AU780783B2 - Method for the production of glycerol by recombinant organisms - Google Patents

Description

P/00/01 I Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A DIVISIONAL PATENT

ORIGINAL

TO BE COMPLETED BY APPLICANT Name of Applicant: E.l. DU PONT DE NEMOURS AND COMPANY and GENENCOR NTERNATIONAL, INC S Actual Inventors: Bulthuis, Gatenby, Haynie, Hsu, Lareau, R.D.

Address for Service: CALLINAN LAWRIE, 711 High Street, Kew, Victoria 3101, Australia Invention Title: METHOD FOR THE PRODUCTION OF GLYCEROL BY RECOMBINANT ORGANISMS The following statement is a full description of this invention, including the best method of performing it known to 01/03/02.SW12610cov.let, 1

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 Asreromonas gracilis for protection against high external 20 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 (DAR1,GPDI) 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 DARI 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) Mol. Cell. Biol., 14:4135) teach the cloning of GPDI 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. Chem., 271:13875). Like the genes encoding G3DPH, 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)) *o 20 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 25 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 ofG3P phosphatase to the host which required careful control of its expression levels to avoid growth inhibition.

22/02 '05 12:51 FAX 61 3 9859 1588 CALLINAN LAWRIE MELB AUS PATENT OFFICE I1007 SUMMARY OF THE INVENTION The present invention provides a method for the production of glycerol from a recombinant microorganism comprising: transforming a suitable host cell with an expression cassette comprising a gene encoding aNADH-dependent glycerol-3-phosphate dehydrogenase enzyme or a NADPH-dependent glycerol-3- phosphate dehydrogenase enzyme; and a gene encoding a polypeptide glycerol-3-phosphate phosphatase (EC 3.1.3.21) enzyme; (ii) culturing the transformed host cell of in the presence of at least one carbon source selected from the group consisting ofmonosaccharides, oligosaccharides, polysaccharides, and single-carbon substrates, whereby glycerol is produced; and 15 (iii) recovering the glycerol produced in (ii) 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-3phosphatase activities for the production of glycerol.

20 The invention also provides a method for the production of glycerol from a recombinant microorganism comprising: .n transforming a suitable host cell with an expression cassette comprising: :0 a gene encoding aNADH-dependent glycerol-3-phosphate 25 dehydrogenase enzyme or a NADPH-dependent glycerol-3-phosphate dehydrogenase enzyme; and a gene encoding a glycerol kinase enzyme; (ii) culturing the transformed host cell of 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 (iii) recovering the glycerol produced in (ii).

22'02/05.at]2610.apceips,2 3 COMS ID No: SBMI-01131978 Received by IP Australia: Time 12:51 Date 2005-02-22 -4- 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 Int'l. Depository Reference Designation Date of Deposit Escherichia coli pAH21/DH5x ATCC 98187 26 September 1996 (containing the GPP2 gene) Escherichia coli (pDARI A/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, MD 20852 U.S.A. The designation is the accession number of the deposited material.

Applicants have provided 23 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 S 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 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 28V6ff1 41051 9.spei.doc.4 4aproduction 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 a. encoded by GUT2 (GenBank Z47047x23), or glpD (GenBank G147838), or glpABC (GenBank M20938).

The terms "glycerol-3-phosphate phosphatase", "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 U18813xll).

The term "glycerol kinase" refers to a polypeptide responsible for an enzyme S 20 activity that catalyzes the conversion of glycerol to glycerol-3-phosphate, or glycerol-3- S phosphate to glycerol, depending on reaction conditions. Glycerol kinase is encoded by GUT1 (GenBank Ull11583x19).

The terms "GPD1", "DARI", "OSGI", "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.

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.

2806/01.cf10519.speci.doc.a The terms "GPPI", "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 and refer to a gene that encodes a cytosolic glycerol-3-phosphatase and is characterized by the base sequence given as SEQ ID The term "GUTI" 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 monosaccharides, oligosaccharides, polysaccharides, and one-carbon substrates or mixtures thereof.

20 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 non-coding) and following noncoding) 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 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 20 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.IX SSC, 0. 1% SDS, 65 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-6phosphate 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 glycerol production An alternative pathway for glycerol production from DHAP has been suggested (Wang et al., (1994) J. Bact. 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 dihydroxyacetone 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.

S"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.

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, GPD1 has been isolated from Saccharomyces and has the base sequence given by SEQ ID NO: I.

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, 11 and 12 corresponding to the genes GPDI, 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 20 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 [GenBank Z38060 or U18813xl glucose- -phosphatase (EC 3.1.3.10) [GenBank M33807]; glucose-6-phosphatase (EC 3.1.3.9) [GenBank U00445]; fructose-l,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 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 1 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), Biol. Chem., 260:15646-15654).

Host cells 20 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 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, 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 CYC1, HIS3, GAL1, GAL10, ADHI, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, and TPI (useful for expression in Saccharomyces); 15 AOXI (useful for expression in Pichia); and lac, trp, XPL, XPR, T7. tac, and trc, (useful for expression in E. coli).

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.

S. 20 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, using calcium-permeabilized cells, electroporation, or by transfection using a recombinant phage virus (Sambrook et al., supra).

In the present invention AH21 and DARI cassettes were used to transform the E. coli 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, cornsteep liquor, sugar beet molasses, and barley malt. Additionally, the carbon substrate may also be one-carbon substrates such as carbon'dioxide, or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated.

Glycerol production from single carbon sources 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 15 eventually the three carbon product, glyceraldehyde-3-phosphate. Likewise, the S* 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.

,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, SDon 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 e *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 OC 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, cyclic adenosine 2':3'-monophosphate, may also be incorporated into the reaction media. Similarly, the use of agents known to modulate enzymatic activities 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 where the range of pH 6.0 to pH 8.0 is preferred for the initial condition.

15 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 20 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 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.

Identification and recovery of glvcerol S.o. 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 Patent No. 2,986,495).

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 (Wang, et al. (1994), J. Bact. 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. coli strain such as BB20 (Cronan et al. (1974), J. Bact., 118:598), alleviating the need to use antibiotic selection and its prohibitive cost in large-scale 15 fermentations.

A related strategy can be used for expression and selection in i osmoregulatory mutants of S. cerevisiae (Larsson et al. (1993), Mol. Microbiol., 10:1101- 11 These osgl mutants are unable to grow at low water potential and show a decreased capacity for glycerol production and reduced G3PDH activity.

20 The osg 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.

*e ~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.

The meaning of abbreviations is as follows: means hour(s), "min" means minute(s), "sec" means second(s), means day(s), "mL" means milliliters, 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 (garB10fhuA22 ompF627 fadL701 relAl pit-lO spoTI tpi-l mcrBl) (Anderson et al., (1970), J. Gen. Microbiol., 62:329).

(tonA22 AphoA8fadL701 relA1 glpR2 glpD3 pit-10 gpsA20 spoTI T2R) S.:(Cronan et al., J. Bac., 118:598).

S* 15 DH5a (deoR endA gyrA96 hsdRI 7 recAl relAl supE44 thi-1 A(lacZYAargFV 69) phi80lacZAMI5 (Woodcock et al., (1989), Nucl. Acids Res..

17:3469).

Identification of Glvcerol 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 Shodex SH-1011 column (8 mm x 300 mm; Waters, Milford, MA) 25 equipped with a Shodex SH-1011P precolumn (6 mm x 50 mm), temperaturecontrolled at 50 OC, using 0.01 N H2SO 4 as mobile phase at a flow rate of 0.5 mL/min. When quantitative analysis was desired, samples were injected onto a a Shodex SH-1011 column (8 mm x 300 mm; Waters, Milford, MA) equipped with a Shodex SH-1011 P precolumn (6 mm x 50 mm), temperature-controlled at 50 using 0.01 N H 2

SO

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 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 0.25 um 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 OC, 20 OC/min to 240 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 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 mM, bis-Tris or 50 mM MES); MgCI 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 pL, 200 mM), 50 mM MES, 10 mM MgCI 2 pH 6.5 buffer. The final phosphatase assay volume was 0.5 mL. The enzyme-containing sample was 15 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 20 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 standard curve that was prepared by using a stock inorganic phosphate solution (0.65 mM) and preparing 6 standards with final inorganic phosphate *i concentrations ranging from 0.026 to 0.130 ptmol/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/HCl, pH buffer with 5 mM DTT,in a total volume of 1.0 mL at 30 oC. 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 uL 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 glvcerol 3-phosphatase for increase of glycerol production in E. coli The Saccharomyces cerevisiae chromosomeV lamda clone 6592 Gene Bank, accession U18813x 1) 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-Xbal 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 Srfl site to generate the plasmids pAHIS containing GPP2.

The plasmid pAHI5 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 pBlueScriptlI 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 pAH 19 containing the GPP2 gene was inserted into pPHOX2 to create plasmid 20 pAH21. The pAH21/ DH5a is the expression plasmid.

Plasmids for the over-expression of DARI in E. coli DARI 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 Ncol is the DARI initiator methionine. At the 3' end of DARI a BamHI 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 pDARIA.

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 with SEQ ID NO:21) was inserted into the NcoI site of pDARIA to create Plasmid pAH40 contains the new RBS and DARI gene in the correct orientation for expression from the tre promoter of pTrc99A (Pharmacia, Piscataway, NJ). The NcoI-BamHI fragement from pDARIA and a, 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, WI) to create plasmid pAH42. The plasmid pAH42 contains a chloramphenicol resistant gene.

Construction of expression cassettes for DARI and GPP2 Expression cassettes for DARI and GPP2 were assembled from the individual DAR1 and GPP2 subclones described above using standard molecular biology methods. The BamHI-PstI fragment from pAHI9 containing the S 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 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 pAHI9 containing the GPP2 gene, was inserted into the BamHI-PstI site of pAH40 to create pAH48. Plasmid pAH48 contains the DARI 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 DH5c using standard molecular biology techniques. The transformants were verified by its DNA RFLP pattern.

20 EXAMPLE 1 PRODUCTION OF GLYCEROL FROM E. COLI TRANSFORMED WITH G3PDH GENE Media S 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 2

HPO

4 3.0 g KH 2

PO

4 1.0 g NH 4 CI, 0.5 g NaCI, 1 mL 20% MgSO 4 .7H 2 0, 8.0 g glucose, 40 mg casamino acids, 0.5 ml 1% thiamine hydrochloride. 100 mg ampicillin.

Growth Conditions Strain AA200 harboring pDARIA or the pTrc99A vector was grown in aerobic conditions in 50 mL of media shaking at 250 rpm in 250 mL flasks at 37 oC. At A 60 0 0.2-0.3 isopropylthio-P-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 °C for 48 h. Glycerol production was determined by HPLC analysis of the culture supematants. Strain pDARIA/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 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 10 mL; 1 M MgSO 4 12 mL; 100X trace elements, 12 mL; glucose, 10 mL; 1% thiamine, 10 mL; 100 mg/mL L-proline, 10 mL; 2.5 mM FeCl 3 5 mL; mixed phosphates buffer, 2 mL (5 mL 0.2 M NaH 2 PO4+ 9 mL 0.2 M K 2 HPO4), and pH to 7.0. The 100X traces elements for phoA medium /L contained: ZnSO 4 .7 H 2 0, 0.58 g; MnSO 4

.H

2 0, 0.34 g; CuSO 4

H

2 0, 0.49 g; CoCl2.6 HzO, 0.47 g; H 3 B0 3 0.12 g, NaMoO 4 .2 H 2 0, 0.48 g.

Shake Flasks Experiments The strains pAH21/DH5a (containing GPP2 gene) and (control) were grown in 45 mL of media (phoA media, 50 ug/mL carbenicillin, and 1 ug/mL vitamin B 12 in a 250 mL shake flask at 37 OC. 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/DH5a 20 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 DARI Growth for demonstration of increased glycerol production by E. coli DH5a-containing pAH43 proceeds aerobically at 37 OC in shake-flask cultures (Erlenmeyer. flasks, liquid volume 1/5th of total volume).

Cultures in minimal media/l% 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 (HCI), contained per liter: 12.6 g (NH 4 2

SO

4 13.7 g K 2

HPO

4 0.2 g yeast extract (Difco), I g NaHCO 3 5 mg vitamin B 12 5 mL Modified Balch's Trace-Element Solution (the composition of which can be found in Methods for General and Molecular Bacteriology Gerhardt et al., eds, p. 158, American Society for Microbiology, Washington, DC (1994)). The shake-flasks are incubated at 37 OC 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.

EXAMPLE 4 Production of glycerol from D-glucose using recombinant E. coli containing Both GPP2 and DARI Example 4 illustrates the production of glucose from the recombinant E co/i DH5WxpAH48. containing both the GPP2 and DARI genes.

The strain DH5a/pAH48 was constructed as described above in the GENERAL METHODS.

Pre-Culture DH5cr/pAH48 were pre-cultured for seeding into a fermentation run.

Components and protocols for the pre-culture are listed below.

Pre-Culture Media

KH

2

PO

4 30.0 g/L 0%0Citric acid :MgSO 4 .7H,)O 2.0 gIL 98% H,S0 4 2.0 mL/L Ferric ammonium citrate 0.3 gIL CaCb*)2H,)O 0.2 gIL Yeast extract 5.0 gIL Trace metals 5.0 mL/L Glucose 10.0 gIL Carbenicillin 100.0 mg/L The above media components were mixed together and the pH adjusted to 6.8 with NH 4 OI-. The media was then filter sterilized.

Trace metals were used according to the following recipe: 2 5 Citric acid. monohydrate 4.0 gIL MgSO 4 .71-12 3.0 gIL MnS04-H-)O 0.5 gIL NaCI 1.0 gIL FeSO4.7H-)0 0. 1 g/L CoC12-6H-)O 0. 1 g/L CaCI 2 0. 1 g/L ZnSO 4 .7H-,O 0. 1 gIL

CUSO

4 .5 H 1 0O 10 mg/L AIK(S0 4 2 '12H 2 0O 10 mg/L

H

3 130 3 10 mgIL Na 2 MoO 4 2H 2 0O 10 mg/L NiSO4-6H-)O 10 mgIL Na 2 SeO 3 10 mgIL Na-)W0 4 .2H 2 0 10 mgIL Cultures were started from seed culture inoculated from 50 .L 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 fermenter.

Fermentation growth Vessel stirred tank fermenter Medium

KH

2

PO

4 6.8 g/L Citric acid 2.0 g/L MgSO4-7H 2 0 2.0 g/L 98% H 2

SO

4 2.0 mL/L Ferric ammonium citrate 0.3 g/L CaCl2-2H 2 0 0.2 g/L 15 Mazu DF204 antifoam 1.0 mL/L The above components were sterilized together in the fermenter vessel.

The pH was raised to 6.7 with NH 4 0H. 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.

20 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 S. 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 1.

Table 1 EFT OD550 [Glucose] [Glycerol] Total Glucose Total Glycerol (hr) (AU) Fed Produced (g) 0 0.8 9.3 6 4.7 4.0 2.0 49 14 8 5.4 0 3.6 71 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 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 S22.2 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 38.2 9.8 5.1 36.1 512 233 *o e oooo* go *o*,oo* EDITORIAL NOTE APPLICATION NUMBER- 18854/02 The following Sequence Listing pages 22 to 49 are part of the description. The claims pages follow on pages 50 to 51.

SEQUENCE LISTING GENERAL INFORMATION:

APPL:

(A)

(B)

(C)

(D)

(E)

(F)

(G)

(H)

(I)

ICANT:

NAME: E. I. DU PONT DE N1 STREET: 1007 MARKET STRE CITY: WILMINGTON STATE: DELAWARE COUNTRY: U.S.A.

POSTAL CODE (ZIP): 19898 TELEPHONE: 302-892-8112 TELEFAX: 302-773-0164 TELEX: 6717325 EMOURS AND COMPANY

ET

ADDRESSEE: GENENCOR INTERNATIONAL, INC.

STREET: 4 CAMBRIDGE PLACE 1870 SOUTH WINTON ROAD CITY: ROCHESTER STATE: NEW YORK COUNTRY: U.S.A.

POSTAL CODE (ZIP): 14618 o *ooooo (ii) TITLE OF INVENTION: METHOD FOR THE PRODUCTION OF GLYCEROL BY RECOMBINANT

ORGANISMS

(iii) NUMBER OF SEQUENCES: (iv) COMPUTER READABLE FORM: MEDIUM TYPE: DISKETTE, 3.5 INCH COMPUTER: IBM PC COMPATIBLE OPERATING SYSTEM: MICROSOFT WORD FOR WINDOWS SOFTWARE: MICROSOFT WORD VERSION 7.OA CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: 60/03602 FILING DATE: NOVEMBER 13, 1996

CLASSIFICATION:

(vii) ATTORNEY/AGENT INFORMATION: NAME: FLOYD, LINDA AXAMETHY REGISTRATION NUMBER: 33,692 REFERENCE/DOCKET NUMBER: CR-9981-P1 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1380 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear

CTTTAATTTT

ACACCCCCCC

AGATTAAACT

TCTTTGAAGG

ACTACTATTG

ATAGTACAAA

AATACTAGAC

GCTAATCCAG

CATCAATTTT

GCTATCTCCT

TACATCACTG

GAAGTCGCTC

AGAGGCGAGG

TTCCACGTTA

GTTGTTGCCT

GCCATCCAAA

TCTAGAGAAG

GCTGGTGGTA

GAATGTGAAA

GTTCACGAAT

TACCAAATCG

GATCTACATG

TTCGAGGCTC

(ii) MOLECULE TYPE: DNA (genomi (xi) SEQUEN4CE DESCRIPTION: SEQ CTTTTATCTT ACTCTCCTAC ATAAGACATC CCTCCACAAA CACAAATATT GATAATATAA TAACTTCCGG CCACTTGAAT GCTGGTAGAA CTGCCGAAAA GCCTTTCAAG GTTACTGTGA CCAAGGTGGT TGCCGAAAAT TGTAAGGGAT TGTGGGTGTT CGAAGAAGAG ATCAATGGTG ATCAAAACGT GAAATACTTG CCTGGCATCA ACTTGATTGA TTCAGTCAAG GATGTCGACA .c) TG CC CCG TAT

GTCTAAAGGG

AGGAACTAGG

AAGAACACTG

GCAAGGACGT

GTGTCATCGA

TAGGTTGTGG

GAGTCGGTTT

AAACATACTA

GAAACGTCAA

AGGAGTTGTT

GGTTGGAAAC

TTTACAACAA

AAGATTAGAT

TTCTATATCA

CTGTAGCCAA

TTTTGAAGTT

TATTCAATGT

GTCTGAAACA

CGACCATAAG

AGATGTTGCT

TTTCGTCGA.A

GGGTGAGATC

CCAAGAGTCT

GGTTGCTAGG

GAATGGCCAA

ATGTGGCTCT

CTACCCAATG

TTATTGGAGA

TATTCATAAA

TTGAAAGGTC

GGTGCTAA.AG

GGTGCTCTAT

ACAGT TGCTT

GTTCTAAAGG

GGTATCTCCA

GGTCTAGGCT

ATCAGATTCG

GCTGGTGTTG

CTAATGGCTA

TCCGCTCAAG

GTCGAAGACT

AAGAACCTGC

AAGATAACAT

TTAGCATTAT

ID NO: 1:

AAGAAACAAT

AGATGTCTGC

AGAGAAGTTC

TTGGATCTGG

ACCCAGAAGT

AAAAATTGAC

CTCTACCCGA

TCATCGTTTT

ATGTTGATTC

GTGTCCAATT

CTGGTGCTAA

ACCACATTCC

CCTTGTTCCA

TCTGTGGTGC

GGGGTAACAA

GTCAAATGTT

CTGATTTGAT

CTTCTGGTAA

GTTTAATTAC

TCCCATTATT

CGGACATGAT

ATCATACTTC

GTCATTTCTC

TGTATATTGT

TGCTGCTGAT

CTCTTCTGTT

TAACTGGGGT

TTTCGCTCCA

TGAAATCATA

CAATTTGGTT

CAACATTCCA

ACACGTCAGA

GCTATCCTCT

CATTGCCACC

AAAGGATTTC

CAGACCTTAC

TTTGAAGAAC

CGCTTCTGCT

TTTCCCAGAA

CACCACCTGC

GGACGCCTGG

CTGCAAAGAA

TGAAGCCGTA

TGAAGAATTA

CCCCACTTTT

ATAACTACTT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 2946 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

U.

C

GAATTCGAGC

AGCGTCAATC

GTGGTAACGC

AGTACGTGTG

TATAAGATGA

AACGACATAT

ACTGTGACGA

AGCCTATGTG

GAAACCAAAA

GATAATACCC

AACTCCGGTT

CCCAGGTAAC

CAGCAATTCG

ACCATCATAT

TCAGTCATCA

TCGAAACAAT

GCCGATGGGT

GATTAATCTA

TTTTTGGTTT

TTTTCCTTCC

GATTTTTTTT

CCCTTTCCTT

ATACACATTC

CTGCAA.ATAC

TTGCCTCATC

ATCCGGATAA

TGTATACCCA

CTATTATAGT

TATCAACTCT

CAATCACCAA

GAATGAAGAA

TGCTTTAATG

ATTTTATCGG

CGTGCGCGAT

GGAGGGCGAA

CGCCTTAGCC

TCATTACCGA

AAGACGACGA

TGCTGAGGGG

TTGTTCAGCA

TACTTTTTTT

ACTAAGCTTT

TTATATATTA

TTCCTTCGCT

CTTAAGCGA.A

ACCACCCAGC

ACCTACGCTA

CAACGGCAGT

ATGAGGAGCG

GGGGAGAGTT

TTTTTTATTA

GGTCGTCCCT

AGAAAACAAA

AACGGTATGC

AACATCCGAG

GAGCTAATCC

AATAAAACTG

TCTAGCCATA

GTTTGTTTTC

TGGCTCTGCC

AAGAGTGTTT

GCTCTTCTCT

TCTTCTTGCC

TTCCTTGATT

ATTTTTAAGT

CCCCTTCCTT

CGCATCCGGT

CTGAAGTGCT GATTACCTTC AGGTAGACTT

AGCACTAGGA

TGGCCGGAAT

GAATATATCT

CCTGATCGTG

TCGTGCAAAT

TGTAATAAGC

TTTTTCCCAT

TACTAGCCCT

CCTAGGGTAT

CACCCGCGCC

TGAGCCATCA

GAG CAAGGAA

GCCATCATGC

CTTCACATGA

ATTGGTTATA

AGCTTACGGA

ACCCTGTCAT

TTTTTTTCTT

TATCCTTGGG

TTATGTATTT

ATCAATGCTT

GTTATATACT

CATCTTGACC

TGATAGAGAT

CGGCAACATC

TCGG TAT CGT

ACCTAGACCT

AACAGACGCA

AAACAAGCAC

TTGCTAATTT

AACCCTGACT

ATCTCACTCT

TTCCTCAACC

CCCACCCCAC

TTACCATCAC

AAGCGTGTAT

TGAAGAAGGT

TTACGCTTTT

CCTATTGCCA

TCTAGTATTT

GTTACTTTTT

TTCTTCTTTC

TGGTAGATTC

GCTGTCAGAA

CGTCGTGCAT

CATCAACCCC

AATATAGTAC

CCTAGAATTG

AAAGATGTGA

TAGTGGCAAA

GCAGCAAGTA

GAATGGGGAA

AGAATTTAAA

TCGTTTCTAT

GTACGTTACA

CAGGCACCGC

CCGTTGATGA

CGTCACCATC

CTTCTAAGAT

TTGAGTATGC

GCGGCGAGGT

TTGTTATTCC

TTTTTTTTTT

TTCTAGTTTT

TACTCCTTTA

AATTCTCTTT

GATTAACAAG

ATAAAATTTT

120 180 240 300 360 420 480 540 600 660 720 '780 840 900 960 1020 1080 1140 1200 1260 1320 1380 GCCTTCAAGA TCTACTTTCC TAAGAAGATC ATTATTACAA ACACAACTGC ACTCAAAGAT

GACTGCTCAT

ATCGGACTCT

TGGTTCTGGT

TTCCCATATC

AAATCTGACG

CCTGCCCCAT

CCTTGTTTTC

CGTGGCCCCT

TGTGCAATTG

TGGTGCAAAC

CCAACTACCA

GCTGTTCCAC

TGCCGGTGCC

GGGTAACAAT

TAGAATGTTT

AGATCTGATC

GACCGGTAAG

GATAATCACA

CCCAATTATT

CGGAGATGAT

TCTGATCTTT

CAACTACTAC

AATCTATCAT

TTTACATATC

TAATCGCCAT

ACTAATATCA

GCCGTGTCAA

AACTGGGGGA

TTCGAGCCAG

GATATCATAA

AATCTAGTGG

AACATCCCTC

CATGTAAGGG

CTATCCTCCT

TTGGCACCGG

AAGGATTATC

AGACCTTACT

TTGAAGAACG

GCCTCCGCAG

TTCCCAGAAT

ACCACCTGCT

TCAGCCTTGG

TGCAGAGAAG

CGAGGCAGTC

TGAAGAGCTA

CCTGTTGCCT

TAGTAACATT

TAACGTTAAT

ACATCACCGT

AACCTTTTCT

TTGTACATTT

CCACCATCGC

AGGTGAGAAT

ATACAAGACA

CCGATCCTGA

ATCAATTTTT

CCATCTCGTG

ATGTTACTGA

AAG TGGCCAA

AAGGTGATGG

TCCACGTCAA

TCGTGGCACT

CCATTCAAAG

CCAAAGTCGA

CAGGCGGTAG

AAGCAGAAAA

TTCACGAGTG

TACCAGATAG

GACATCGATG

CTTTTTCCCC

ACTACAGTTA

TTCTATATAT

TAATGAAAGA

GTTATCTATA

GAAACGTGCG

CAAAGTCATT

GTGGGTTTTT

CCAGAACGTT

TCTTTTACAC

ACCAAACATA

TCTAAAAGGG

TGAGTTAGGA

GGAGCATTGG

CAAGGATGTA

TGTCATCGAT

TGCATGTGGT

GCTGGGTTTA

GACCTACTAT

AAACGTCAAG

GGAATTGCTT

GCTACAAACA

TCTACAACAA

ACGAATAGAC

CAACCAATTT

TTATAATTTT

ACATAACTAC

TACGACACCC

GCCCTTAAAG

AACAGCACAA ACACTGTCAT GAGGACCATC

CCCTTCAAGG

GCGGAAAACA

GATGAAAAGA

AAPATATCTAC

TCCATCAAGG

GTCAAACAAT

TTCGAGTTGG

ATCCAATGTG

TCCGAAACCA

GATCATAAGA

GATGTTGCTG,

TTCGTAGAAG

GGTGAAATTA

CAAGAATCCG

GTTGCCACAT

AACGGTCAAT

TIGTGAGTTGA

CGTCCGCATG

AC TCT CCCCC

ATCATTATAC

CTATTCTCTT

CATTATACAC

TGTACACTAA

CTGTTTCTTC

CTATCAGAAG

TTACAGTGAT

CAGAATTGCA

TCGGCGACGA

CCAATATTGA

GTGCTGACAT

TGCAAGGCCA

GCTCCAAGGG

GCGCACTATC

CCGTGGCTTA

'TTTTGAAATT

GTATATCCAT

GTATGGGATG

TCAAGTTCGG

CTGGTGTTGC

ACATGGCCAA

CCGCCCAAGG

CCCAAGAATT

GAAGACCTAC

CCCCTCCCCC

ACAAGTTCTA

TTTCTTTAAG

GCTATTATCG

CACAATTAAA

GAGCTTTTCA

1440 1500 1560 1620 1680 1740 1800 1860 192o 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2 640C 2700 2760 2820 2880 2940 2946

CTGCAG

INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 3178 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (qenomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

CTGCAGAACT

ATGCGCAAGG

ATACTTATAT

AACTTTTATT

TAAACTTAAA

CCCTTATCTT

GTTTTCGGTA

GACGCT.GTAC

CATGGTGCAA

GTCTGGACAA

GATGTGCCCT

TTGCCTCGGG

AGAAGGCCTT

AGCGTAAACA

CCATCTACAG

TTGGCGGTTC

AGGCTCCCAT

TTAACGACTC

TCTTGATCTA

GTGCCGAGGC

TCAATGCCAC

TGCCGGACTC

TCATGGACCC

ACTCCCCGAA

TTTTACCTTG

TCGTCTGCTC

GCATCAGCGA

ACAGCACCAG

TCAGTTTTGG

AGAGAACAGC

GACCGTGCTA

ACGAGAAGAA

TGGATGACTA

TTCCCCACCG

GACGCATCAA

AGATGCTGCG

AACGTCGTCC

CTGGGAGTTC

TCTTATCAAC

TGTGCCCATC

GTGACCAATA

ACCTTATGTC

CAGGGGAAAT

CACAAATAGG

TTGCCATCAC

GAGCTGCCGG

GCCAAGGTGA

CCGCTCCACC

TTCGACGTGT

ACCAGGGGAC

AAATCTACCA

TCCAAGGCAC

ACTGCCC CT C

CTCGCGGTTA

ATCACTGCAC

TTTTCTCTGC

TTCACAACCC

GAACTTTGGT

TGCTACAAGA

TGCAGCTGGT

TAGGCCGTTA

GGCAGGTCTC

TGATCATCGG

TCAATGTGGC

AGATGATTCA

AACTGGATCT

ACCTGTGCAC

TCTATATGGG

ACCTACTGTC

AGGCCTCGCT

CCATCACGGG

TCAAAGACCC

AGCTTGTCAG

TTTTGCAAAT

AGATCAAGTC

TTGGCGTTCA

TCAGAACCTC

GCACCACAGA

GAAAGAAGCT

TAATTCCTTT

TCCGATACGT

CGCACGCTAA

CTAAACGAAG

CTAAATACGT

GCCATGGCCA

GTGCACAATG

TAGACGAGAC

TGGCGGGGCC

CCTTGTTGAA

CGGTGGGGTG

GGTCATCGAG

GGTGCTACCA

CTGTAAATTC

CAAATCCGCC

TGTGTACCAT

TGTGGAGAAC

AACTTCTGGT

AATCAACGCT

GGACCGCAAC

GACTTTCAAT

CATCGTATTG

TGATGGCAGA

CATCCCACTA

GAATTGTTTC

TTAGCAACAC

TATCCCACCC

AAATCGTATT

GACTCTCCCT

ACTAATATAT

CAGCCACGGG

ACCCGAGCTA

CTGCTGGACC

ACGGGGACAG

AAGGGGGATT

CGGTACT TAG

GCACTCAACG

ATTCTGATCC

TACGATTTCT

ACCGTGGAGA

GATGGGTCCT

GGCGCTACCG

AAGGTTATCG

AAATGTGTGG

CCATCCGGTC

CAAATCTCCG

CCCTCTTTTT

GTGATGTTCT

AAGCAAGTCC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 11.40 1200 1260 1320 1380 1440 1500 CACCTGGCAG GTCCCGTACA

CCAAAACTTG

GCTTACCACA

GCGTTTGAAC

TGTCGAGGTA

CCGGGACGTT

GGGCCCATAC

CCCGCTAAAC

GAAAATGGTC

GGATATGGGT

GCAGGGCAAA

AAAAAAT CAT

GACAATTTAA

GCCACTTTAG

CAAAAATTGA

GAGACTAATG

AGTGACGCCA

GACAACTCCA

ATCCCATCTA

TTGTTGGACG

GTCCTTGCCG

CAGAAAACCC

ATATCGAATT

TGGTCAGAGA

TGGTAAGATC

AATGGACTAC

GATTCCACAA

GGACGCAAAA

ACTACTTGGT

CCATGGAAAA

GCGAGGAGAA

TAAAGTATTC

CAAGATTCC

TTATGGGTGA

TGAACTTCAT

-GATAACATTC

AACAATAATA

TGGAAGAGTT

GGTAATAGAC

CTATTTCCAA

CGTTTTAATT

TAATATTCTT

ATGGAAAATT

AAATTTTCAA

AATAGTACCA

ACGTGTAATG

CATAGTGTCA

ATTTTTCTTG

AGTTACAAAA

tATGCCTACA

CCCCGTGAAA

TCCACGTACA

CCACTTCTTG

TTACAGACAA

CCTGAAACCT

CTATGTGGCT

TCAAAACTAC

TAAACTGCCT

CAACTTGGTC

CATGCAGTAC

CTTCTTGGAC

TGAGTTCAAT

CCAAGGACGT

ACAAGAGTAA

ATAATGGTGG

AAAGTAAACT

TCTACTACTA

TACATAATAT

ATCCCCTTTA

CAAACGGTCC

TTGCTAGTCA

QT TT TTAT CA

TTTAGAACGC

GC CATCAT TA

TTGTTTTTCA

GTCAAATCGT

TTTATCGTTT

GAGGCTGATA

AGAGAAGACG

ATCCCCGCAG

TTCACTTCGG

ATGGCTGAGG

TGTCACACAA

TTATTGGCTC

GGAACCCGTT

TTG TCCT TAG

AATTTTGATA

GAATATTGTA

GCCAAGGAAG

TGGTCGGAGA

TTCGGTGTCT

TAATAATGGT

TAATGGCAAT

AAAAAAA CTA

CAATTGATCT

AATCTATATA

TCTC TAG TCT

TGGTGCATAC

TAAACCCTTT

GATCCATGTT

CCAATATTCA

ATGTGCCTGT

ATATAATGTT

AATAAAATCT

TCACTGTTGT

TTCAAGATAT

TGCTAAGTGC

ACGGGAAGAA

ATAATGGCCT

AAACAGTCGA

GAGATATTAA

AAAACTACCA

CCTCTATCAT

CCGACAAGGA

CTTTCAGATA

GAACTCCCTT

CTTTGAATGC

AAAAGAGGCA

AAATCGATCA

AATGATGATA

GAAATCGCTA

CAAAAATATA

TCAAATTATG

ATCATTGCTG

AGTTTTATCA

GCAATACATA

CATAAAACAA

TCCTATCTGC

CATTGTGTTC

ATGGTTAACC

TAGTATCAAT

CGATAAATGG

CAATTTTTTG

CTTGAAAGAA

ATGGGCTGGT

GGGCTCTGCC

AATTACTATT

CAAAGTTGTC

GCTTGCTGGT

TTTATCATCA

TTGCGAATTT

AAATAACGTA

TCCATTCACA

GGACTTCCTT

CGTGCATGCC

GTGGGAACTT

TGATAGTTAA

ATAATAATAA

TTATTACCTA

TGAAGAAAAA

ACCTTCCTAG

GTAGACTTCC

TAAAATATAG

TTTATGGTGC

TACGTAGACA

CTTGACAACC

AAGGTCTTTA

ACTCCAAATA

GGATATGTTA

ATGACTAAGA

TTCTTGTAAT

CTACAGCACT

GTCAGACCTT

ACTCAGGGCG

GCAGGTGGTA

GAAGTTGGCG

GCAGAAGAAT

AAAATGTCCA

TTCAAAGAAT

ATCTACTCTA

ATCGGTGAGT

TTAAGAAGAA

ACCGTCAAAG

GAAAAAACTG

GGGTGACAAA

TGATAGTAAT

TTTTCCTTAA

AAAAAAAAGA

TGTTTATATT

GTTTTAATAT

AAACACTAAA

TCGCTACTTG

TCATCGTCGA

TTCACCAGTG

GCTTATATTT

CGACGGTGTT

TTTTTGGTAA

CACTCGAG

1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3178 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 816 base pairs TYPE: nucleic acid STRANDEDNESS: sinqie TOPOLOGY: linear (ii I MOLECULE TYPE: DNA (qenornic) (xi) SEQUENCE DESCRIPTION: SEQ ID 00:4: 4

ATGAAACGTT

GCAATGCCTT

GACGGTACCA

GACAAGCCTT

GATGCCATTG

GGTGAA.ATCC

TGTAATGCTT

GACATGGCCA

GCCAATGATG

TTGGGTTTCC

GCACCAGCTG

ACTTTCGATT

TCTATCAGAG

TACTTATACG

(2)

ATGGGATTGA

GGTACCATTA

AAACCTTATT

TGACCACAAA

TCATCATCTC

ACTTCGATGC

CCAAGTTCGC

CAGAAAAGTA

TGAACGCCTT

AGAAATGGTT

TCAAGCAAGG

CAATTAATGA

GTATTGCTGC

TGGACTTCTT

TCGGTGAATA

ACCTTTATCT

TCAACCAGCC

CGAACACGTT

TCCAGACTTT

CGG TGAACAC

GCCAAAGGAA

CGACATTTTG

TAAGCCTCAC

ACAAGACCCA

TGGTAAGGCT

GAAGGAAAAG

CAACGCTGAA

TCAATGTTTT AAAATATATC AGAACAACAA

TTGAAAATCA

ATTGCTGCTT

ATTCACATCT

GCTGATGAAG

TCCATCGAAG

AAATGGGCTG

AAGATCAAGA

CCAGAACCAT

TCCAAATCTA

GCTGGCTGTA

GGTTGTGACA

ACCGATGAAG

AAGCAAATAT

ACGCCGCTCT

TCTGGAGAGA

CTCACGGTTG

AATACGTTAA

TTCCAGGTGC

TCGCCACCTC

GACCAGAATA

ACTTAAAGGG

AGGTTGTTGT

AAATCGTTGG

TCATTGTCAA

TCGAATTGAT

ACAAACCATC

ATTCGATGTT

TTTCGGTAAA

GAGAACTTAC

CAAGCTAGAA

TGTCAAGTTG

TGGTACCCGT

CTTCATCACC

TAGAAACGGT

CTTTGAAGAC

TAT TGCTACC

GAACCACGAA

CTTTGATGAC

120 180 240 300 360 420 480 540 600 660 720 780 816 CTAAGGATGA CTTGTTGAA.A TGGTAA INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 753 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CTACTAAACC TCTATCTTTG AAAGTTAACG CCGCTTTGTT CGACGTCGAC TCATCTCTCA ACCAGCCATT GCTGCATTCT GGAGGGATTT CGGTAAGGAC TCGATGCTGA ACACGTTATC CAAGTCTCGC ATGGTTGGAG AACGTTTGAT

GCCT.TTGCTA

G-AAATTCCGG

AACGCTTTGA

ATGGCACAAA

kATGATGTCA

GGATATCCGA

CCAGCAGGTA

TTCGACTTGG

ATCAGAGTTG

AGT TCG CTCC

TCAAGTACGG

AC6CTCTACC

AATGGTTCGA

AACAGGGTAA

TcAATGAGCA

TTGCCGCCGG

ACTTCCTAAA

GCGGCTACAA

AGACTTTGCC

TGAAAAATCC

AAAAGAGAAA

GCATCTGGGA

GCCTCATCCA

AGACCCTTCC

AAAAGCCGCC

GGAAAAAGGC

TGCCGAAACA

AATGAAGAGT

ATTGMAGTCC

TGGGCTGTGG

ATCAGGAGAC

GAACCATATC

AAATCTAAGG

GGTTGTAAGA

TGTGACATCA

GACGAAGTTG

ATGTTAACAA

CAGGTGCAGT

CAACTTCCGG

CAAAGTACTT

TGAAGGGCAG

TAGTAGTATT

TCATTGGTAT

TTGTCAAAAA

AATTCATTTT

ATTAGAAGCT

TAAGCTGTGC

TACCCGTGAT

CATTACCGCT

GAATGGCTTA

TGAAGACGCT

TGCCACTACT

CCACGA.ATCC

TGACGACTAC

240 300 360 420 480 .5410 600 660 720 753 I.

S*

TTATATGCTA AGGACGATCT GTTGAAATGG TAA INFORMATION FOR SEQ ID NO:6: i) SEQUENCE CHARACTERISTICS: LENGTH: 2520 base pairs TYPE: nucleic acid STRANDEDNESS: sinole TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (qenomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: TGTATTGGCC ACGATAACCA CCCTTTGTAT

GACTTTTATT

G TA? T TCT GAGGO CT GA

AGACAGCCAA

CGAACCATAT

ATGTTTCCCT

CAGCGCCTTT

TTACGAAGTG

TGCATTCTGT

TCAGCATCGA

CGTGAAACAC

AAACAACGTA

TrCTTCTAATT CTG CAT TGAC

GACTTTTAGA

AAAATATACC

CTCTCTTCCG

ACACTAGTTT

ACTACGTCCC

TCAACAGATG

AGGGCAAGAT

CAAACGCCGG

TGTAAAAACA

GGAGTAAAAC

AAAAAA.ATTG

ACGGATAAGG

ATGTGGTTTG

ACTTGTAGTA

AAAACAAGAA

GCTTATCGCC

GGGCCAGGAC

TGGGGTGTCT

TGACATCAAA

ACTGTTTTTG TTTTTCACAT TAACAAGAAT CTACCCATAC CATCAATTAA AGGGTGTGGA AA~AAAAA AGGAAAAGGA TGTAATAAAA TGTGGGGGGA AGTTGTGGCC GGAACTATAC TTCTCCAAAC GTTACATATT CAGAGCCGTA TGTCCAAAAT AGTATTGATG TAGGAACGAC GTTTCAAAAC. ACC 4AATTGh GGCCTAAGGA GACCCT&AC ACCAGCGGAA AGCCCATCT

GGTAAATAAC

AGGCCATTTC

GTAGCATAGT

AAGGAAAAAA

TGCCTGTTCT

AAATAGTTAT

CCGATCAAGC

AATGGAAGAT

CTCATCCAGA

ATATTCA&ZT,

AGCCOCAGC'

TTCTGCAGAA

120 180 240 300 360 420 4.80 GGCTATGCCA TTCAAGAAAC CAAATTCCTA AAAATCGAGG -AATTGGAkCTT-GGACTTCCAT

AACGAACCCA

CTGGTGAACG

GAACGTGTAG

AGAGAAACCA

GTTTGGAACG

GATAGGCAAC

TGTTCCAAGC

AACGACCTGA

GCGTTCGTTT

AAGTACGACA

GAAATTGTGT

AAGCTACACG

CAGGGCTGTC

GCTGCAAAAT

TTGATCTCCC

TACGGTGGCC

GTGGCTGGTG

GATGTCGGAC

TTTAGTGGCC

TCTCAATTCA

GCCAGGGCTA

GACTTTTTAG

GTGGATGGCG

CCCTGTGTCA

GCAGCCAATA

GTTAAGAAAT

CATCCAAACC

AAGTATTGGG

CACGAACAGG

CGTTGAAGTT

TCGTCCAATG

CAAACGGTCT

CAATTCTGTG

ACACCAGAAC

TGCAGCTTAG

TGCGCTGGTT

TGTTCGGCAC

CTGACGTAAC

ACGAGTTGCT

CCTCATCTCA

ATTCGCCAAA

TGGGCGACCA

GTACTTATGG

AACATGGCGC

AAAAACCAGA

CTGTGGTCCA

CGATTGCATC

TATTCGCTCC

CTACTGCCTC

TCTTGAAGGC

AGGAAATTTC

GGATGTCGAG

AAGTCAGAAG

TGGCTTTCAA

GGGTCTTTTA

TTAAGATATT

AAGTTGCCGT

TTCTAGAAAA

CCCCAAACCG

CCTTGCCTCA

CCCACCTTAC

GTCCCGCCGC

GATCAAAATC

ACAGAAGACT

CCTCGACAAT

TGTGGACACA

CAACGCTTCC

GGAATTTTGG

ATACTACGGT

AACAGTACTG

AAGCGCATCC

TACCGGTTGC

ACTGACGACT

ATTGAGCAAG

ATGGCTACGT

TACGGTTCCT

CTATTGGGAC

CCACATCGCC

AATGAGTTCT

CGACGTCACA

GTCTAATGAA

GTCTCCGACA

GGATGTGAAC

CAATGGAATG

CAGAAGTGAA-

GGAAAGATCC

CTTCCAATAA

GGTTGGGTTG

AGTTTGCTCT

AAGGTAATAT

ACAGGAAAAC

GTTAGAGACA

GGATTGCCAT

GAGCCTCTGT

TGGCTGATTT

AGAACTGGAT

GGTATTGACA

GACTTTGGCA

CGAGATCTAG

ATGGTGGGGC

TTTTTACTGT

CTAGCATTTT

CCACATTTTG

GATAATTTAC

GATTCTGGTG

CCAGATGCCA

AGAGCTGCCG

GACGCGTTTG

TATGAAAAGT

GTcATGCAAA

GCGGAATGTA

GAGCGCCCAT

GAGAAAAACG

tCGACGATG AAkGGTTGGC

CAACATAAAT

AGTGCCATCC

CTCTGCAGAC

GCATGGGTAT

CAATTGTTAA

AATGGCAAAA

TGCTCTCCAC

GTACCAAGGC

ACCAATTAAC

TTATGAACCT

AGAACCTGAT

TTCCTGATTG

TCAAGAGAAA

AACTCGCTTA

ACAATACGGG

GGTTCCCACA

CATTAGAGGG

GATTGATCGA

GCGTAGTTTT

GAGCCACCAT

TGGAAGGTGT

GTGAAGGTTC

CGCCCCTGTC

TTCAAGCCGA

CCGCATTGGG

TATGGAAGGA

AACAAATATC

CTGAAAGGAG

TGAAGGACAT

GCAGAAATTA

TATCAACAGC

AGCAAACATG

CTACGGTATT

CACTAGCGTC

GTATTTCTCC

GTATGAGGAG

TAAACAAAAG

CTCCACTTTA

TCACATGCCC

GATAATGGAA

CCTGCCCATA

CAAACCCGGT

GACCAAAAAA

TtTGCAAGAG

TTCCGTCGCT

TAAATCAGAG

CGTCCCCGCA

AATGGGGATG

TTGCTTTCAA

CAAAGACAGG

GGTTCTGGCA

TATCCTAGGT

GGCAGCCATT

CCTACACGAT

ACCAGAGGCT

AAAGCATTGG

AGAAGGTGAA

840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560, 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 AATTTCTATT AACAATGTAA INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 391 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Met Ser Ala Ala Ala Asp Arg Leu Asn Leu Thr Ser Gly His Leu Asn 1 5 10 Ala Gly Arg Lys Arg Ser Ser Ser Ser Val Ser Leu Lys Ala Ala Glu 25 Lys Pro Phe Lys Val Thr Val Ile Gly Ser Gly Asn Trp Gly Thr Thr 35 40 Ile Ala Lys Val Val Ala Glu Asn Cys Lys Gly Tyr Pro Glu Val Phe .50 55 Ala Pro Ile Val Gin Met Trp Val Phe Glu Glu Glu Ile Asn Gly Glu 70 75 Lys Leu Thr Glu lie Ile Asn Thr Arg His Gin Asn Val Lys Tyr Leu 85 90 Pro Gly Ile Thr Leu Pro Asp Asn Leu Val Ala Asn Pro Asp Leu Ile 100 105 110 we Asp Ser Val Lys Asp Val Asp Ile Ile Val Phe Asn Ile Pro His Gin 115 120 125 Phe Leu Pro Arg Ile Cys Ser Gin Leu Lys Gly His Val Asp Ser His 130 135 140 Val Arg Ala Ile Ser Cys Leu Lys Gly Phe Glu Val Gly Ala Lys Gly 145 150 155 160 Val Gin Leu Leu Ser Ser Tyr Ile Thr Glu Glu Leu Gly Ile Gin Cys 165 170 175 Gly Ala Leu Ser Gly Ala Asn Iie Ala Thr Glu Val Ala Gin Glu His 180 185 190 Trp Ser Glu Thr Thr Val Ala Tyr His Ile Pro Lys Asp Phe Arg Gly 195 200 205 Glu Gly Lys Asp Val Asp His Lys Val Leu Lys Ala Leu Phe His Arg 210 215 220 Pro Tyr Phe His Val Ser Val Ile Glu Asp Val Ala Gly Ile Ser Ile 225 230 235 240 Cys Gly Ala Leu Lys Asn Val Val Ala Leu Gly Cys Gly Phe Val Glu 245 250 255 Gly Leu Gly Trp Gly Asn Asn Ala Ser Ala Ala Ile Gin Arg Val Gly 260 265 270 Leu Gly Glu Ile Ile Arg Phe Gly Gin Met Phe Phe Pro Glu Ser Arq 275 280 285 Glu Glu Thr Tyr Tyr Gin Glu Ser Ala Gly Val Ala Asp Leu Ile Thr 290 295 300 Thr Cys Ala Gly Gly Arg Asn Val Lys Val Ala Arg Leu Met Ala Thr 305 310 315 320 Ser Gly Lys Asp Ala Trp Glu Cys Glu Lys Glu Leu Leu Asn Gly Gin 325 330 335 Ser Ala Gin Gly Leu Ile Thr Cys Lys Glu Val His Glu Trp Leu Glu 340 345 350 Thr Cys Gly Ser Val Glu Asp Phe Pro Leu Phe Glu Ala Val Tyr Gin 355 360 365 Ile Val Tyr Asn Asn Tyr Pro Met Lys Asn Leu Pro Asp Met Ile Glu 370 375 380 Glu Leu Asp Leu His Glu Asp S. 385 390 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 384 amino acids TYPE: amino acid STRANDEDNESS: unknown 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 1 5 10 His Pro Ile Arg Arg Ser Asp Ser Ala Val Ser Ile Val His Leu Lys 25 Arg Ala Pro Phe Lys Val Thr Val Ile Gly Ser Gly Asn Trp Gly Thr 40 Thr Ile Ala Lys Val Ile Ala Glu Asn Thr Glu Leu His Ser His Ile 55 Phe Glu Pro Glu Val Arq Met Trp Val Phe Asp Glu Lys Ile Gly Asp 70 75 Giu Asn Leu Thr Asp Ile Ile Asn Thr Arg His GiLn Asn Val Lys Tyr 90 Leu Pro Asn Ile Asp Leu Pro His Asn Leu Val Ala Asp Pro Asp Leu 100 105 110 Leu His Ser Ile Lys Giy Ala Asp Ile Leu Val Phe Asn Ile Pro His 115 120 125 Gin Rhe Leu Pro Asn Ile Val Lys Gin Leu Gin Giy His Val Ala Pro 130 135 140 His Val Arg Ala Ile Ser Cys Leu Lys Gly Phe Giu Leu Giy Ser Lys 145 150 155 160 Gly Val Gin Leu Leu Ser Ser Tyr Val Thr Asp Giu Leu Gly Ile Gin 165 170 175 Cys Giy Ala Leu Ser Giy Ala Asn Leu Ala Pro Giu Val Ala Lys Giu 180 185 190 .His '1rp Ser Giu Thr Thr Val Ala Tyr Gin) Leu Pro Lys Asp Tyr Gin *195 200 205 Giy Asp Giy Lys Asp Val Asp His Lys Ile Leu Lys Leu Leu Phe His *210 215 220 Arg Pro Tyr Phe His Val Asn Val Ile Asp Asp Val Ala Gly Ile Ser 225 230 235 240 Ile Ala Gly Ala Leu Lys Asn Val Val Ala Leu Ala Cys Gly Phe Val 245 250 255 Giu Gly Met Gly Trp Gly Asn Asn Ala Ser Ala Ala Ile Gin Arg Leu 0260 265 270 G!ly Leu Gly Gu Ile Ile Lys Phe Gly Arcq Met Phe Phe Pro Glu Ser 275 280 285 00Lys Val Giu Thr Tyr Tyr Gin Giu Ser Ala Gly Val Ala Asp Leu Ile 290 295 300 Thr Thr Cys Ser Gly Gly Arg Asn Val Lys Val Ala Thr Tyr Met Ala 305 310 315 320 Lys Thr Gly Lys Ser Ala Leu Giu Ala Giu Lys Giu Leu Leu Asn Gly 325 330 335 Gin Ser Ala Gin Gly Ile Ile Thr Cys Arg Giu Val His Glu Trp Leu 340 345 350 Gin Thr Cys Giu Leu Thr Gin Giu Phe Pro Ilie Ile Arg Giy Ser Leu 355 360 365 Pro Asp Ser Leu Gin Gin Arq Pro His Gly Arq Pro Thr Gly Asp Asp 370 375 380 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 614 amino acids TYPE: amino acid STRANDEDNESS: unknown 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 1 5 10 Val Ser Arq Arg Asp Leu Leu Asp Arg Leu Asp Thr H is Gin Phe 25 Asp Val Leu Ile Ile Gly Gly Gly Ala Thr Gly Thr Gly Cys Ala Leu 40 Ala Ala Thr Arq Gly Leu Asn Val Ala Leu Vai Glu Lys, Gly Asp .50 55 Phe Ala Ser Gly Thr Ser Ser Lys Ser Thr Lys Met Ile His Gly Gly 70 75 Val Arg Tyr Leu Glu Lys Ala Phe Trp Glu Phe Ser Lys Ala Gin Leu 90 Asp Leu Va Ile Giu Aia Leu Asn Glu Arg Lys His Leu Ile Asn Thr 100 105 110 *.*Ala Pro His Leu Cys Thr Val Leu Pro Ile Leu Ile Pro Ile Tyr Ser 115 120 125 Thr Trp Gin Val Pro Tyr Ile Tyr Met Gly Cys Lys Phe Tyr Asp Phe 130 135 140 Phe GIly Gly Ser Gin Asn Leu Lys Lys Ser Tyr Leu Leu Ser Lys Ser 145 150 155 160 Ala Thr Val Glu Lys Ala Pro Met Leu Thr Thr Asp Asn Leu Lys Ala 165 170 175 Ser Leu Val Tyr His Asp Gly Ser Phe Asn Asp Ser Arq L~u Asn Ala 180 185 190 Thr Leu Ala Ile Thr Gly Val Glu Asn Gly Ala Thr Val Leu Ile Tyr 195 200 205 Vai Glu Val Gin Lys Leu Ile Lys Asp Pro Thr Ser Gly Lys Val Ile 210 215 220 Gly Ala Glu Ala Arq Asp Val Glu Thr Asn Giu Leu Val Ar Ile Asn 225 230 235 240 Ala Lys Cys Val Val 245 Asn Ala Thr Gly Pro Tyr Ser Asp Ala Ile Leu 250 255 Gin Met Asp Arq Asn Pr 260 Asn Ser Lys le Lys Sei 0 r 4 LY s Tyr 305 Arg Thr Ala Pro Leu 385 Ala Gly Ala Leu I Trp J 465 Ser I Ile I Ser L Mel 29( Sei Val Asp Aso VaI 370 Val Thr Leu Glu Lys 450 rhr .ys le .eu 275 t Val 0 r Pro Met Ile Ile 355 Lvs Arg Gin Ile Glu 435 Pro Gin I Met S Cys G Ala A 515 Ly Phe Pro 340 Gin Arq Asp Gly rhr 420 Thr -ys sn )er lu ,00 sp Pro 3 Asp Phe 325 Leu Asp Glu Pro Va I 405 lie Val His Tyr Asn 485 Phe I Lys C Ser Met 310 Leu Lys lie Asp Arg 390 Vai Ala Asp rhr Val 470 ['yr ?he Uu Se Th

II

29

GI'

Pr GIr LeL Val 375 Thr Ara Gi Lys Arg 455 Ala Leu ksn r Gly r Phe 280 e Gly 5 y Leu a Trp i Val I Lys 360 Leu Ile Ser Gly I Val 440 Asp I Leu I Val G Glu S 5 Asn V 520 Le 26.

As Va Le.

Gin Pro 345 Glu Ser Pro Lis Lys 425 lal :le ~eu ;In er 05 'al L Pr 5 n Gi.

L Hit i Asl I G15 330 Glu Leu Ala Ala Phe 410 Tro Glu Lys Ala Asn 490 Met lie o Asp n Ile S lie 2 Val 315 Lys Asn Gin Trp Asp 395 Leu Thr Val Leu I Gin I 475 Tyr G Glu A Tyr S Se Se Va Arg Val Pro His Ala 380 Gly Phe Thr ;ly Ila 260 Isn ;ly LSf br r Pro Val 285 L Leu Thr Leu Met Tyr 365 Gly Lys Thr Tyr Gly I 445 Gly I Tyr Thr A Lys L 5 Ser G 525 Le 27 Me Pr Se] Ala Pro 350 lie Val Lys Ser krg 230 Phe la [is Lrg eu lu u Asn 0 t Asp 0 Ser r Asp Gly 335 Thr Glu Arg Gly Asp 415 Gin His Glu Leu 1 4 Ser 495 Pro 1 Glu A Asp Pro Phe Gly 320 Thr Glu Phe Pro Ser 400 Asn Asn lu er 180 er ~eu sn Asn Leu 530 Val Asn Phe Aso Thr 535 Phe Arq Tyr Pro Thr Ile Gly Glu Leu 545 Len As n Ser Gin (2 Lys Tyr Ser Met Gin Tyr Glu Tyr Cys Arg Thr Pro Len Asp Phe 550 555 560 Len Arg Arg Thr Arg Phe Ala Phe Leu Asp Ala Lys Gin Ala Leu 565 1570 575 Ala Val His Ala Thr Val Lys Val Met Gly Asp Giu Phe Asn Trp, 580 585 590 Gin Lys Lys Arg Gin Trp Gin Len Gin Lys Thr Val Asn Phe Ile 595 600 605 Gly Arg Phe Gly Val 610 INFORMATION FOR SEQ ID NO:1O: SEQUENCE CHARACTERISTICS: LENGTH: 339 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown ft *4 4. ft ft.

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID ft ft **vft

S

Met Asn Gin Arg Asn Gly Len Cys Len ValI Pro Gin Gly Glu 145 Th r Trp As n 50 Gin ValI Len Ala Asp 130 Len Al a Gly Ala Ser ValI Met Gin 115 Gin Ala 5 Ala Asp Phe Len Ser Pro Gly Pro Gly Ala Ile Pro Len Al a 70 His Asp Arg Len Len 150 Ser Thr Gin Pro 55 Thr Val1 Ala Len Ala 135 Pro Met Thr Val 10 Len Ala Arg 25 His Ile Ala 40 Asp Val Pro Ala Len Ala Phe Gly Glu 90 Arq Len Val 105 Len Gin Asp 120 Val Ile Ser Thr Ala Ile Ile Gly Ala Asn Gly His Thr Len Gin Phe Pro Asp Ala Ser Arg 75 Val Len Arg Trp Ala Thr Val Ala Arg 125 Gly Pro Thr 140 Ser Len Ala 155 Gly Gin Arq Thr As n Gin Lys 110 Gln Phe Ser Ser Val1 Asp Len Ile Ile Gly Ala Ala Thr Tyr Val Arg His Len Lys Len Len Lys Asp 160 Gin Thr Phe Ala Asp Asp Leu Gin Gin Leu Leu His Cys Gly Lys Ser 165 170 175 Phe Arq Val Tyr Ser Asn Pro Asp Phe Ile Gly Val Gin Leu Gly Gly 180 185 190 Ala Val Lys Asn Val Ile Ala Ile Gly Ala Gly Met Ser Asp Gly Ile 195 200 205 Gly Phe Gly Ala Asn Ala Arg Thr Ala Leu Ile Thr Arg Gly Leu Ala 210 215 220 Glu Met Ser Arg Leu Gly Ala Ala Leu Gly Ala Asp Pro Ala Thr Phe 225 230 235 240 Met Gly Met Ala Gly Leu Gly Asp Leu Val Leu Thr Cys Thr Asp Asn 245 250 255 Gin Ser Arq Asn Arq Arg Phe Gly Met Met Leu Gly Gin Gly Met Asp 260 265 270 *bVal Gin Ser Ala Gin Giu Lys Ile Gly Gin Val Val Glu Giv Tyr Arq 275 80285 Asn Thr Lys Giu Val Ara Giu Leu Ala His Arq Phe Gly Val Glu Met 290 295 300 Pro Ile Thr Glu Gu Ile Ty r Gin Val Leu Tyr Cys Gly Lys Asn Ala 30 310 31532 Arq Giu Ala Ala Leu Thr Leu Leu Gly Arq Ala Arq Lys Asp Glu Arg 325 330 335 .Ser Ser His INFORMATION FOP. SEQ ID NO:ll: SEQUENCE CHARACTERISTICS: LENGTH: 501 amino acids *9 TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l11: Met Giu Thr Lys Asp Leu Ile Val Ile Gly Gly Gly Ile Asn Gly Ala 1 5 10 Gly Ile Ala Ala Asp Ala Ala Gly Arq Gly Leu. Ser Val Leu Met Leu 25 Giu Ala Gin Asp Leu Ala Cys Ala Thr Ser Ser Ala Ser Ser Lys Leu 40 Ile His Gly Giy Leu Arg Tyr Leu Glu His Tyr Gin Phe Arg Leu Val S55 37 Ser Giu Ala Leu Ala Glu Arg Glu Val Leu Leu Lys Met Ala Pro His 70 0S@@ 0SOe *9 4e 9 0@S 9 .9 9 0 69 *9 9

C*

990.09 9 9.

99.

9 9 9 9 9 99900S 0 99*9 0 0S69 Ile Pro Lys Ser Trp 145 Arg Arg Lys Trp Gly 225 Thr Phe Val1 Ile Ser 305 Asp Asp Al a Ala Arq ValI 130 ValI Lys Glu Lys Val1 210 Ile Gin Val Giu As n 290 Arg Asp Ile Phe Trp Th r 115 Le u Asp Gly As n Tyr 195 Lys Arg Lys Ile Tyr 275 Tyr Asp Glu His Pro Met 100 Ser Lys Asp Gly Gly 180 Se r Gin Le u Gin Pro 260 Lys Leu Asp Ser Asp 340 Met Ile Leu Pro Al a Giu 165 Leu Trp Phe Ile Ala 245 T rp Gly Le u Ile Asp 325 Glu Arg Arg Pro Glu Arg 150 Val Trp Gin Phe Lys 230 Tyr Met Asp Asn Val1 310 Ser As n Phe Ile Gly Ile 135 Leu Le u Ile Al a Asp 215 Gly Ile Asp Pro Val 295 Trp Pro Gly Arg Gly Se r 120 Lys Val1 Thr ValI Arg 200 Asp Ser Le u Glu Ly s 280 Tyr Th r Gin Lys Le u Le u 105 Thr Arg Le u Arg Giu 185 Gly Gly His Gin Phe 265 Al a As n Tyr Al a Al a 345 Pro 90 Phe Gly Gly Al a Th r 170 Al a Le u Met Ile As n 250 Ser Val Th r Se r Ilie 330 Pro 75 His Met Leu Phe As n 155 Arg Glu Val His Val 235 Giu Ile Lvs His Gly 315 Thr Le u Arg Ty r Arg Glu 140 Al a Al a Asp Asn Le u 220 Val Asp Ile Ile Phe 300 Val1 Arg Le u Pro Asp Phe 125 Tyr Gin Thr.

Ile Ala 205 Pro Pro Lys Gly Glu 285 Lys Arg Asp Se r His His 110 Gly Ser Met Se r Asp 190 Thr Ser Arq Arg Thr 270 Glu Lys Pro Tyr Val 350 Le u Leu Ala Asp Val1 Ala 175 Thr Gly Pro Val Ile 255 Thr Ser Gin Leu Thr 335 Phe Arg Gly As n Cys Val1 160 Arg Gly Pro Tyr His 240 Val Asp Glu Le u Cys 320 Leu Gly Gly Lys Leu Thr Thr Tyr Arg Lys Leu Ala Giu His Aia Leu Glu Lys Leu Thr Pro Tyr Tyr Gin Gly Ile Gly Pro Ala Trp Thr Lys Giu Ser 370 375 380 Val Leu Pro Gly Gly Ala Ile Giu Giy 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 405 410 415 Tyr Ala Arq Thr Tyr Gly Ser Asn Ser Giu Leu Leu Leu Gly Asn Ala 420 425 430 Gly Thr Val Ser Asp Leu Gly Giu Asp Phe Gly His Glu Phe Tyr Glu 435 440 445 Ala Giu Leu Lys Tyr Leu Val Asp His Giu Trp Vai Arg Arg Ala Asp 450 455 460 *Asp Ala Leu Trp Arg Arg Thr Lys Gin Giy Met Trp Leu Asn Ala Asp 465 470 475 480 eg..Gin Gin Ser Arq Val Ser Gin Trp Leu Vai Glu Tyr Thr Gin Gin Arq 0485 490 495 Leu Ser Leu Ala Ser 500 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 542 amino acids TYPE: amino acid STRANDEDNESS: unknown Se.(D) TOPOLOGY: unknown M4OLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 000 0000 Met Lys Thr Arg Asp Ser Gin Ser Ser Asp Val Ile Ile Ile Gly Gly i 5 10 Gly Ala Thr Gly Ala Gly Ile Ala Arg Asp Cys Ala Leu Arq Gly Leu 25 Arg Val Ile Leu Vai Glu Arg His Asp Ile Ala Thr Gly Ala Thr Gly 40 Arq Asn His Gly Leu Leu His Ser Gly Ala Arq Tyr Ala Val Thr Asp 55 Ala Giu Ser Ala Arg Glu Cys Ile Ser Giu Asn Gin Ile Leu Lys Arg 70 75 le Ala Arg His Cys Vai Giu Pro Thr Asn Gly Leu Phe Ile Thr Leu 90 Pro Glu Asp Asp Leu Ser Phe Gin Ala Thr Phe Ile Arg Ala Cys Glu 100 105 110 Glu Ile Asp 145 Ala Leu Leu Ala Arg 225 Asn Leu Ile Asp Thr 305 Asp Asp Gly Val Ala Glu 130 Gly Lys Ile Thr Gly 210 Met Gln Val Asp Ile 290 Arg Asp His Lys Cys 370 Gly 115 Pro Thr Glu Arg Gly 195 Ile Phe His Pro Tyr 275 Leu Ile Asp Ala Leu 355 Arg Ile Ala Val His Glu 180 Glu Trp Pro Val Gly 260 Asn Leu Leu Pro Glu 340 Met Lys Ser Val Asp Gly 165 Gly Thr Gly Ala Ile 245 Asp Glu Arg Arg Ser 325 Arg Thr Leu Ala Asn Pro 150 Ala Ala Gin Gln Lvs 230 Asn Thr Ile Glu Ala 310 Gly Asp Tyr Gly Glu Pro 135 Phe Val Thr Ala His 215 Gly Arg Ile Asp Gly 295 Tyr Arg Gly Arg Asn 375 Ala 120 Ala Arg Ile Val Leu 200 Ile Ser Cys Ser Asp 280 Glu Ser Asn Leu Leu 360 Thr Ile Leu Leu Leu Cys 185 His Ala Leu Arg Leu 265 Asn Lys Gly Leu Asp 345 Met Arg Asp Ile Thr Thr 170 Gly Ala Glu Leu Lys 250 Ile Arg Leu Val Ser 330 Gly Ala Pro Pro Gly Ala 155 Ala Val Pro Tyr Ile 235 Pro Gly Val Ala Arg 315 Arg Phe Glu Cys Gin Ala 140 Ala His Arg Val Ala 220 Met Ser Thr Thr Pro 300 Pro Gly Ile Trp Thr 380 Gin 125 Val Asn Glu Val Val 205 Asp Asp Asp Thr Ala 285 Val Leu Ile Thr Ala 365 Thr Ala Lys Met Val Arg 190 Val Leu His Ala Ser 270 Glu Met Val Val Ile 350 Thr Ala Arg Ile Val Pro Leu Asp 160 Thr Gly 175 Asn His Asn Ala Arg Ile Arg Ile 240 Asp Ile 255 Leu Arg Glu. ,Va l Ala Lys Ala Ser 320 Leu Leu 335 Thr Gly Asp Ala Asp Leu Ala Leu Pro Gly Ser Gin Glu Pro Ala Glu Val Thr Leu Arg Lys Val 385 390 395 400 Ile Ser Leu Pro Ala Pro Leu Arg Gly Ser Ala Val Tyr Arq His Gly 405 410 415 Asp Arg Thr Pro Ala Trp Leu Ser Glu Gly Arg Leu His Arg Ser Leu 420 425 430 Val Cys Giu Cys Giu Ala Val Thr Ala Gly Glu Val Gin Tyr Ala Val 435 440 445 Giu Asn Leu Asn Val Asn Ser Leu Leu Asp Leu Arq Arg Arq Thr Arg 450 455 460 Val Gly Met Gly Thr Cys Gin Gly Giu Leu Cys Ala Cys Arg Ala Ala 465 470 475 480 Gly Leu Leu Gin Arq Phe Asn Val Thr Thr Ser Ala Gin Ser Ile Giu 485 490 495 Gin Leu Ser Thr Phe Leu Asn Giu Arg Trp Lys Gly Val Gin Pro Ile Ala Tro Gly Asp Ala Leu Arq Giu Ser Giu Phe Thr Arq Tro) Val Tyr 0 .515 520 525 Gin Gly Leu Cys Gly Leu Glu Lys Giu Gin Lys Asp Ala Leu 530 535 540 0*00* INFORMATION FOR SEQ ID NO:J.3: *0..0SEQUENCE CHARACTERISTICS: LENGTH: 250 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown i MOLECULE TYPE: protein 1 x, SEQUENCE DESCRIPTION: SEQ ID NO:13: Met Gly Leu Thr Thr Lys Pro Leu Ser Leu Lys Val Asn Ala Ala Leu 1 5 10 Phe Asp Val Asp Gly Thr Ile Ile Ile Ser Gin Pro Ala Ile Ala Ala 25 Phe Trp Arci Asp Phe Gly Lys Asp Lys Pro Tyr Phe Asp Ala Glu His 40 Val Ile Gin Val Ser His Giy Trp Arg Thr Phe Asp Ala Ile Ala Lys 55 Phe Ala Pro Asp Phe Ala Asn Giu Glu Ty r Val Asn Lys Leu Giu Ala 70 75 Giu Ilie Pro Val Lys Tyr Gly Giu Lys Ser Ile Glu Val Pro Gly Ala 90 Val Lys Leu Cys Asn Ala Leu Asn Ala Leu Pro Lys Glu Lys Trp Ala 100 105 110 Val Ala Thr Ser Gly Thr Arg Asp Met Ala Gin Lys Trp Phe Glu His 115 120 125 Leu Gly Ile Arg Arg Pro Lys Tyr Phe Ile Thr Ala Asn Asp Val Lys 130 135 140 Gin Gly Lys Pro His Pro Glu Pro Tyr Leu Lys Gly Arg Asn Gly Leu 145 150 155 160 Gly Tyr Pro Ile Asn Glu Gln Asp Pro Ser Lys Ser Lys Val Val Val 165 170 175 Phe Glu Asp Ala Pro Ala Gly Ile Ala Ala Gly Lys Ala Ala Gly Cys 180 185 190 Lys Ile Ile Gly Ile Ala Thr Thr Phe Asp Leu Asp Phe Leu Lys Glu 195 200 205 Lys Gly Cys Asp Ile Iie Val Lys Asn His Glu Ser Ile Arg Val Gly 210 215 220 Gly Tyr Asn Ala Glu Thr Asp Glu Val Glu Phe Ile Phe Asp Asp Tyr 225 230 235 240 Leu Tyr Ala Lys Asp Asp Leu Leu Lys Trp 245 250 INFORMATION FOR SEQ ID NO:14: S(i) SEQUENCE CHARACTERISTICS: LENGTH: 271 amino acids TYPE: amino acid STRANDEDNESS: unknown 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 1 5 10 Ile Gin Thr Ile Ala Met Pro Leu Thr Thr Lys Pro Leu Ser Leu Lys 25 Ile Asn Ala Ala Leu Phe Asp Val Asp Gly Thr Ile Ile Ile Ser Gin .40 Pro Ala Ile Ala Ala Phe Trp Arg Asp Phe Gly Lys Asp Lys Pro Tyr 55 Phe Asp Ala Glu His Val Ile His Ile Ser His Gly Trp Arg Thr Tyr 70 75 Asp Ala Ile Ala Lys Phe Ala Pro Asp Phe Ala Asp Glu Glu Tyr Val 90 Asn Lys Leu Glu Gly Glu Ile Pro Glu Lys Tyr Gly Glu His Ser Ile 100 105 110 Glu Val Pro Gly Ala Val Lys Leu Cys Asn Ala Leu Asn Ala Leu Pro 115 120 125 Lys Glu Lys Trp Ala Val Ala Thr Ser Gly Thr Arg Asp Met Ala Lys 130 135 140 Lys Trp Phe Asp Ile Leu Lys Ile Lys Arg Pro Glu Tyr Phe Ile Thr 145 150 155 160 Ala Asn Asp Val Lys Gin Gly Lys Pro His Pro Glu Pro Tyr Leu Lys 165 170 175 Gly Arg Asn Gly Leu Gly Phe Pro Ile Asn Glu Gin Asp Pro Ser Lys 180 185 190 Ser Lys Val Val Val Phe Glu Asp Ala Pro Ala Gly Ile Ala Ala Gly 195 200 205 Lys Ala Ala Gly Cys Lys Ile Val Gly Ile Ala Thr Thr Phe Asp Leu 210 215 220 Asp Phe Leu Lys Glu Lys Gly Cys Asp Ile Ile Val Lys Asn His Glu 225 230 235 240 Ser Ile Arg Val Gly Glu Tyr Asn Ala Glu Thr Asp Glu Val Glu Leu 245 250 255 Ile Phe Asp Asp Tyr Leu Tyr Ala Lys Asp Asp Leu Leu Lys Trp 260 265 270 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 709 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Phe Pro Ser Leu Phe Arg Leu Val Val Phe Ser Lys Arg Tyr Ile 1 5 10 Phe Arg Ser Ser Gin Arg Leu Tyr Thr Ser Leu Lys Gin Glu Gin Ser 25 Arg Met Ser Lys Ile Met Glu Asp Leu Arg Ser Asp Tyr Val Pro Leu 40 Ile Ala Ser Ile Asp Val Gly Thr Thr Ser Ser Arg Cys Ile Len Phe Asn Arg Ser Ala Thr Ala Gly Lys Phe Leu 130 Leu Lys 145 Leu Val Thr Ilie Ile Cys Arg Arg 210 Thr Arg 225 Asp Arg Thr Tyr Leu Cys Asp Thr 290 Asp Val 305 Lys Tyr Ile His Trp Se r Pro Pro 115 Lys Phe Asn As n Met 195 Thr Thr Gin Phe Thr 275 T rp Thr Asp Met Gly Gin Lys Gly Ala Arg 100 Ile Phe Ile Gin Pro Lys Vai Val 165 Ser Glu 180 Gly Ile Gly Lys Ile Lys Leu Gin 245 Ser Cys 260 Lys Aia Leu Ile Asn Ala Asn Giu 325 Pro Gin 340 Asp 70 Lys Gin Ser Gin Pro 150 Gin Arg Ala Pro Ile 230 Leu Ser Tyr Tyr Ser 310 Len Ile 55 Val1 Ile Thr Ala Leu 135 Giy Cys Val1 As n Ile 215 Val1 Arg Lys Giu Gin 295 Arq Len ValI Ser Gly Pro Giu 120 Asp Trp Len Al a Met 200 Val1 Ara Gin Len Giu 280 Len Thr Gin Ser Lys His Val Ser 90 Asn Ala 105 Gly Tyr Leu A sp Val Gin Ala Ser 170 Asn Gly 185 Arg Glu Asn Tyr Asp Lys Lys Thr 250 Arg Trp 265 Asn Asp Thr Lys Giy Phe Phe Trp 330 Ser Ser 345 Gin Ile 75 Gly Leu Gly Asp Ala Ile Phe His 140 Cys His 155 Ser Leu Len Pro Thr Thr Gly Ile 220 Trp Gin 235 Gly Len Phe Len Leu Met Gin Lys 300 Met Asn 315 Gly Ile Gin Tyr Arg Arg Ile Lys 110 Gin Gin 125 Asn Gin Pro Gin Len Ser Pro Tyr 190 Ile Len 205 Vai Trp Asn Thr Pro Len Asp Asn 270 Phe Gly 285 Ala Phe Len Ser Asp Lys Ser Thr Pro Ser Thr Ser Thr Lys Pro Thr Lys Len 160 Len Gin 175 Lys Val Trp Ser Asn Asp Ser Vai 240 Len Ser 255 Gin Pro Thr Val Val Ser Thr Len 320 Asn Len 335 Gin Tyr Tyr Gly Asp Phe 350 Gly Val Gly 385 Ala Gly Phe Ser Val 465 Asp Phe Ala Ile Leu 545 Asp Ser Gin Pro Ala 625 Val Ile Leu 370 Asp Ala Thr Trp Lys 450 Val Val Val Arg Ala 530 Lvs Phe Val Ile Thr 610 Phe Lys Pro 355 Arg Gin Lys Lys Phe 435 Pro Gin Gly Pro Ala 515 Arg Ala Leu Leu Gin 595 Ala Lys Lys Asp Asp Ser Cys Lys 420 Pro His Trp Pro Ala 500 Thr Ala Met Glu Ala 580 Ala Glu Asp Trp Trp Leu Ala Thr 405 Leu His Phe Leu Ile 485 Phe Ile Ala Ser Glu 565 Val Asp Cys Val Val 645 Ile Val Ser 390 Tyr Ile Leu Ala Arg 470 Ala Ser Met Val Ser 550 Ile Asp Ile Thr Asn 630 Phe Met Lys 375 Met Gly Ser Gin Leu 455 Asp Ser Gly Gly Glu 535 Asp Ser Gly Leu Ala 615 Glu Tyr Glu 360 Arg Val Thr Gin Glu 440 Glu Asn Thr Leu Met 520 Gly Ala Asp Gly Gly 600 Leu Arg Asn Leu Leu Gin Cys 410 Gly Gly Ser Arg Pro 490 Ala Gin Cys Gly Thr 570 Ser Cys Ala Leu Met 650 His Pro Leu 395 Phe Ala Gly Val Leu 475 Asp Pro Phe Phe Glu 555 Tyr Arg Val Ala Trp 635 Glu Asp Ser 365 Ile Gin 380 Ala Tyr Leu Leu Leu Thr Gin Lys 445 Ala Val 460 Ile Asp Ser Gly Tyr Trp Thr Thr 525 Gin Ala 540 Gly Ser Glu Lys Ser Asn Lys Val 605 Ile Ala 620 Lys Asp Lys Asn Pro Gly Lys Tyr Thr 430 Pro Ala Lys Gly Asp 510 Ala Arg Lys Ser Glu 590 Arg Ala Leu Glu Lys Cys Pro Asn 415 Leu Glu Gly Ser Val 495 Pro Ser Ala Asp Pro 575 Val Arg Asn His Gin 655 Thr Leu Gly 400 Thr Ala Leu Ala Glu 480 Val Asp His Ile Arg 560 Leu Met Ser Met Asp 640 Ile Ser Pro Glu Ala His Pro Asn Leu Lys Ile Phe Arg Ser Glu Ser Asp 660 665 670 Asp Ala Glu Arg Arg Lys His Trp Lys Tyr Trp Glu Val Ala Val Glu 675 680 685 Arg Ser Lys Gly Trp Leu Lys Asp Ile Glu Gly Glu His Glu Gin Val 690 695 700 Leu Glu Asn Phe Gin 705 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: single .i TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GCGCGGATCC AGGAGTCTAG AATTATGGGA TTGACTACTA AACCTCTATC T 51 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GATACGCCCG GGTTACCATT TCAACAGATC GTCCTT 36 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: TTGATAATAT AACCATGGCT GCTGCTGCTG ATAG 34 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GTATGATATG TTATCTTGGA TCCAATAAAT CTAATCTTC 39 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID CATGACTAGT AAGGAGGACA ATTC 24 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: CATGGAATTG TCCTCCTTAC TAGT 24 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: CTAGTAAGGA GGACAATTC 19 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: CATGGAATTG TCCTCCTTA 19 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 15 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "PRIMER" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: GATCCAGGAA ACAGA INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 15 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "PRIMER" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID CTAGTCTGTT TCCTG Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification, they are to be interpreted as specifying the presence of the stated features, integers, steps or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, component or group thereof.

This is a divisional application of AU 54307/98, the disclosure of which is incorporated herein by way of reference.

01/03/02.swl 2610spec.49

Claims (9)

1. A method for the production of glycerol from a recombinant microorganism comprising: transforming a suitable host cell with an expression cassette comprising a gene encoding a NADH-dependent glycerol-3-phosphate dehydrogenase enzyme or a NADPH-dependent glycerol-3-phosphate dehydrogenase enzyme; and a gene encoding a polypeptide glycerol-3-phosphate phosphatase (EC

3.1.3.21) enzyme; (ii) culturing the transformed host cell of 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 (iji) recovering the glycerol produced in (ii). 15 2. A method according to any one of Claim 1, wherein the suitable host cell is selected from the group consisting of bacteria, yeast, and filamenitous fungi. 3. A method according to Claim 2, wherein the suitable host cell is selected from the group consisting of Citrobacter, Enterobacter, Clostridium, Kleibsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, 20 Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella, Bacillus, Streptomyces, and Pseudomonas. c 4. A method according to Claim I or Claim 2, wherein the suitable host cell is E. coli or Saccharomyces.

5. A method according to any one of Claims 1 to 4, wherein the carbon source is 25 glucose.

6. A method according to any one of Claims 1 to 5, wherein the gene encoding a NADH-dependent glycerol-3-phosphate dehydrogenase enzyme corresponds to the amino acid sequence given in SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:10 and wherein the amino acid sequence encompasses amino acid substitutions, deletions or insertions that do not alter the functional properties of the enzyme.

7. A Eschericha coll pAH21/DH5a containing the glycerol-3-phosphate phosphatase gene and identified by the designation ATCC 98187.

8. An Escherichia coli pDARIA/AA200 containing the glycerol-3-phosphate dehydrogenase gene and identified by the designation ATCC 98248. 22/02i/. 1 s2610. COMS ID No: SBMI-01131978 Received by IP Australia: Time 12:51 Date 2005-02-22 .2.2/02 '05 12:52 FAX 61 3 9859 1588 CALLINAN LAWRIE MELB AUS PATENT OFFICE 0 009

9. A method according to any one of Claims 1 to 6, wherein the gene encoding a polypeptide glycerol-3-phosphatase (EC 3.1.3.21) enzyme is selected from the group consisting of the amino acid sequence given in SEQ ID NO:13 and SEQ ID NO:14 and wherein the amino acid sequence may encompass amino acid substitutions, deletions or additions that do not alter the function of the enzyme. A method according to Claim 1 substantially as herein described with reference to any one of the Examples.

11. An Escherichia coli pAH21/DH5a containing the glycerol-3-phosphate phosphatase gene and identified by the designation ATCC 98187, substantially as herein described with reference to any one of the Examples.

12. An Escherichia coli pDARlA/AA200 containing the glycerol-3-phosphate dehydrogenase gene and identified by the designation ATCC 98248, substantially as herein described with reference to any one of the Examples. 15 DATED this 22 day of February, 2005 EJ. DU PONT DE NEMOURS AND COMPANY and GENENCOR INTERNATIONAL, INC. By their Patent Attorneys: CALLINAN LAWRIE 9 9 99* 9 9* 9 f 99* 9 9. 9 .9* 9 9 99 f ft *o f ft 2/O2,05i,atl2610.apooipse.Sl COMS ID No: SBMI-01131978 Received by IP Australia: Time 12:51 Date 2005-02-22