CA2624764C - Method for the production of glycerol by recombinant organisms - Google Patents

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.

Description

TITLE
METHOD FOR THE PRODUCTION OF GLYCEROL
BY RECOMBINANT ORGANISMS
FIELD OF INVENTION
The present invention relates to the field of molecular biology and the use of recombinant organisms for the production of desired compounds. More specifically it describes the expression of cloned genes for glycerol-3-phosphate dehydrogenase (G3PDH) and glycerol-3-phosphatase (G3P phosphatase), either separately or together, for the enhanced production of glycerol.
BACKGROUND
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.
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 yeasts. 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 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 (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.
The gene encoding glycerol-3-phosphate dehydrogenase (DAR 1,GPD1) has been cloned and sequenced from Saccharomyces diastaticus (Wang et al., (1994),.! Boa. 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.
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, (1993)). Albertyn et al., (1994) Mot ('ell. 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.
As with G3DPH, 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. ('hem., 271:13875). Like the genes encoding G3DPFI, it appears that GPP2 is osmotically-induced.
There is no known art that teaches glycerol production from recombinant organisms with G3PDH/G3P phosphatase expressed together or separately. Nor is there known art that teaches glycerol production from any wild-type organism with these two enzyme activities that does not require applying some stress (salt or an osmolyte) to the cell. Eustace ((1987), Can. J. Microbiol., 33:112-117)) teaches away from achieving glycerol production by recombinant DNA
techniques. By selective breeding techniques, these investigators created a hybridized yeast strain that produced glycerol at greater levels than the parent strains; however, the G3PDH activity remained constant or slightly lower.
A microorganism capable of 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.
The problem to be solved, therefore, is how 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. This process has not previously been described for a recombinant organism and required the isolation of genes encoding the two enzymes and their subsequent expression. A surprising and unanticipated difficulty encountered was the toxicity of G3P phosphatase to the host which required careful control of its expression levels to avoid growth inhibition.

SUMMARY OF THE INVENTION
The present invention provides a method for the production of glycerol from a recombinant organism comprising: (i) transforming a suitable host cell with an expression cassette comprising either or both (a) a gene encoding a glycerol-3-phosphate dehydrogenase enzyme;
(b) a gene encoding a glycerol-3-phosphate phosphatase enzyme;
(ii) 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, or mixtures thereof whereby glycerol is produced; and (iii) recovering the glycerol. Glucose is the most preferred carbon source.
The invention further provides transformed host cells comprising expression cassettes capable of expressing glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase activities for the production of glycerol.
BRIEF DESCRIPTION OF BIOLOGICAL
DEPOSITS AND SEQUENCE LISTING
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:
Depositor Identification Intl. Depository Reference Designation Date of Deposit Escherichia coli pAH21/DH5a ATCC 98187 26 September 1996 (containing the GPP2 gene) Escherichia coli (pDAR1A/AA200) ATCC 98248 6 November 1996 (containing the DAR1 gene) "ATCC" refers to the American Type Culture Collection international depository located at 12301 Parklawn Drive, Rockville, Is/1D 20852 U.S.A. The designation is the accession number of the deposited material.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides 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), The microorganism is contacted with a carbon source and glycerol is isolated from the conditioned media. The genes may be incorporated into the host microorganism separately or together for the production of glycerol.
As used herein the following terms may be used for interpretation of the claims and specification.
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 by several genes including GPD1 (GenBank Z74071x2), 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 0147838), or glpABC (GenBank M20938).
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 to glycerol. G3P phosphatase is encoded by GPP1 (GenBank Z47047x125), or GPP2 (GenBank U18813x11).
The term "glycerol kinase" refers to a polypeptide responsible for an enzyme activity that catalyzes the conversion of glycerol to glycerol-3-phosphate.
or glycerol-3-phosphate to glycerol, depending on reaction conditions.
Glycerol kinase is encoded by GUT I (GenBank U11583x19).
The terms "GPD1", "DAR1", "OSG I", "D2830". and "YDL022W" will be used interchangeably and refer to a gene that encodes a cytosolic glycerol-phosphate dehydrogenase and is characterized by the base sequence given as SEQ ID NO:l.
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.
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.

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-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.
The terms "GPP2", "HOR2" and "YER062C" are used interchangeably = 5 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.
The term "GUT 1" refers to a gene that encodes a cytosolic glycerol kinase =
and is characterized by the base sequence given as SEQ ID NO:6.
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.
The terms "polypeptide" and "protein" are used herein interchangeably.
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 mono-saccharides, oligosaccharides, polysaccharides, and one-carbon substrates or mixtures thereof.
The terms "host cell" and "host organism" refer to a microorganism capable of receiving foreign or heterologous genes and expressing those genes to produce an active gene product.
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.
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.
"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.
As used herein, 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 . 10 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 (0.1X SSC, 0.1% SDS, 65 C), with the sequences exemplified herein.
The term "expression" refers to the transcription and translation to gene product from a gene coding for the sequence of the gene product.
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 are 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.

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.
The term "genetically altered" refers to the process of changing hereditary material by transformation or mutation.
Representative enzyme pathway 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).
Alternate pathways for alveerol production An alternative pathway for glycerol production from DHAP has been suggested (Wang et al., (1994).1 Boa. 176:7091-7095). In this proposed pathway DHAP could be dephosphorylated by a specific or non-specific phosphatase to give dihydroxyacetone, which could then be reduced to glycerol by a dihydroxy-acetone reductase. Dihydroxyacetone reductase is known in prokaryotes and in Schizosaccharomyces pombe, and cloning and expression of such activities together with an appropriate phosphatase could lead to glycerol production.
Another alternative pathway for glycerol production from DHAP has been suggested (Redkar (1995), Experimental Mycology, 19:241, 1995). In this pathway DHAP is isomerized to glyceraldehyde-3-phosphate by the common glycolytic enzyme triose phosphate isomerase. Glyceraldehyde-3-phosphate is dephosphorylated to glyceraldehyde, which is then reduced by alcohol dehydrogenase or a NADP-dependent glycerol dehydrogenase activity. The cloning and expression of the phosphatase and dehydrogenase activities from Aspergillus nidulans could lead to glycerol production.
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Genes encoding G3PDH and G3P phosphatase The present invention provides genes suitable for the expression of G3PDH and G3P phosphatase activities in a host cell.
Genes encoding G3PDH are known. For example, GPD I 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).
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 a 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.
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).
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 to glycerol.
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.-) [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 X12545 or J03207] or phosphotidyl glycero phosphate phosphatase (EC 3.1.3.27) [GenBank M23546 =
and M23628].
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.
The skilled artisan will appreciate that, although glycerol kinase catalyzes the degradation of glycerol in nature, the same enzyme will be able to function in the synthesis of glycerol, converting glycerol-3-phosphate to glycerol under the appropriate reaction energy conditions. Evidence exists for glycerol production through a glycerol kinase. Under anaerobic or respiration-inhibited conditions, Trypanosoma brucei gives rise to glycerol in the presence of Glycerol-3-P and ADP. The reaction occurs in the glycosome compartment (Hammond, (1985), J. Biol. Chem., 260:15646-15654).
Host cells 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.
Vectors and expression cassettes 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 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, NY, 1989)).

_ 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.
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 CYC I, HIS3, GAL I, GAL 10, ADH1, PGK, PH05, GAPDH, ADC, TRP1, URA3, LEU2, ENO. and TPI (useful for expression in Saccharomyces);
A0X1 (useful for expression in Pichia); and lac, trp, XPL, A.PR, T7, tac, and trc, (useful for expression in E. coil).
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.
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 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).
In the present invention AH21 and DAR1 cassettes were used to transform the E. coil DH5a as fully described in the GENERAL METHODS and EXAMPLES.
Media and Carbon Substrates 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, comsteep 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 (Yamada = etal. (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.
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.
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For example, methylotrophic yeast are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al. (1993), Microb.
Growth Cl 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.
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.
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 Typically cells are grown at 30 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 2':3'-mono-phosphate, 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.
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.
Reactions may be performed under aerobic or anaerobic conditions where anaerobic or microaerobic conditions are preferred.
Identification and purification of G3PDH and G3P phosphatase The levels of expression of the proteins G3PDH and G3P phosphatase are measured by enzyme assays. G3PDH activity assay relies on the spectral properties of the cosubstrate, NADH, in the DHAP conversion to G-3-P. NADH
has intrinsic UV/vis absorption and its consumption can be monitored spectro-photometrically at 340 rim. 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.
Identification and recovery of glycerol Glycerol 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 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.
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. Patent No.

2,986.495).
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Selection of transformants by complementation In the absence of a functional gpsA-encoded G3PDH, E. coli cells are unable to synthesize G3P, a condition which leads to a block in membrane biosynthesis. Cells with such a block are auxotrophic, requiring that either glycerol or G3P be present in the culture media for synthesis of membrane phospholipids.
A cloned heterologous wild-type gpsA gene is able to complement the chromosomal gpsA mutation to allow growth in media lacking glycerol or G3P
= 5 (Wang, et al. (1994), .I. Bad. 176:7091-7095). Based on this complementation strategy, growth of gpsA-defective cells on glucose would only occur if they = possessed a plasmid-encoded gpsA, allowing a selection based on synthesis of G3P from DHAP. Cells which lose the recombinant gpsA plasmid during culture would fail to synthesize G3P and cell growth would subsequently be inhibited.
The complementing G3PDH activity can be expressed not only from gpsA. but also from other cloned genes expressing G3PDH activity such as GPD1, GPD2, GPD3, GUT2, glpD, and glpABC. These can be maintained in a gpsA-defective E. coil strain such as BB20 (Cronan et al. (1974),J. Bad., 118:598), alleviating the need to use antibiotic selection and its prohibitive cost in large-scale fermentations.
A related strategy can be used for expression and selection in osmoregulatory mutants of S. cerevisiac (Larsson et al. (1993), Mol.
Microbial..
10:1101-1111). These osg I mutants are unable to grow at low water potential and show a decreased capacity for glycerol production and reduced G3PDH activity.
The osgl salt sensitivity defect can be complemented by a cloned and expressed G3PDH gene. Thus, the ability to synthesize glycerol can be used simultaneously as a selection marker for the desired glycerol-producing cells.
EXAMPLES
GENERAL METHODS
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).
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, DC. (1994) or in Biotechnology: A Textbook of Industrial Microbiology (Thomas D. Brock, Second Edition (1989) Sinauer = Associates, Inc., Sunderland, MA). All reagents and materials used for the growth and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, WI), DIFCO Laboratories (Detroit, MI), GIBCO/BRL (Gaithersburg, MD), or Sigma Chemical Company (St. Louis. MO) unless otherwise specified.

= ' WO 98/21340 It..
!Ilia', 11.4l1L7..1 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.
Cell strains 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 or from Life Technologies, Gaithersburg, MD).
AA200 (garBIO jhuA22 ompF627.fadL701 re/A] pit-10 spoT1 tpi-1 phoM510 mcrB1) (Anderson et al., (1970),]. Gen. Microbiol., 62:329).
BB20 (tonA22 dphoA8 fadL70.1 relAl glpR2 glpD3 pit-I0 gpsA20 spoT1 T2R) (Cronan et al., J. Bact., 118:598).
DH5cc (deoR endAl gyrA96 hsdR17 reek' relA I supE44 thi-I daacZYA-argFV169) phi8OlacZAM15 F-) (Woodcock et al., (1989), Nucl. Acids Res., 17:3469).
Identification of Glycerol 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 RI detection. Samples were injected onto a ShodexTm SH-1011 column (8 mm x 300 mm; Waters, Milford MA) equipped with ShodexTmSH-1011P precolumn (6 mm x 50 mm), temperature-controlled at 50 C, using 0.01 N H2SO4 as mobile phase at a flow rate of 0.5 mL/min. When quantitative analysis was desired, samples were injected onto a ShodexTm SH-1011 column (8 mm x 300 mm; Waters, Milford, MA) equipped with a ShodexTM SH-1011P precolumn (6 mm x 50 mm), temperature-controlled at 50 C, using 0.01 N H2SO4 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 glycerol (RI detection) and glucose (RI detection) were 17.03 min and 12.66 min, respectively.
Glycerol was also analyzed by GC/MS. Gas chromatography with mass spectrometry detection for and quantitation of glycerol was done using a DB-WAX column (30 m, 0.32 mm I.D., 0.25 urn film thickness, J & W Scientific, Folsom, CA), at the following conditions: injector: split, 1:15; sample volume:
1 uL; temperature profile: 150 C intitial temperature with 30 sec hold, 40 C/min to 180 C, 20 C/min to 240 C, hold for 2.5 min. Detection: El Mass Spectrometry (Hewlett Packard 5971, San Fernando, CA), 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. GPP
= 5 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-a-glycerol phosphate. or d,l-a-glycerol phosphate. The final concentrations of the reagents in the assay are: buffer (20 mM, bis-Tris or 50 mM MES); MgC12 (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 4, 200 mM), 50 mM MES, 10 mM MgCl2, 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 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 nip 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 gmol/mL.
Spectrophotometric Assay for Glycerol 3-Phosphate Dehydrogenase (G3PDH) Activity 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/HC1, pH 7.5 buffer with 5 mM DTT,in a total volume of 1.0 mL at 30 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 III. 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.
PLASMID CONSTRUCTION AND STRAIN CONSTRUCTION
Cloning and expression of _glycerol 3-phosphatase for increase of glycerol production in E. coil The Saccharomyces cerevisiae chromosomeV lamda clone 6592 ( Gene Bank, accession U18813x 1 I) 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 Smal site at the 3' end. The product was subcloned into pCR-Script (Stratagene, Madison, WI) at the Sill 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 BarnHI-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-Pstl fragment from pAH19 containing the GPP2 gene was inserted into pPHOX2 to create plasmid pAH21. The pAH21/ DI-15a is the expression plasmid.
Plasmids for the over-expression of DAR1 in E. coli 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 Ncol site at the 5' end of DAR1 where the ATG within Ncol is the DAR1 initiator methionine. At the 3' end of DAR1 a BamH1 site is introduced following the translation terminator. The PCR fragments were digested with Ncol + BamHI and cloned into the same sites within the expression plasmid pTrc99A
(Pharmacia, Piscataway, NJ) to give pDAR1A.
In order to create a better ribosome binding site at the 5' end of DAR1, an SpeI-RBS-Ncol 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 (Phamiacia, Piscataway, NJ). The Ncol-BamHI fragement from pDAR1A and a second set of Spel-RBS-NcoI linker obtained by annealing synthetic primers (SEQ ID NO:22 with SEQ ID NO:23) was inserted into the Spei-BamH1 site of pBC-SK+
(Stratagene. Madison, WI) to create plasmid pAH42. The plasmid pAH42 contains a chloramphenicol resistant gene.

=Y
Construction of expression cassettes for DAR1 and GPP2 Expression cassettes for DAR1 and GPP2 were assembled from the individual DAR1 and GPP2 subclones described above using standard molecular biology methods. The BamHI-Pstl fragment from pAH19 containing the ribosomal binding site (RBS) and GPP2 gene was inserted into pAH40 to create pAH43. The BamHI-Pstl fragment from pAH19 containing the RBS and GPP2 gene was inserted into pAH42 to create pAH45.
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 Xbal-Pstl 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, NJ).
Transformation of E. coli All the plasmids described here were transformed into E. coli DH5a using standard molecular biology techniques. The transformants were verified by its DNA RFLP pattern.

PRODUCTION OF GLYCEROL FROM E. COLI

Media 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 Na2HPO4, 3.0 g KH,PO4, 1.0 g NH4C1, 0.5 g NaC1, 1 mL 20% MgSO4.7H20, 8.0 g glucose, 40 mg casamino acids, 0.5 ml 1% thiamine hydrochloride. 100 mg ampicillin.
Growth Conditions 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 C. At A600 0.2-0.3 isopropylthio-P-D-galactoside was added to a final concentration of 1 nilvl and incubation continued for 48 h. For anaerobic growth samples of induced cells were used to fill Falcon n4 #2054 tubes which were capped and gently mixed by rotation at 37 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.

PRODUCTION OF GLYCEROL FROM E. COLI
TRANSFORMED WITH G3P PHOSPHATASE GENE (GPP2) Media Synthetic phoA media was used in shake flasks to demonstrate the increase of glyceol 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: I M MgSO4, 12 mL; 100X trace elements, 12 mL;
50% glucose, 10 mL; 1% thiamine, 10 mL; 100 mg/mL L-proline, 10 mL;
2.5 mM FeC13, 5 mL; mixed phosphates buffer, 2 mL (5 mL 0.2 M NaH2PO4+
9 mL 0.2 M K1HPO4), and pH to 7Ø The 100X traces elements for phoA
medium /L contained: ZnSO4 .7 H20, 0.58 g; MnSO4.H20, 0.34 g; CuSO4.5 1-170, 0.49 g; CoCl2.6 I-120, 0.47 g: H3B03, 0.12 g, NaMo04.2 H20, 0.48 g.
Shake Flasks Experiments The strains pAH21/DH5a (containing GPP2 gene) and pPHOX2/DH5ct (control) were grown in 45 mL of media (phoA media, 50 ug/mL carbenicillin, and 1 ug/mL vitamin Bp) in a 250 mL shake flask at 37 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. pAH2I/DH5ot produced 0.2 g/L glycerol after 24 h.

Production of glycerol from D-elucose using recombinant E. coli containing both GPP2 and DAR1 Growth for demonstration of increased glycerol production by E. coli D1-15a-containing pAH43 proceeds aerobically at 37 C in shake-flask cultures (Erlenmeyer flasks, liquid volume 1/5th of total volume).
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 (NCI), contained per liter: 12.6 g (NH4)2SO4, 13.7 g K2HPO4, 0.2 g yeast extract (Difco), 1 g NaHCO3, 5 mg vitamin B12, 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, DC (1994)). The shake-flasks are incubated at 37 C with vigorous shaking for overnight, after which they are sampled for GC analysis of the supernatant. The pAH43/DH5a showed glycerol production of 3.8 g/L after 24 h.

Production of glycerol from D-glucose using recombinant E. coli containing Both GPP2 and DAR1 Example 4 illustrates the production of glucose from the recombinant E. coli DH5a/pAH48, containing both the GPP2 and DAR1 genes.
The strain DH5a/pAH48 was constructed as described above in the GENERAL METHODS.
Pre-Culture DH5a/pAH48 were pre-cultured for seeding into a fermentation run.
Components and protocols for the pre-culture are listed below.
Pre-Culture Media KH2PO4 30.0 g/L
Citric acid 2.0 g/L
MgSO4=7H20 2.0 g/L
98% H2SO4 2.0 mL/L
Ferric ammonium citrate 0.3 g/L
CaC12.21-120 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
The above media components were mixed together and the pH adjusted to 6.8 with NH40H. The media was then filter sterilized.
Trace metals were used according to the following recipe:
/5 Citric acid, monohydrate 4.0 g/L
MgSO4=71420 3.0 g/L
MnSO4=H20 0.5 g/L
NaC1 1.0 g/L
FeSO4=7H20 0.1 g/L
CoC12.6H10 0.1 g/L
CaCl2 0.1 g/L
ZnSO4=7H20 0.1 g/L
CuSO4=5 1120 10 mg/L
A1K(SO4)2=12H20 10 mg/L
H3B03 10 mg/L
=
Na2Mo04=2H20 10 mg/L
NiSO4.61-I20 10 mg/L
Na2Se03 10 mg/L
Na2W04-2H20 10 mg/L

Cultures were started from seed culture inoculated from 50 pl frozen stock (15% glycerol as cryoprotectant) to 600 mL medium in a 2-L Erlenmeyer flask. Cultures were grown at 30 C in a shaker at 250 rpm for approximately 12 h and then used to seed the fennenter.
Fermentation growth Vessel 15-L stirred tank fermenter Medium K1-12PO4 6.8 g/L
Citric acid 2.0 g/L
MgSO4=7H20 2.0 g/L
98% H2SO4 2.0 mL/L
Ferric ammonium citrate 0.3 g/L
CaC1r2H,0 0.2 g/L
Mazu DF204 antifoam 1.0 mL/L
The above components were sterilized together in the ferrnenter vessel.
The pH was raised to 6.7 with NH4OH. 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 The temperature was controlled at 36 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 The results of the fermentation run are given in Table I.

Table 1 EFT 0D550 [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 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: E.I. DU PONT DE NEMOURS AND COMPANY
GENENCOR INTERNATIONAL, INC.
(ii) TITLE OF INVENTION: METHOD FOR THE
PRODUCTION OF
GLYCEROL BY RECOMBINANT
ORGANISMS
(iii) NUMBER OF SEQUENCES: 25 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: E. I. DU PONT DE NEMOURS AND COMPANY
(B) STREET: 1007 MARKET STREET
(C) CITY: WILMINGTON
(D) STATE: DELAWARE
(E) COUNTRY: U.S.A.
(F) POSTAL CODE (ZIP): 19898 (A) ADDRESSEE: GENENCOR INTERNATIONAL, INC.
(B) STREET: 4 CAMBRIDGE PLACE

(C) CITY: ROCHESTER
(D) STATE: NEW YORK
(E) COUNTRY: U.S.A.
(F) POSTAL CODE (ZIP): 14618 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: DISKETTE, 3.5 INCH
(B) COMPUTER: IBM PC COMPATIBLE
(C) OPERATING SYSTEM: MICROSOFT WINDOWS 95 (D) SOFTWARE: MICROSOFT WORD VERSION 7.0A
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,624,764 (B) FILING DATE: NOVEMBER 10, 1997 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
00 APPLICATION NUMBER: US 60/030,602 (B) FILING DATE: NOVEMBER 13, 1996 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: ALLEGRO, LISA
(B) REFERENCE/DOCKET NUMBER: 34711-3083 (ix) TELECOMMUNICATIONS INFORMATION:
(A) TELEPHONE: 416-865-0040 (B) TELEFAX: 416-865-7380 VM) 98/21340 PCT/US97/20293 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1380 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

GCTATCTCCT GTCTAAAGGG TTTTGAAGTT GGTGCTAAAG GTGTCCAATT GCTATCCTCT 600 .

:

(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2946 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID 11O:2:

AGCGTCAATC CTGCAAATAC ACCACCCAGC AGCACTAGGA TGATAGAGAT AATATAGTAC .120 . .

GATTAATCTA TTGTTCAGCA GCTCTTCTCT ACCCTGTCAT TCTAGTATTT TTTTTTTTTT 1080 ' . .

. .

GGGTAACAAT GCCTCCGCAG CCATTCAAAG GCTGGGTTTA GGTGAAATTA tCAAGTTCGG 2280 CAACTACTAC TAGTAACATT.ACTACAGTTA TTATAATTTT CTATTCTCTT TTTCTTTAAG 2760 .TTTACATATC ACATCACCGT TAATGAAAGA TACGACACCC TGTACACTAA CACAATTAAA 2380 ,TAATCGCCAT AACCTTTTCT CTTATCTATA GCCCTTAAAG CTGTTTCTTC GAGCTTTTCA 2940 =
=
CTGCAG 2946' (2) INFORMATION FOR SEQ ID NO:3:
(1) SEQUENCE CHARACTERISTICS:
. (A) LENGTH: 3178 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GATGTGCCCT AGATGCTGCG, ACCAGGGGAC TCAATGTGGC CCTTGTTGAA AAGGGGGATT 660 =
VA) 98/21340 PCT/US97/20293 CAGAAAACCC TATGCCTACA GAGGCTGATA TTCAAGATAT CTTGAAAGAA CTACAGCACT 1560 1 ' GGACGCAAAA CTATGTGGCT TTATTGGCTC AAAACTACCA TTTATCATCA AAAATGTtCA 1920 GCGAGGAGAA CAACTTGGTC AATTTTGATA CTTTCAGATA TCCATTCACA ATCGGTGAGT" 2100 TTATGGGTGA TGAGTTCAAT TGGTCGGAGA AAAAGAGGCA GTGGGAACTT GAAAAAACTG 2280 .

GATAACATTC ACAAGAGTAA TAATAATGGT AATGATCATA ATAATAATAA TGATAGTAAT 2400.

AAATTTTCAA GTTTTTATCA GATCCATGTT TCCTATCTGC CTTGACAACC TCATCGTCGA 2880' AATAGTACCA TTTAGAACGC CCAATATTCA CATTGTGTTC AAGGTCTTTA TTCACCAGTG 2940 ' _ = .

(2) INFORMATION FOR SEQ ID NO:4:
.(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 816 base pairs õ
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

TGTAATGCTT TGAACGCCTT GCCAAAGGAA AAATGGGCTG TCGCCACCTC TGGTACCCGT 420.

(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 753 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:' =
=

GAAATTCCGG TCAAGTACGG TGAAAAATCC ATTGAAGTCC CAGGTGCAGT TAAGCTGTGC 30,0 CCAGCAGGTA TTGCCGCCGG,AAAAGCCGCC GGTTGTAAGA TCATTGGTAT TGCCACTACT 600 (2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2520 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

CGTGAAACAC CAAACGCCGG.TGACATCAAA ACCAGCGGAA AGCCCATCTT TTCTGCAGAA 720 ,9 õ

AACGAACCCA CGTTGAAGTT CCCCAAACCG GGTTGGGTTG AGTGCCATCC GCAGAAATTA 840 ' =
GAACGTGTAG CAAACGGTCT CCCACCTTAC AAGGTAATAT'GCATGGGTAT AGCAAACATG 960 , GATAGGCAAC TGCAGCTTAG ACAGAAGACT GGATTGCCAT TGCTCTCCAC GTATTTCTCC 1140 GATGTCGGAC CGATTGCATC TACGGTTCCT GATTCTGGTG GCGTAGTTTT CdTCCCCGCA 1860 GACTTTTTAG AGGAAATTTC CGACGTCACA TATGAAAAGT CGCCCCTGTC GdTTCTGGCA 2100 ' =
GCAGCCAATA TGGCTTTCAA GGATGTGAAC GAGCGCCCAT TATGGAAGGA CCTACACtAT 2260 , CATCCAAACC TTAAGATATT CAGAAGTGAA TCCGACGATG CTGAAAGGAG AAAGCATTGG 2400 VVC) 98/21340 PCl/US97/20293 (2) INFORMATION FOR SEQ ID NO:?:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 391 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:?:
Met Ser Ala Ala Ala Asp Arg Leu Asn Leu Thr Ser Gly His Leu Asn Ala Gly Arg Lys Arg Ser Ser Ser Ser Val Ser Leu Lys Ala Ala Glu =

Lys,Pro Phe Lys Val Thr Val Ile Gly Ser Gly Asn Trp Gly Thr Thr Ile Ala Lys Vol Val Ala Glu Asn Cys Lys Gly Tyr Pro Glu Val Phe Ala Pro Ile Val Gin Met Trp Val Phe Glu Glu Glu Ile Asn Gly Glu Lys Leu Thr Glu Ile Ile Asn Thr Arg His Gin Asn Vol Lys Tyr Leu Pro Gly Ile Thr Leu Pro Asp Asn Leu Val Ala Asn Pro Asp Leu Ile Asp Ser Val Lys Asp Val Asp Ile Ile Val Phe Asn Ile Pro His Gin Phe Leu Pro Arg Ile Cys Ser Gin Leu Lys Gly. His Val Asp Ser His Val Arg Ala Ile Ser Cys Leu Lys Gly Phe Glu Val Gly Ala Lys Gly . Val Gin Leu Leu Ser Ser Tyr Ile Thr Glu Glu Leu Gly Ile Gin Cys Gly Ala Leu Ser Gly Ala Asn Ile Ala Thr Glu Val Ala Gin Glu His 180 185 190 ' Trp Ser Glu Thr Thr Val Ala Tyr His Ile Pro Lys /kap Phe Arg Gly ' Glu Gly Lys Asp Val Asp His Lys Vol Lou Lys Ala Leu Phe His Arg . .
.
210 215 22.0 , Pro Tyr Phe His Val Ser Val Ile Glu Asp Val Ala Gly Ile Ser Ile Cys Gly Ala Leu Lys Asn Val Val Ala Lou Gly Cys Gly Phe Vol Glu Gly Leu Gly Trp Gly Asn Asn Ala Ser Ala Ala Ile Gin Arg Val Gly Leu Gly Glu Ile Ile Arg Phe Gly Gin Met Phe Phe Pro Glu Ser Arg Glu Glu Thr Tyr Tyr Gin Glu Ser Ala Gly Vol Ala Asp Lou Ile Thr Thr Cys Ala Gly Gly Arg Asn Val Lys Val Ala Arg Leu Met Ala Thr Ser Gly Lys Asp Ala Trp Glu Cys Glu Lys Glu Leu Leu Asn Gly Gin Ser Ala Gin Gly Leu Ile Thr Cys Lys Glu Val His Glu Trp Leu Glu Thr Cys Gly Ser Val Glu Asp Phe Pro Leu Phe Glu Ala Vol Tyr Gin Ile Vol Tyr Asn Asn Tyr Pro Met Lys Asn Leu Pro Aso Met Ile Glu Glu Leu Asp Lou His Glu Asp (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 384 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Thr Ala His Thr Asn Ile Lys Gin His Lys His Cys His Glu Asp His Pro Ile Arg Arg Ser Asp Ser Ala Val Ser Ile Val His Leu Lys Arg Ala Pro Phe Lys Vol Thr Vol Ile Gly Ser Gly Asn Trp Gly Thr Thr Ile Ala Lys Vol Ile Ala Glu Asn Thr Glu Leu His Ser His Ile Phe Glu Pro Glu Vol Arq Met Trp Val Phe Asp Glu Lys Ile Gly Asp 3?
:

Glu Asn Leu Thr Asp Ile Ile Asn Thr Arg His Gin Asn Val Lys Tyr Leu Pro Asn Ile Asp Leu Pre His Asn Leu Vol Ala Asp Pro Asp Leu Leu His Ser Ile Lys Gly Ala Asp Ile Leu Val Phe Asn Ile Pro His Gin Phe Leu Pro Asn Ile Val Lys Gin Leu Gin Gly His Val Ala Pro His Val Arg Ala Ile Ser Cys Leu Lys Gly Phe Glu Leu Gly Ser Lys Gly Val Gin Leu Leu Ser Ser Tyr Val Thr Asp Glu Leu Gly Ile Gin Cys Gly Ala Leu Ser Gly Ala Asn Leu Ala Pro Glu Vol Ala Lys Glu His Trp Ser Giu Thr Thr Vol Ala Tyr Gin Leu Pro Lys Asp Tyr Gin Gly Asp Gly Lys Asp Val Asp His Lys Ile Leu Lys Leu Leu Phe His Arg Pro Tyr Phe His Val Asn Val Ile Asp Asp Val Ala Gly Ile Ser Ile Ala Gly Ala Leu Lys Asn Val Val Ala Leu Ala Cys Gly Phe Val Glu Gly Met Gly Trp Gly Asn Asn Ala Ser Ala Ala Ile Gin Arg Leu Gly Leu Gly Glu Ile Ile Lys Phe Gly Arg Met Phe Phe Pro Glu Ser.

Lys Val Glu Thr Tyr Tyr Gin Glu Ser Ala Gly Val Ala Asp Leu Ile Thr Thr Cys Ser Gly Gly Arg Asn Val Lys Val Ala Thr Tyr Met Ala Lys Thr Gly Lys Ser Ala Leu Glu Ala Glu Lys Glu LOU =Leu Asn Gly Gin Ser Ala Gin Gly Ile Ile-Tht Cys Arg Glu Val His Glu Trp Leu Gin Thr Cys Glq Leu Thr Gin Glu Phe Pro Ile Ile Arg Gly Ser Lou =
. Pro Asp Ser Leu Gin Gln Arg Pro His Gly Arg Pro Thr Gly Asp Asp WO 98/21340 . PCIYUS97/20293 ;2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 614 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Met Thr Arg Ala Thr Trp Cys Asn Ser Pro Pro Pro Leu His Arg Gin Val Ser Arg Arg Asp Leu Leu Asp Arg Leu Asp Lys Thr His Gin Phe Asp Val Leu Ile Ile Gly Gly Gly Ala Thr Gly Thr Gly Cys Ala Leu Asp Ala Ala Thr Arg Gly Leu Asn Val Ala Leu Val Glu Lys Gly Asp Phe Ala Ser Gly Thr Ser Ser Lys Ser Thr Lys Met Ile His Giy Gly Val Arg Tyr Leu Glu Lys Ala Phe Trp Glu Phe Ser Lys Ala Gin Leu Asp Leu Val Ile Glu Ala Leu Asn Glu Arg Lys His Leu Ile Asn Thr Ala Pro His Leu Cys Thr Val Leu Pro Ile Leu Ile Pro Ile Tyr Ser Thr Trp Gin Val Pro Tyr Ile Tyr Met Gly Cys Lys Phe Tyr Asp Phe Phe Gly Gly Ser Gin Asn Leu Lys Lys Ser Tyr Leu Leu Ser Lys Ser Ala Thr Val Glu Lys Ala Pro Met Leu Thr Thr Asp Asn Leu Lys Ala Ser Leu Val Tyr His Asp Gly Ser Phe Asn Asp Ser Arg Leu Asn Ala Thr Leu Ala Ile Thr Gly Val Glu Asn Gly Ala Thr Val Leu Ile Tyr Val Glu Val Gin Lys Leu Ile Lys Asp Pro Thr Sr Gly Lys Val Ile Gly Ala Glu Ala Arg Asp Val Glu Thr Asn Glu Leu Val Arg Ile Asn _ .

MO) 98/21340 Ala Lys Cys Val Val Asn Ala Thr Gly Pro Tyr Ser Asp Ala Ile Leu 245 250 . 255. = .
Gin Met Asp Arg Asn Pro Ser Gly Leu Pro Asp Ser Pro Leu Asn Asp Asn Ser Lys Ile Lys Ser Thr Phe Asn Gin Ile Ser Val Met Asp Pro Lys Met Val Ile Pro Ser Ile Gly Val His Ile Val Leu Pro Ser Phe Tyr Ser Pro Lys Asp Met Gly Leu Leu Asp Val Arg Thr Ser Asp Gly Arg Val Met Phe Phe Leu Pro Trp Gin Gly Lys Val Leu Ala Gly Thr Thr Asp Ile Pro Leu Lys Gin Val Pro Glu Asn Pro Met Pro Thr Glu Ala Asp Ile Gin Asp Ile Leu Lys Glu Leu Gin His Tyr Ile Glu Phe Pro Val Lys Arg Glu Asp Val Leu Ser Ala Trp Ala Gly Val Arg Pro Leu Val Arg Asp Pro Arg Thr Ile Pro Ala Asp Gly Lys Lys Gly Ser Ala Thr Gin Gly Val Val Arg Ser His Phe Leu Phe Thr Ser Asp Asn Gly Leu Ile Thr Ile Ala Gly Gly Lys Trp Thr Thr Tyr Arg Gin Met Ala Glu Glu Thr Val Asp Lys Val Val Glu Val Gly Gly Phe His Asn Leu Lys Pro Cys His Thr Arg Asp Ile Lys Leu Ala Gly Ala Glu Glu Trp Thr Gln Asn Tyr Val Ala Leu Leu Ala Gin Asn Tyr His Leu Ser Ser Lys Met Ser Asn Tyr Leu Val Gin Asn Tyr Gly Thr Arg Ser Ser Ile Ile Cys Glu Phe Phe Lys Glu Ser Met Glu Asn Lys Leu Pro Leu Ser Leu Ala Asp Lys Glu Asn Asn Val Ile Tyr Ser Ser Glu Glu Asn Asn Leu Val Asn Phe Asp Thr Phe Arg Tyr Pro Phe Thr Ile Gly Glu _ .

Leu Lys Tyr Ser Met Gln Tyr Glu Tyr Cys Arg Thr Pro Leu Asp Phe Leu Leu Arg Arg Thr Arg Phe Ala Phe Leu Asp Ala Lys Glu Ala Leu Asn Ala Val His Ala Thr Val Lys Val Met Gly Asp Glu Phe Asn Trp Ser Glu Lys Lys Arg Gln Trp Glu Leu Glu Lys Thr Val Asn Phe Ile 595 600 . 605 Gln Gly Arg Phe Gly Val (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 339 amino acids (B) TYPE: amino acid (C) STRANDEDEESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:10:
Met Asn Gln Arg Asn Ala Ser Met Thr Val Ile Gly Ala Gly Ser Tyr Gly Thr Ala Leu Ala Ile Thr Leu Ala Arg Asn Gly His Glu Val Val Leu Trp Gly His Asp Pro Glu His Ile Ala Thr Leu Glu Arg Asp Arg . .
Cys Asn Ala Ala Phe Leu Pro Asp Val Pro Phe Pro Asp Thr Leu His Leu Glu Ser Asp Leu Ala Thr Ala Leu Ala Ala Ser Arg Asn Ile Leu Val Val Val Pro Ser His Val Phe Gly Glu Val Leu Arg Gln Ile Lys = 85 90 95 Pro Leu Met Arg Pro Asp Ala Arg Leu Val Trp Ala Thr Lys Gly Leu Glu Ala Glu Thr Gly Arg Leu Leu Gln Asp Val Ala Arg Glu Ala Leu Gly Asp Gln Ile Pro Leu Ala Val Ile Ser Gly Pro Thr Phe Ala Lys Glu Leu Ala Ala Gly Leu Pro Thr Ala Ile Ser Leu Ala Ser Thr Asp .
.
. .

VA) 98,a1340 Gin Thr Phe Ala Asp Asp Leu Gin Gin Leu Leu His Cys Gly Lys Ser Phe Arg Val Tyr Ser Asn Pro Asp Phe Ile Gly Val Gin Leu Gly Gly Ala Val Lys Asn Val Ile Ala Ile Gly Ala Gly Met Ser Asp Gly Ile = Gly Phe Gly Ala Asn Ala Arg Thr Ala Leu Ile Thr Arg Gly Leu Ala Glu Met Ser Arg Leu Gly Ala Ala Leu Gly Ala Asp Pro Ala Thr Phe Met Gly Met Ala Gly Leu Gly Asp Leu Val Leu Thr Cys Thr Asp Asn Gin Ser Arg Asn Arg Arg Phe Gly Met Met Leu Gly Gin Gly Met Asp Val Gin Ser Ala Gin Glu Lys Ile Gly Gin Val Val Glu Gly Tyr Arg Asn Thr Lys Glu Val Arg Glu Leu Ala His Arg Phe Gly Val Glu Met.

Pro Ile Thr Glu Glu Ile Tyr Gin Val Leu Tyr Cys Gly Lys Ash Ala Arg Glu Ala Ala Leu Thr Leu Leu Gly Arg Ala Arg Lys Asp Glu Arg Ser Ser His (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 501 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Met Glu Thr Lys Asp Leu Ile Val Ile Gly Gly Gly Ile Asn Gly Ala Gly Ile Ala Ala Asp Ala Ala Gly Arg Gly Leu ger Val Leu Met Leu = Glu Ala Gin Asp Leu Ala Cys Ala Thr Ser Ser Ala Ser Ser Lys Leu Ile His Gly Gly Leu Arg Tyr Leu Glu His Tyr Glu Phe Arg Leu Val .
.
Ser Glu Ala Leu Ala Glu Arg Glu Val Leu Leu Lys Met Ala Pro His Ile Ala Phe Pro Met Arg Phe Arg Leu Pro His Arg Pro His Leu Arg Pro Ala Trp Met Ile Arg Ile Gly Leu Phe Met Tyr Asp His Leu Gly Lys Arg Thr Ser Leu Pro Gly Ser Thr Gly Leu Arg Phe Gly Ala Asn Ser Val Leu Lys Pro Glu Ile Lys Arg Gly Phe Glu Tyr Ser. Asp Cys Trp Val Asp Asp Ala Arg Leu Val Leu Ala Asn Ala Gin Met Val Val Arg Lys Gly Gly Glu Val Leu Thr Arg Thr Arg Ala Thr Ser Ala Arg Arg Glu Asn Gly Lou Trp Ile Val Glu Ala Glu Asp Ile Asp Thr Gly Lys Lys Tyr Ser Trp Gin Ala Arg Gly Leu Val Asn Ala Thr Gly Pro Trp Val Lys Gin Phe Phe Asp Asp Gly Met His Leu Pro Ser Pro Tyr Gly Ile Arg Leu Ile Lys Gly Ser His Ile Val Val Pro Arg Val His Thr Gin Lys Gin Ala Tyr Ile Leu Gin Asn Glu Asp Lys Arg Ile Val Phe Val Ile Pro Trp Met Asp Glu Phe Ser Ile Ile Gly Thr Thr Asp Val Glu Tyr Lys Gly Asp Pro Lys Ala Val Lys Ile Glu Glu Ser Glu Ile Asn Tyr Leu Leu Asn Val Tyr Asn Thr His Phe Lys Lys Gin' Lou Ser Arg Asp Asp Ile Val Trp Thr Tyr Ser Gly Val Arg Pro Leu Cys .305 310 315 320 Asp Asp Glu Ser Asp Ser Pro Gin Ala Ile Thr Arg Asp Tyr Thr Leu Asp Ile His Asp Glu Asn Gly Lys Ala Pro Lou Leu Ser Val Phe Gly =
Gly Lys Lou Thr Thr Tyr Arg Lys Leu Ala Glu His Ala Leu G1u Lys Leu Thr Pro Tyr Tyr Gin Gly Ile Gly Pro Ala Trp Thr Lys Glu Ser =

Val Leu Pro Gly Gly Ala Ile Glu Gly Asp Arg Asp Asp Tyr Ala Ala 385 390 . = 395 400 = Arg Leu Arg Arg Arg Tyr Pro Phe Leu Thr Glu Ser Leu Ala Arg His Tyr Ala Arg Thr Tyr Gly Ser Asn Ser Glu Lou Lou Leu Gly Asn Ala =

Gly Thr Val Ser Asp Leu Gly Glu Asp Phe Gly His Glu Phe Tyr Glu Ala Glu Leu Lys Tyr Leu Vol Asp His Glu Trp Vol Arg Arg Ala Asp Asp Ala Leu Trp Arg Arg Thr Lys Gin Gly Met Trp Leu Asn Ala Asp Gin Gln Ser Arg Val Ser Gin Trp Leu Val Glu Tyr Thr Gin Gin Arg Leu Ser Leu Ala Ser (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 542 amino acids (3) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Met Lys Thr Arg Asp Ser Gin Ser Ser Asp Val Ile Ile Ile Gly Gly Gly Ala Thr Gly Ala Gly Ile Ala Arg Asp Cys Ala Leu Arg Gly Leu Arg Val Ile Leu Val Glu Arg His Asp Ile Ala Thr Gly Ala Thr Gly Arg Asn His Gly Leu Lou His Ser Gly Ala Arg Tyr Ala Vol Thr Asp Ala Glu Ser Ala Arg Glu Cys Ile Ser Glu Asn Gin Ile Lou Lys Arg Ile Ala Arg His Cys Val Glu Pro Thr Asn Gly Leu Phe Ile Thr Leu . . .
.
' Pro Glu Asp Asp Leu Ser Phe Gin Ala Thr Phe Ile Arg Ala Cys Glu .Glu Ala Gly Ile Ser Ala Glu Ala Ile Asp Pro Gin Gin Ala Arg Ile Ile Glu Pro Ala Val Asn Pro Ala Lou Ile Gly Ala Val Lys Vol Pro Asp Gly Thr Vol Asp Pro Phe Arg Lou Thr Ala Ala Asn Met Leu Asp Ala Lys Giu His Gly Ala Val Ile Lou Thr Ala His Glu Val Thr Gly Leu Ile Arg Glu Gly Ala Thr Val Cys Gly Val Arg Val Arg Asn His Leu Thr Gly Glu Thr Gin Ala Lou His Ala Pro Val Val Val Asn Ala Ala Gly Ile Trp Gly Gin His Ile Ala Glu Tyr Ala Asp Leu Arg. Ile Arg Met Phe Pro Ala Lys Gly Ser Leu Leu Ile Met Asp His Arg Ile Asn Gin His Val Ile Asn Arg Cys Arg Lys Pro Ser Asp Ala Asp Ile Leu Val Pro Gly Asp Thr Ile Ser Leu Ile Gly Thr Thr Ser Leu Arg Ile Asp Tyr Asn Glu Ile Asp Asp Asn Arg Val Thr Ala Glu Glu Val Asp Ile Leu Leu Arg Glu Gly Glu Lys Leu Ala Pro Val Met Ala Lys Thr Arg Ile Leu Arg Ala Tyr Ser Gly Val Arg Pro Leu Val Ala Ser Asp Asp Asp Pro Ser Gly Arg Asn Lou Ser Arg Gly Ile Val Leu Leu Asp His Ala Glu Arg Asp Gly Leu Asp Gly Phe Ile Thr Ile Thr Gly Gly Lys Leu Net Thr Tyr Arg Leu Met Ala Glu Trp Ala Thr Asp Ala Val Cys Arg Lys Leu Gly Asn Thr Arg Pro Cys Thr Thr Ala Asp Leu Ala Leu Pro Gly Ser Gin Glu Pro Ala Glu Val Thr Leu Arg Lys Val WO 98/21340 PC17/US97a0293 Ile Ser Leu Pro Ala Pro Leu Arg Sly Ser Ala Val Tyr Arg His Gly Asp Arg Thr Pro Ala Trp Leu Ser Glu Gly Arg Lou His Arg Ser Leu Val Cys Glu Cys Glu Ala Val Thr Ala Gly Glu Val Gin Tyr Ala Val Glu Asn Leu Asn Val Asn Ser Leu Leu Asp Leu Arg Arg Arg Thr Arg =

Val Gly Met Gly Thr Cys Gin Gly Glu Leu Cys Ala Cys Arg Ala Ala Gly Leu Leu Gin Arg Phe Asn Val Thr Thr Ser Ala Gin Ser Ile Glu Gin Leu Ser Thr Phe Leu Asn Glu Arg Trp Lys Gly Val Gin Pro Ile Ala Trp Gly Asp Ala Leu Arg Glu Ser Glu Phe Thr Arg Trp Val Tyr Gin Gly Leu Cys Gly Leu Glu Lys Glu Gin Lys Asp Ala Leu (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 250 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met Gly Leu Thr Thr Lys Pro Leu Ser Leu Lys Val Asn Ala Ala Leu Phe Asp Val Asp Gly Thr Ile Ile Ile Ser Gin Pro Ala Ile Ala Ala 20 . 25 30 Phe Trp Arg Asp Phe Gly Lys Asp Lys Pro Tyr Phe Asp Ala Glu His Val Ile Gin Val Ser His Gly Trp Arg Thr Phe Asp Ala Ile Ala Lys Phe Ala Pro Asp Phe Ala Asn Glu Glu Tyr Val Asn Lys Lou Glu Ala Glu Ile Pro Val Lys Tyr Gly Glu Lys Ser Ile Glu Val Pro Gly Ala õ -MN) 98/21340 Val Lys Leu Cys Asn Ala Leu Asn Ala Leu Pro Lys Glu Lys Trp Ala Val Ala Thr Ser Gly Thr Arg Asp Met Ala Gin Lys Trp Phe Glu His Leu Gly Ile Arg Arg Pro Lys Tyr Phe Ile Thr Ala Asn Asp Val Lys Gin Gly Lys Pro His Pro Glu Pro Tyr Leu Lys Gly Arg Asn Gly Leu Gly Tyr Pro Ile Asn Glu Gin Asp Pro Ser Lys Ser Lys Val Val Val Phe Glu Asp Ala Pro Ala Gly Ile Ala Ala Gly Lys Ala Ala Gly Cys Lys Ile Ile Gly Ile Ala Thr Thr Phe Asp Leu Asp Phe Leu Lys Glu Lys Gly Cys Asp Ile Ile Vol Lys Aan His Glu Ser Ile Arg Val'Cly Gly Tyr Asn Ala Glu Thr Asp Glu Val Glu Phe Ile Phe Asp Asp Tyr Leu Tyr Ala Lys Asp Asp Leu Leu Lys Trp (2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 271 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Met Lys Arg Phe Asn Val Leu Lys Tyr Ile Arg Thr Thr Lys Ala Asn Ile Gin Thr Ile Ala Met Fro Leu Thr Thr Lys Pro Leu Ser Leu Lys Ile Asn Ala Ala Leu Phe Asp Val Asp Gly Thr Ile Ile Ile Ser Gin Pro Ala Ile Ala Ala Phe Trp Arg Asp Phe Gly Lys Asp Lys Pro Tyr Phe Asp Ala Glu His Val Ile His Ile Ser His Gly Trp Arg Thr Tyr _ =
WO 98/21340 ¨ PCTIUS9/a0293 Asp. Ala Ile Ala Lys Phe Ala Pro Asp Phe Ala Asp Glu Glu Tyr Val Asn Lys Leu Glu Gly Glu Ile Pro Glu Lys Tyr Gly Glu His Ser Ile = Glu Val Pro Gly Ala Val Lys Leu Cys Asn Ala Leu Asn Ala Leu Pro = Lys Glu Lys Trp Ala Val Ala Thr Ser Gly Thr Arg Asp Met Ala Lys Lys Trp Phe Asp Ile Leu Lys Ile Lys Arg Pro Glu Tyr Phe Ile Thr Ala Asn Asp Val Lys Gin Gly Lys Pro His Pro Glu Pro Tyr Leu Lys Gly Arg Asn Gly Leu Gly Phe Pro Ile Asn Glu Gin Asp Pro Ser Lys Ser Lys Val Val Val Phe Glu Asp Ala Pro Ala Gly Ile Ala Ala Gly Lys Ala Ala Gly Cys Lys Ile Val Gly Ile Ala Thr Thr Phe Asp Leu Asp Phe Leu Lys Glu Lys Gly Cys Asp Ile Ile Val Lys Asn His Glu Ser Ile Arg Val Gly Glu Tyr Asn Ala Glu Thr Asp Glu Val Glu Leu Ile Phe Asp Asp Tyr Leu Tyr Ala Lys Asp Asp Leu Leu Lys Trp (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 709 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
. Met Phe Pro Ser Leu Phe Arg Leu Val Val Phe Ser Lys Arg Tyr Ile Phe Arg Ser Ser Gin Arg Leu Tyr Thr Ser Leu Lys Gin Glu Gin Ser Arg Met Ser Lys Ile Met Glu Asp Leu Arg Ser Asp Tyr Val Pro Leu =

Ile Ala Ser Ile Asp Val Giy Thr Thr Ser Ser Arg Cys Ile Leu Phe Asn Arg Trp Gly Gin Asp Val Ser Lys His Gin Ile Glu Tyr Ser Thr Ser Ala Ser Lys Gly Lys Ile Gly Val Ser Gly Lou Arg Arg Pro Ser Thr Ala Pro Ala Arg Glu Thr Pro Asn Ala Gly Asp Ile Lys Thr Ser Gly Lys Pro Ile Phe Ser Ala Glu Gly Tyr Ala Ile Gin Glu Thr Lys Phe Leu Lys Ile Glu Glu Leu Asp Leu Asp Phe His Asn Glu Pro Thr Leu Lys Phe Pro Lys Pro Gly Trp Val Glu Cys His Pro Gin Lys Lou Leu Val Asn Val Val Gin Cys Lou Ala Ser Ser Leu Leu Set Lou Gin Thr Ile Asn Ser Glu Arg Val Ala Asn Gly Leu Pro Pro Tyr Lys Val Ile Cys Met Gly Ile Ala Asn Met Arg Glu Thr Thr Ile Leu Trp Ser Arg Arg Thr Gly Lys Pro Ile Val Asn Tyr Gly Ile Val Trp Asn Asp Thr Arg Thr Ile Lys Ile Val Arg Asp Lys Trp Gin Asn Thr Ser Val Asp Arg Gin Leu Gin Leu Arg Gin Lys Thr Gly Leu Pro Leu Lou Ser Thr Tyr Phe Ser Cys Ser Lys Leu Arg Trp Phe Leu Asp Asn Glu Pro Lou Cys Thr Lys Ala Tyr Glu Glu Asn Asp Leu Met Phe Gly Thr Val Asp Thr Trp Leu Ile Tyr Gin Leu Thr Lys Gin Lys Ala Phe Val Ser Asp Val Thr Asn Ala Ser Arg Thr Gly Phe Met Asn Lou Ser Thr Leu Lys Tyr Asp Asn Glu Leu Lou Glu Phe Trp Glv Ile Asp Lys Asn Leu Ile His Met Pro Glu Ile Val Ser Ser Ser Gin Tyr Tyr Gly Asp Phe VM) 98a1340 PCT/US97/20293 Gly Ile Pro Asp Trp Ile Met Glu Lys Leu His Asp Ser Pro Lys Thr Val Leu Arg Asp Leu Val Lys Arg Asn Leu Pro Ile Gin Gly Cys Leu Gly Asp Gin Ser Ala Ser Met Val Gly Gin Leu Ala Tyr Lys Pro Gly =
Ala Ala Lys Cys Thr Tyr Gly Thr Gly Cys Phe Leu Leu Tyr Asn Thr Gly Thr Lys Lys Leu Ile Ser Gln His Gly Ala Leu Thr Thr Leu Ala Phe Trp Phe Pro His Leu Gin Glu Tyr Gly Gly Gin Lys Pro Glu Leu Ser Lys Pro His Phe Ala Leu Glu Gly Ser Val Ala Val Ala Gly Ala Val Val Gin Trp Leu Arq Asp Asn Leu Arg Leu Ile Asp Lys Ser Glu Asp Val Gly Pro Ile Ala Ser Thr Val Pro Asp Ser Gly Gly Val Val Phe Val Pro Ala Phe Ser Gly Leu Phe Ala Pro Tyr Trp Asp Pro Asp Ala Arg Ala Thr Ile Met Gly Met Ser Gin Phe Thr Thr Ala Ser His Ile Ala Arg Ala Ala Val Glu Gly Val Cys Phe Gin Ala Arg Ala Ile Leu Lys Ala Met Ser Ser Asp Ala Phe Gly Glu Gly Ser Lys Asp Arg Asp Phe Leu Glu Glu Ile Ser Asp Val Thr Tyr Glu Lys Ser Pro Leu Ser Val Leu Ala Val Asp Gly Gly Met Ser Arg Ser Asn Glu Val Met Gin Ile Gin Ala Asp Ile Leu Gly Pro Cys Val Lys Val Arg Arg Ser Pro Thr Ala Glu Cys Thr Ala Leu Gly Ala Ala Ile Ala Ala Asn Met Ala Phe Lys Asp Val Asn Glu Arg Pro Leu Trp Lys Asp Leu His Asp .

Val Lys Lys Trp Val Phe Tyr Asn Gly Met Glu Lys Asn Glu Gin Ile . _ Ser Pro Glu Ala His Pro Asn Leu Lys Ile Phe Arg Ser Glu Ser Asp ' Asp Ala Glu Arg Arg Lys His Trp Lys Tyr Trp Glu Val Ala Val Glu Arg Ser Lys Gly Trp Leu Lys Asp Ile Glu Gly Glu His Glu Gin Val Leu Glu Asn Phe Gin (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NC:19:

(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs =
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

(2) INFORMATION FOR SEQ ID NO':21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
CATGGAATTG TCCTCCTTAC TAGT '24 (2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs =
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

VM) 98/21340 (2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "PRIMER"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "PRIMER"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

Claims (5)

1. A transformed host cell for the production of glycerol comprising:
(a) an exogenous polynucleotide encoding a polypeptide having glycerol-3-phosphate phosphatase activity and an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 and 14; and (b) an exogenous gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity and an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11, and 12.

2. The transformed host cell of claim 1, wherein the exogenous polynucleotide of (a) is selected from the group consisting of SEQ ID NOs: 5 and 6.

3. The transformed host cell of claim 1, wherein the exogenous polynucleotide of (a) is selected from the group consisting of SEQ ID NOs: 1, 2, 3, and 4.

4. The transformed host cell of any of claims 1-3, wherein the transformed host cell is a Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus , Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella, Bacillus, Streptomyces, or Pseudomonas cell.

5. The transformed host cell of claim 4, wherein the transformed host cell is an Escherichia coli cell.