.0001. PCR
and Southern blot (E.M. Southern 1975 J. Mol. Biol. 98:503-517)
analyses were used to determine the nature of the chromosomal
integration events in the transformants. Western blot analysis (H.
Towbin, et al. (1979) Proc. Natl. Acad. Sci. 76:4350) 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+NAD(+).fwdarw.dihydroxyacetone+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 (C.-T. Tang, et al. (1997) J.
Bacteriol. 140:182). 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 7
Construction of E. coli Strain with GLPK and GLDA Gene
knockouts
[0134] 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: 42 and SEQ ID
NO: 43, which incorporate terminal Sph1 and Xba1 sites,
repectively, 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 bp 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 (Genecor
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. The Sph1/Xba1 fragment from
pQN8 containing the kan-interupted gldA was subcloned between the
Sph1 and Xba1 sites of pGP704, using E. coli SY327 as host, to
generate pQN9. E. coli RJF10 (see EXAMPLE 5) was transformed with
pQN9 and selected on kan. Since the pGP704 backbone cannot
replicate in most E. coli hosts, transformants arise by integration
of the plasmid, (or portions of it) into the chromosome. Double
crossover integration events are determined by identifying those
transformants which are kan resistant and ampicillin sensitive. PCR
and Southern blot analyses are used to determine the nature of the
chromosomal integration events in the transformants. Western blot
analysis is used to determine whether glycerol dehydrogenase, the
product of gldA, is produced in the transformants. Activity assays
are used to determine whether glycerol dehydrogenase activity
remains in the transformants. The kan marker is removed from the
chromosome using the Cre-producing plasmid pJW168, as described in
EXAMPLE 5.
Example 8
Consumption of Glycerol Produced from D-Glucose by Recombinant E.
coli Containing both GPP2 and DAR1 with and without Glycerol Kinase
(GLPK) Activity
[0135] EXAMPLE 8 illustrates the consumption of glycerol by the
recombinant E. coli FM5/pAH48 and RJF10/pAH48. The strains
FM5/pAH48 and RJF10/pAH48 were constructed as described above in
the GENERAL METHODS.
Pre-Culture
[0136] FM5/pAH48 and RJF10/pAH48 were pre-cultured for seeding a
fermenter in the same medium used for fermentation, or in LB
supplemented with 1% glucose. Either carbenicillin or ampicillin
were used (100 mg/L) for plasmid maintenance. The medium for
fermentation is as described in EXAMPLE 4.
[0137] Cultures were started from frozen stocks (15% glycerol as
cryoprotectant) in 600 mL medium in a 2-L Erlenmeyer flask, grown
at 30.degree. C. in a shaker at 250 rpm for approximately 12 h, and
used to seed the fermenter.
Fermentation Growth
[0138] A 15-L stirred tank fermenter with 5-7 L initial volume was
prepared as described in EXAMPLE 4. Either carbenicillin or
ampicillin were used (100 mg/L) for plasmid maintenance.
Environmental Conditions to Evaluate Glycerol Kinase (GlpK)
Activity
[0139] The temperature was controlled at 30.degree. C. and the air
flow rate controlled at 6 standard liters per minute. Back pressure
was controlled at 0.5 bar. Dissolved oxygen tension was controlled
at 10% by stirring. Aqueous ammonia was used to control pH at 6.7.
The glucose feed (60% glucose) rate was controlled to maintain
excess glucose until glycerol had accumulated to at least 25 g/L.
Glucose was then depleted, resulting in the net metabolism of
glycerol. Table 2 shows the resulting conversion of glycerol.
TABLE-US-00006 TABLE 2 Conversion of glycerol by FM5/pAH48 (wt) and
RJF10/pAH48 (glpK) rate of glycero consumption Strain number of
examples g/OD/hr FM5/pAH48 2 0.095 .+-. 0.015 RJF10/pAH48 3 0.021
.+-. 0.011
[0140] As is seen by the data in Table 2, the rate of glycerol
consumption decreases about 4-5 fold where endogenous glycerol
kinase activity is eliminated.
Environmental Conditions to Evaluate Glycerol Dehydrogenase (GldA)
Activity
[0141] The temperature was controlled at 30.degree. C. and the air
flow rate controlled at 6 standard liters per minute. Back pressure
was controlled at 0.5 bar. Dissolved oxygen tension was controlled
at 10% by stirring. Aqueous ammonia was used to control pH at 6.7.
In the first fermentation, glucose was kept in excess for the
duration of the fermentation. The second fermentation was operated
with no residual glucose after the first 25 hours. Samples over
time from the two fermentations were taken for evaluation of GlpK
and GldA activities. Table 3 summarizes RJF10/pAH48 fermentations
that show the effects of GldA on selectivity for glycerol.
TABLE-US-00007 TABLE 3 GldA and GlpK activitities from two
RJF10/pAH48 fermentations Time Overall selectivity Fermentation
(hrs) GldA GlpK (g/g) 1 25 - - 42% 46 - - 49% 61 + - 54% 2 25 + -
41% 46 ++ - 14% 61 ++ - 12%
[0142] As is seen by the data in Table 3, the presence of glycerol
dehydrogenase (GlDA) activity is linked to the conversion of
glycerol under glucose-limited conditions; thus, it is anticipated
that eliminating glycerol dehydrogenase activity will reduce
glycerol conversion.
Sequence CWU 1
1
* * * * *