AU2005203028A1 - Method for the production of glycerol by recombinant organisms - Google Patents

Description

P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A DIVISIONAL PATENT

ORIGINAL

TO BE COMPLETED BY APPLICANT Name of Applicant: Actual Inventors: Address for Service: Invention Title: E.I. Du Pont De Nemours and Company and Genencor International, Inc.

B.A. Bulthuis; A.A. Gatenby; S.L. Haynie; A. K-H Hsu; and R.D.

Lareau CALLINAN LAWRIE, 711 High Street, Kew, Victoria 3101, Australia 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 us:- 12/07/05,eh15120.cov,1 n TITLE SMETHOD 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

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0, recombinant organisms for the production of desired compounds. More specifically it 0 describes the expression of cloned genes for glycerol-3-phosphate dehydrogenase (G3PDH) C, and glycerol-3-phosphatase (G3P phosphatase), either separately or together, for the enhanced 0 10 production of glycerol.

N BACKGROUND Glycerol is a compound in great demand by industry for use in cosmetics, liquid soaps, food, pharmaceuticals, lubricants, anti-freeze solutions, and in numerous other applications. The esters of glycerol are important in the fat and oil industry.

Not all organisms have a natural capacity to synthesize glycerol. However, the biological production of glycerol is known for some species of bacteria, algae, and yeasts.

The bacteria Bacillus licheniformis and Lactobacillus lycopersica synthesize glycerol.

Glycerol production is found in the halotolerant algae Dunaliella sp. and Asteromonas gracilis for protection against high external salt concentrations (Ben-Amotz et al., (1982) Experientia 38:49-52). Similarly, various osmotolerant yeasts synthesize glycerol as a protective measure. Most strains of Saccharomyces produce some glycerol during alcoholic fermentation, and this can be increased physiologically by the application of osmotic stress (Albertyn et al., (1994) Mol. Cell. Biol. 14, 4135-4144). Earlier this century glycerol was produced commercially with Saccharomyces cultures to which steering reagents were added such as sulfites or alkalis. Through the formation of an inactive complex, the steering agents block or inhibit the conversion of acetaldehyde to ethanol; thus, excess reducing equivalents (NADH) are available to or "steered" towards dihydroxyacetone phosphate (DHAP) for reduction to produce glycerol. This method is limited by the partial inhibition of yeast growth that is due to the sulfites. This limitation can be partially overcome by the use of alkalis which create excess NADH equivalents by a different mechanism. In this practice, the alkalis initiated a Cannizarro disproportionation to yield ethanol and acetic acid from two equivalents of acetaldehyde.

The gene encoding glycerol-3-phosphate dehydrogenase (DAR1,GPD1) has been cloned and sequenced from Saccharomyces diastaticus (Wang et al., (1994), J. Bact.

t) 176:7091-7095). The DAR1 gene was cloned into a shuttle vector and used to transform 0 E. coli where expression produced active enzyme. Wang et al., supra, recognizes that DAR1 is regulated by the cellular osmotic environment but does not suggest how the gene might be used to enhance glycerol production in a recombinant organism.

Other glycerol-3-phosphate dehydrogenase enzymes have been isolated. For example, sn-glycerol-3-phosphate dehydrogenase has been cloned and sequenced from S. cerevisiae 00 0 (Larason et al., (1993) Mol. Microbiol., 10:1101, (1993)). Albertyn et al., (1994) Mol. Cell.

Biol., 14:4135) teach the cloning of GPD1 encoding a glycerol-3-phosphate dehydrogenase C from S. cerevisiae. Like Wang et al., both Albertyn et al., and Larason et al. recognize the

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0 10 osmo-sensitvity of the regulation of this gene but do not suggest how the gene might be used CN 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)) teaches away from achieving glycerol production by recombinant DNA techniques. By selective breeding techniques, these investigators created a hybridized yeast strain that produced glycerol at greater levels than the parent strains; however, the G3PDH activity remained constant or slightly lower.

A microorganism capable of producing glycerol under physiological conditions is industrially desirable, especially when the glycerol itself will be used as a substrate in vivo as part of a more complex catabolic or biosynthetic pathway that could be perturbed by osmotic stress or the addition of steering agents.

The problem to be solved, therefore, is how to direct carbon flux towards glycerol production by the addition or enhancement of certain enzyme activities, especially G3PDH and G3P phosphatase which respectively catalyze the conversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) and then to glycerol. This process has not previously been described for a recombinant organism and required the isolation of genes encoding the two enzymes and their subsequent expression. A surprising and unanticipated -4t3n difficulty encountered was the toxicity of G3P phosphatase to the host which required careful Scontrol of its expression levels to avoid growth inhibition.

SUMMARY OF THE INVENTION The present invention provides a method for the production of glycerol from a recombinant organism comprising: 0 transforming a suitable host cell with an expression cassette comprising either or

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0 both of C, a gene encoding a glycerol-3-phosphate dehydrogenase enzyme 0 10 a gene encoding a polypeptide glycerol-3-phosphate phosphatase enzyme; CN (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).

Glucose is the most preferred carbon source.

The invention further provides transformed host cells comprising expression cassettes capable of expressing glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase activities for the production of glycerol.

The invention also provides a method for the production of glycerol from a recombinant organism 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 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).

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: O Depositor Identification Int'l. Depository Reference Designation Date of Deposit S Escherichia coli pAH21/DH5a ATCC 98187 26 September 1996 (containing the GPP2 gene) Escherichia coli (pDAR1A/AA200) ATCC 98248 6 November 1996 00 C (containing the DAR1 gene) CI "ATCC" refers to the American Type Culture Collection international depository O located at 12301 Parklawn Drive, Rockville, MD 20852 U.S.A. The designation is the C 5 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 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 production of glycerol.

As used herein the following terms may be used for interpretation of the claims and specification.

The terms "glycerol-3-phosphate dehydrogenase" and "G3PDH" refer to a polypeptide responsible for an enzyme activity that catalyzes the conversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P). In vivo G3PDH may be NADH; NADPH; or FAD-dependent. The NADH-dependent enzyme (EC 1.1.1.8) is encoded by several genes including GPD1 (GenBank Z74071x2), or GPD2 (GenBank Z35169xl), or GPD3 (GenBank G984182), or DAR1 (GenBank Z74071x2). The NADPH-dependent enzyme (EC 1.1.1.94) is n encoded by gpsA (GenBank U321643, (cds 197911-196892) G466746 and L45246). The O FAD-dependent enzyme (EC 1.1.99.5) is encoded by GUT2 (GenBank Z47047x23), or glpD S(GenBank G147838), or glpABC (GenBank M20938).

The terms "glycerol-3-phosphatase", "glycerol-3-phosphatase", "sn-glycerol-3phosphatase",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 00 0 glycerol. G3P phosphatase is encoded by GPP1 (GenBank Z47047xl25), or GPP2 (GenBank l U18813x11).

C The term "glycerol kinase" refers to a polypeptide responsible for an enzyme activity 0 10 that catalyzes the conversion of glycerol to glycerol-3-phosphate, or glycerol-3-phosphate to N glycerol, depending on reaction conditions. Glycerol kinase is encoded by GUT1 (GenBank Ul 1583x19).

The terms "GPD1", "DARI', "OSG1", "D2830", and "YDL022W" will be used interchangeably and refer to a gene that encodes a cytosolic glycerol-3-phosphate dehydrogenase and is characterized by the base sequence given as SEQ ID NO: 1.

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.

The terms "GPP1", "RHR2" and "YIL053W" are used interchangeably and refer to a gene that encodes a cytosolic glycerol-3-phosphatase and is characterized by the base sequence given in SEQ ID NO:4.

The terms "GPP2", "HOR2" and "YER062C" are used interchangeably 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 "GUT1" 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.

tt The terms "carbon substrate" and "carbon source" refer to a carbon source capable of Sbeing 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.

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 00product.

Cc 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 non-coding) the coding region. The terms "native" and "wild-type" gene refer to the gene as found in nature with its own regulatory sequences.

As used herein, the terms "encoding" and "coding" refer to the process by which a gene, through the mechanisms of transcription and translation, produces an amino acid sequence. The process of encoding a specific amino acid sequence is meant to include DNA sequences that may involve base changes that do not cause a change in the encoded amino acid, or which involve base changes which may alter one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. Therefore, the invention encompasses more than the specific exemplary sequences. Modifications to the sequence, such as deletions, insertions, or substitutions in the sequence which produce silent changes that do not substantially affect the functional properties of the resulting protein molecule are also contemplated. For example, alterations in the gene sequence which reflect the degeneracy of the genetic code, or which result in the production of a chemically equivalent amino acid at a given site, are contemplated; thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as 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 t for another, such as lysine for arginine, can also be expected to produce a biologically Sequivalent 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 00 retention of biological activity in the encoded products. Moreover, the skilled artisan Cc recognizes that sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (0.1X SSC, 0.1% SDS, 65 with the sequences exemplified herein.

,IC 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 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, -9t a carbon substrate such as glucose is converted to glucose-6-phosphate via hexokinase in the

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O presence of ATP. Glucose-phosphate isomerase catalyzes the conversion of glucose-6- (Ni phosphate to fructose-6-phosphate and then to fructose-1,6-diphosphate through the action of 6-phosphofructokinase. The diphosphate is then taken to dihydroxyacetone phosphate (DHAP) via aldolase. Finally NADH-dependent G3PDH converts DHAP to glycerol-3phosphate which is then dephosphorylated to glycerol by G3P phosphatase. (Agarwal (1990),

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0, Adv. Biochem. Engrg. 41:114).

c Alternate pathways for glycerol production An alternative pathway for glycerol production from DHAP has been suggested 0 10 (Wang et al., (1994) J. Bact. 176:7091-7095). In this proposed pathway DHAP could be C 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. 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: 1, encoding the amino acid sequence given in SEQ ID NO:7 (Wang et al., supra). Similarly, G3PDH activity has also been isolated from Saccharomyces encoded by GPD2 having the base sequence given in SEQ ID NO:2 encoding the amino acid sequence given in SEQ ID NO:8 (Eriksson et al., (1995) Mol. Microbiol., 17:95).

For the purposes of the present invention it is contemplated that any gene encoding a polypeptide responsible for G3PDH activity is suitable wherein that activity is capable of catalyzing the conversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P). Further, it is contemplated that any gene encoding the amino acid sequence of tn G3PDH as given by SEQ ID NOS:7, 8, 9, 10, 11 and 12 corresponding to the genes GPD1, O 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 00 include GenBank accessions M34393, M20938, L06231, U12567, L45246, L45323, L45324, m L45325, U32164, U32689, and U39682. Genes isolated from fungi include GenBank rC accessions U30625, U30876 and X56162; genes isolated from insects include GenBank 0 10 accessions X61223 and X14179; and genes isolated from mammalian sources include (N 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-3phosphatase (EC [GenBank Z38060 or U18813xl glucose- 1-phosphatase (EC 3.1.3.10) [GenBank M33807]; glucose-6-phosphatase (EC 3.1.3.9) [GenBank U00445]; fructose-1,6-bisphosphatase (EC 3.1.3.11) [GenBank X12545 or J03207] or phosphotidyl glycero phosphate phosphatase (EC 3.1.3.27) [GenBank M23546 and M23628].

Genes encoding glycerol kinase are known. For example, GUT1 encoding the glycerol kinase from Saccharomyces has been isolated and sequenced (Pavlik et al. (1993), Curr. Genet., 24:21) and the base sequence is given by SEQ ID NO:6, which encodes the -11tt amino acid sequence given in SEQ ID NO: 15. The skilled artisan will appreciate that,

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Salthough glycerol kinase catalyzes the degradation of glycerol in nature, the same enzyme will be able to function in the synthesis of glycerol, converting glycerol-3-phosphate to glycerol under the appropriate reaction energy conditions. Evidence exists for glycerol production through a glycerol kinase. Under anaerobic or respiration-inhibited conditions, Trypanosoma brucei gives rise to glycerol in the presence of Glycerol-3-P and ADP. The reaction occurs in

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00 the glycosome compartment (Hammond, (1985), J. Biol. Chem., 260:15646-15654).

c Host cells C 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 C 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.

-12- Vt Initiation control regions, or promoters, which are useful to drive expression of the O G3PDH and G3P phosphatase genes in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genes is suitable for the present invention including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRPl, URA3, LEU2, ENO, and TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, trp, XPL, XPR, T7, tac, and 00 CN trc, (useful for expression in E. coli).

Termination control regions may also be derived from various genes native to the ,I preferred hosts. Optionally, a termination site may be unnecessary; however, it is most O 10 preferred if included.

c 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 DAR1 cassettes were used to transform the E. coli 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).

-13t' These organisms can assimilate single carbon compounds, ranging in oxidation state from 0 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 phosphate to form a 6 carbon sugar that becomes fructose and eventually the three carbon oO product, glyceraldehyde-3-phosphate. Likewise, the serine pathway assimilates the one- Mcr carbon compound into the glycolytic pathway via methylenetetrahydrofolate.

CNi In addition to one and two carbon substrates, methylotrophic organisms are also 0 10 known to utilize a number of other carbon-containing compounds such as methylamine, CI glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeast are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al. (1993), Microb. Growth C1 Compd., [Int. Symp.], 7th, 415-32.

Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly, various species of Candida will metabolize alanine or oleic acid (Sulter et al. (1990), Arch.

Microbiol., 153(5):485-9). Hence, the source of carbon utilized in the present invention may encompass a wide variety of carbon-containing substrates and will only be limited by the choice of organism.

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 -14- Vt known to modulate catabolite repression directly or indirectly, cyclic adenosine O 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 9.0 where the range 00 SofpH 6.0 to pH 8.0 is preferred for the initial condition.

c Reactions may be performed under aerobic or anaerobic conditions where anaerobic or N microaerobic conditions are preferred.

S 10 Identification and purification of G3PDH and G3P phosphatase C The levels of expression of the proteins G3PDH and G3P phosphatase are measured by enzyme assays. G3PDH activity assay relies on the spectral properties of the cosubstrate, NADH, in the DHAP conversion to G-3-P. NADH has intrinsic UV/vis absorption and its consumption can be monitored 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 glycerol Glycerol may be identified and quantified by high performance liquid chromatography (HPLC) and gas chromatography/mass spectroscopy (GC/MS) analyses on the cell-free extracts. Preferred is a method where the fermentation media are analyzed on an analytical ion exchange column using a mobile phase of 0.01N sulfuric acid in an isocratic fashion.

Methods for the recovery of glycerol from fermentation media are known in the art.

For example, glycerol can be obtained from cell media by subjecting the reaction mixture to the following sequence of steps: filtration; water removal; organic solvent extraction; and fractional distillation Patent No. 2,986,495).

Selection oftransformants 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 Vn glucose would only occur if they possessed a plasmid-encoded gpsA, allowing a selection Sbased 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 00 0, (Cronan et al. (1974), J. Bact., 118:598), alleviating the need to use antibiotic selection and its prohibitive cost in large-scale fermentations.

C A related strategy can be used for expression and selection in osmoregulatory mutants S 10 of S. cerevisiae (Larsson et al. (1993), Mol. Microbiol., 10:1101-1111). These osgl mutants C are unable to grow at low water potential and show a decreased capacity for glycerol production and reduced G3PDH activity. The osgl salt sensitivity defect can be complemented by a cloned and expressed G3PDH gene. Thus, the ability to synthesize glycerol can be used simultaneously as a selection marker for the desired glycerol-producing cells.

EXAMPLES

GENERAL METHODS Procedures for phosphorylations, ligations, and transformations are well known in the art. Techniques suitable for use in the following examples may be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989).

Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Techniques suitable for use in the following examples may be found in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society for Microbiology, Washington, DC. (1994) or in Biotechnology: A Textbook of Industrial Microbiology (Thomas D. Brock, Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA). All reagents and materials used for the growth and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, WI), DIFCO Laboratories (Detroit, MI), GIBCO/BRL (Gaithersburg, MD), or Sigma Chemical Company (St. Louis, MO) unless otherwise specified.

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.

-16t) Cell strains

O

SThe 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 (garB10JhuA22 ompF627fadL701 relAl pit-10 spoTI tpi-1 phoM510 mcrBl) 00 0, (Anderson et al., (1970), J. Gen. Microbiol., 62:329).

CN BB20 (tonA22 AphoA8fadL701 relAl glpR2 glpD3 pit-10 gpsA20 spoT1 T2R) (Cronan et al., S 10 J. Bact., 118:598).

(deoR endAl gyrA96 hsdR17 recAl relAl supE44 thi-1 A(lacZYA-argFV169) phi80lacZAM15 (Woodcock et al., (1989), Nucl. Acids Res., 17:3469).

Identification of Glycerol The conversion of glucose to glycerol was monitored by HPLC and/or GC. Analyses were performed using standard techniques and materials available to one of skill in the art of chromatography. One suitable method utilized a Waters Maxima 820 HPLC system using UV (210 nm) and RI detection. Samples were injected onto a Shodex SH-1011 column (8 mm x 300 mm; Waters, Milford, MA) equipped with a Shodex SH-1011P precolumn (6 mm x 50 mm), temperature-controlled at 50 oC, using 0.01 N H 2

SO

4 as mobile phase at a flow rate of 0.5 mL/min. When quantitative analysis was desired, samples were injected onto a Shodex SH-1011 column (8 mm x 300 mm; Waters, Milford, MA) equipped with a Shodex SH-1011P precolumn (6 mm x 50 mm), temperature-controlled at 50 oC, using 0.01 N H 2 SO4 as mobile phase at a flow rate of 0.69 mL/min. When quantitative analysis was desired, samples were prepared with a known amount oftrimethylacetic 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 oC/min to 180 20 °C/min to 240 hold for 2.5 min. Detection: El -17- Vt Mass Spectrometry (Hewlett Packard 5971, San Femando, CA), quantitative SIM using ions O 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 00 substrate used was either 1-a-glycerol phosphate, or d,l-a-glycerol phosphate. The final Cc concentrations of the reagents in the assay are: buffer (20 mM, bis-Tris or 50 mM MES); N1 MgCl 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 C N cuvette. This method involved incubating an enzyme sample in a cuvette that contained mM substrate (50 tL, 200 mM), 50 mM MES, 10 mM MgC12, pH 6.5 buffer. The final phosphatase assay volume was 0.5 mL. The enzyme-containing sample was added to the reaction mixture; the contents of the cuvette were mixed and then the cuvette was placed in a circulating water bath at T 37 oC for 5 to 120 min, the length of time depending on whether the phosphatase activity in the enzyme sample ranged from 2 to 0.02 U/mL. The enzymatic reaction was quenched by the addition of the acid molybdate reagent (0.4 mL). After the Fiske SubbaRow reagent (0.1 mL) and distilled water (1.5 mL) were added, the solution was mixed and allowed to develop. After 10 min, to allow full color development, the absorbance of the samples was read at 660 nm using a Cary 219 UV/Vis spectrophotometer. The amount of inorganic phosphate released was compared to a standard curve that was prepared by using a stock inorganic phosphate solution (0.65 mM) and preparing 6 standards with final inorganic phosphate concentrations ranging from 0.026 to 0.130 pmol/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 7.5 buffer with 5 mM DTT,in a total volume of mL at 30 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 18second substrate, DHAP (50 uL 40 mM DHAP), was then added and the absorbance change O over time was monitored for at least 3 min to determine to determine the gross rate. G3PDH activity was defined by subtracting the background rate from the gross rate.

PLASMID CONSTRUCTION AND STRAIN CONSTRUCTION Cloning and expression of glycerol 3-phosphatase for increase of glycerol production in E.

coli 0, The Saccharomyces cerevisiae chromosomeV lamda clone 6592 Gene Bank, accession U18813xl 1) was obtained from ATCC. The glycerol 3-phosphate phosphatase C (GPP2) gene was cloned by cloning from the lamda clone as target DNA using synthetic 0 10 primers (SEQ ID NO:16 with SEQ ID NO:17) incorporating an BamHI-RBS-XbaI site at the CN1 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 pAH15 containing GPP2. The plasmid 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 pAH19 containing the GPP2 gene was inserted into pPHOX2 to create plasmid pAH21. The pAH21/ DH5a is the expression plasmid.

Plasmids for the over-expression of DAR1 in E. coli DAR1 was isolated by PCR cloning from genomic S. cerevisiae DNA using synthetic primers (SEQ ID NO:18 with SEQ ID NO:19). Successful PCR cloning places an NcoI site at the 5' end of DAR1 where the ATG within NcoI is the DAR1 initiator methionine. At the 3' end of DAR1 a BamHI site is introduced following the translation terminator. The PCR fragments were digested with NcoI BamHI and cloned into the same sites within the expression plasmid pTrc99A (Pharmacia, Piscataway, NJ) to give pDAR1A.

In order to create a better ribosome binding site at the 5' end of DAR1, an SpeI-RBS-NcoI linker obtained by annealing synthetic primers (SEQ ID NO:20 with SEQ ID NO:21) was inserted into the NcoI site ofpDAR1A to create pAH40. Plasmid contains the new RBS and DAR1 gene in the correct orientation for expression from the trc promoter of pTrc99A (Pharmacia, Piscataway, NJ). The NcoI-BamHI fragement from pDAR1A and an second set of SpeI-RBS-NcoI linker obtained by annealing synthetic primers (SEQ ID NO:22 with SEQ ID NO:23) was inserted into the SpeI-BamHI site of pBC-SK+ (Stratagene, Madison, WI) to create plasmid pAH42. The plasmid pAH42 contains a chloramphenicol resistant gene.

-19- V) Construction of expression cassettes for DAR1 and GPP2

O

SExpression 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 pAH19 containing the ribosomal binding site (RBS) and GPP2 gene was inserted into pAH40 to create pAH43. The BamHI-PstI fragment from pAH19 containing the RBS and GPP2 gene was inserted into pAH42 to create

OO

00 The ribosome binding site at the 5' end of GPP2 was modified as follows. A

N

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 0 10 from pAH19 containing the GPP2 gene, was inserted into the BamHI-PstI site of pAH40 to Ci create pAH48. Plasmid pAH48 contains the DAR1 gene, the modified RBS, and the GPP2 gene in the correct orientation for expression from the trc promoter ofpTrc99A (Pharmacia, Piscataway, NJ).

Transformation ofE. coli All the plasmids described here were transformed into E. coli DH5ca using standard molecular biology techniques. The transformants were verified by its DNA RFLP pattern.

EXAMPLE 1 PRODUCTION OF GLYCEROL FROM E. COLI TRANSFORMED WITH G3PDH GENE Media Synthetic media was used for anaerobic or aerobic production of glycerol using E. coli cells transformed with pDAR1A. The media contained per liter 6.0 g Na 2

HPO

4 3.0 g

KH

2

PO

4 1.0 g NH 4 C1, 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 pDAR1A or the pTrc99A vector was grown in aerobic conditions in 50 mL of media shaking at 250 rpm in 250 mL flasks at 37 At A 600 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 supernatants. Strain pDAR1A/AA200 produced 0.38 g/L glycerol after 48 h under anaerobic conditions, and 0.48 g/L under aerobic conditions.

in EXAMPLE 2 SPRODUCTION OF GLYCEROL FROM E. COLI (N 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 Cr^ MgSO 4 12 mL; 100X trace elements, 12 mL; 50% glucose, 10 mL; 1% thiamine, 10 mL; CN 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

HPO

4 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 .5 H 2 0, 0.49 g; CoCl 2 .6 H20, 0.47 g; H 3

BO

3 0.12 g, NaMoO 4 .2 H20, 0.48 g.

Shake Flasks Experiments The strains pAH21/DH5a (containing GPP2 gene) and pPHOX2/DH5a (control) were grown in 45 mL of media (phoA media, 50 ug/mL carbenicillin, and 1 ug/mL vitamin B 1 2 in a 250 mL shake flask at 37 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 produced 0.2 g/L glycerol after 24 h.

EXAMPLE 3 Production of glycerol from D-glucose using recombinant E. coli containing both GPP2 and DAR1 Growth for demonstration of increased glycerol production by E. coli pAH43 proceeds aerobically at 37 °C in shake-flask cultures (erlenmeyer flasks, liquid volume 1/5th of total volume).

Cultures in minimal media/1% glucose shake-flasks are started by inoculation from overnight LB/1% glucose culture with antibiotic selection. Minimal media are: filtersterilized defined media, final pH 6.8 (HC1), contained per liter: 12.6 g (NH 4 )2SO 4 13.7 g

K

2

HPO

4 0.2 g yeast extract (Difco), 1 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 °C with vigorous shaking for -21overnight, after which they are sampled for GC analysis of the supernatant. The SpAH43/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 DAR1 Example 4 illustrates the production of glucose from the recombinant E. coli 00 C DH5a/pAH48, containing both the GPP2 and DAR1 genes.

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

0 10 Pre-Culture c DH5a/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 Citric acid 2.0 g/L MgSO 4 -7H 2 0 2.0 g/L 98% H 2

SO

4 2.0 mL/L Ferric ammonium citrate 0.3 g/L CaCl 2 -2H 2 0 0.2 g/L Yeast extract 5.0 g/L Trace metals 5.0 mL/L Glucose 10.0 g/L Carbenicillin 100.0 mg/L The above media components were mixed together and the pH adjusted to 6.8 with

NH

4 OH. The media was then filter sterilized.

Trace metals were used according to the following recipe: Citric acid, monohydrate 4.0 g/L MgSO 4 -7H 2 0 3.0 g/L MnSO4-H 2 0 0.5 g/L NaCl 1.0 g/L FeSO4.7H 2 0 0.1 g/L CoC12-6H 2 0 0.1 g/L -22tn CaCl 2 0.1 g/L ZnSO 4 -7H 2 0 0.1 g/L CuSO 4 -5 H 2 0 10 mg/L C A1K(S0 4 2 .12H 2 0 10 mg/L

H

3 B0 3 10 mg/L 00Na 2 MoO 4 2H 2 0 10 mg/L m NiSO4-6H 2 0 10 mg/L S Na 2 SeO 3 10 mg/L Na 2

WO

4 -2H 2 0 10 mg/L Cultures were started from seed culture inoculated from 50 PL frozen stock 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 MgSO 4 -7H20 2.0 g/L 98% H 2 S0 4 2.0 mL/L Ferric ammonium citrate 0.3 g/L CaCl 2 -2H 2 0 0.2 g/L 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. Carbenicillin was added at 100 mg/L. Volume after inoculation was 6 L.

-23- V Environmental Conditions For Fermentation 0 The temperature was controlled at 36 °C and the air flow rate was controlled at 6 standard liters per minute. Back pressure was controlled at 0.5 bar. The agitator was set at 350 rpm. Aqueous ammonia was used to control pH at 6.7. The glucose feed (60% glucose monohydrate) rate was controlled to maintain excess glucose.

Results 00 CThe results of the fermentation run are given in Table 1.

c Table 1 N EFT OD550 [Glucose] [Glycerol] Total Glucose Total Glycerol (hr) (AU) Fed Produced (g) N 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 22.2 11.0 12.6 382 24.2 10.8 6.5 26.6 404 178 26.2 10.9 6.8 442 28.2 10.4 10.3 31.5 463 216 30.2 10.2 13.1 30.4 493 213 32.2 10.1 8.1 28.2 512 196 34.2 10.2 3.5 33.4 530 223 36.2 10.1 5.8 548 38.2 9.8 5.1 36.1 512 233

Claims (20)

1. 2. A method according to Claim 1, wherein the expression cassette comprises a gene encoding a glycerol-3-phosphate dehydrogenase activity.
2. 3. A method according to Claim 1, wherein said expression cassette comprises a gene encoding a glycerol-3-phosphate phosphatase activity.
3. 4. A method according to Claim 1, wherein said expression cassette comprising gene encoding a glycerol-3-phosphate phosphatase activity and a glycerol-3-phosphate dehydrogenase activity. A method according to any one of Claims 1 to 4, wherein the suitable host cell is selected from the group consisting of bacteria, yeast, and filamentous fungi.
4. 6. A method according to Claim 5, wherein the suitable host cell is selected from the group consisting of Citrobacter, Enterobacter, Clostridium, Kleibsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella, Bacillus, Streptomyces, and Pseudomonas.
5. 7. A method according to Claim 6, wherein the suitable host cell is E. coli or Saccharomyces. -64- t 8. A method according to any one of Claims 1 to 7, wherein the carbon source is O 0 glucose.
6. 9. A method according to any one of Claims 1 to 8, wherein the gene encoding a ("1 glycerol-3-phosphate dehydrogenase enzyme corresponds to the amino acid sequence given in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11, or SEQ ID 00 NO: 12 and wherein the amino acid sequence encompasses amino acid substitutions, deletions O or insertions that do not alter the functional properties of the enzyme. O 10 10. A method according to any one of Claims 1 to 9, wherein the gene encoding a O CNI glycerol-3-phosphatase enzyme corresponds to the amino acid sequence given in SEQ ID NO:13 or 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.
7. 11. A method according to any one of Claims 1 to 10, wherein the gene encoding a glycerol kinase enzyme corresponds to the amino acid sequence given in SEQ ID NO:15 and wherein said amino acid sequence may encompass amino acid substitutions, deletions or additions that do not alter the function of said enzyme.
8. 12. A transformed host cell comprising a gene encoding a glycerol-3-phosphate dehydrogenase activity.
9. 13. A transformed host cell comprising a gene encoding a glycerol-3-phosphate phosphatase activity.
10. 14. A method for selecting for glycerol-3-phosphate dehydrogenase gene expression by complementation comprising supplying glycerol or glycerol-3-phosphate to a strain auxotrophic for glycerol or glycerol-3-phosphate by virtue of a mutation in glycerol-3- phosphate dehydrogenase gene of the strain. A method for selecting for glycerol-3-phosphate dehydrogenase gene expression by complementation comprising supplying salt to a strain osmosensensitive by virtue of a mutation in gene for glycerol-3-phosphate dehydrogenase of the strain. S16. A Escherichia coli pAH21/DH5a containing the glycerol-3-phosphate Sphosphatase gene and identified by the designation ATCC 98187. 17 An Escherichia coli pDARIA/AA200 containing the glycerol-3-phosphate dehydrogenase gene and identified by the designation ATCC 98248. 00 C 18. A method according to any one of Claims 1 to 9, wherein the gene encoding a Cc polypeptide glycerol-3-phosphatase (EC 3.1.3.21) enzyme is selected from the group N1 consisting of the amino acid sequence given in SEQ ID NO:13 and SEQ ID NO:14 and S 10 wherein the amino acid sequence may encompass amino acid substitutions, deletions or CN additions that do not alter the function of the enzyme.
11. 19. A transformed host cell comprising a plasmid containing a gene encoding a polypeptide glycerol-3-phosphate phosphatase (EC 3.1.3.21). A method for the production of glycerol from a recombinant organism 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 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).
12. 21. A method according to Claim 20, wherein the gene encoding a glycerol kinase enzyme is set forth in the amino acid sequence given in SEQ ID NO:15 and wherein said amino acid sequence may encompass amino acid substitutions, deletions or additions that do not alter the function of said enzyme.
13. 22. A method according to Claim 20 or Claim 21, wherein the suitable host cell is selected from the group consisting of bacteria and fungi. -66- O 23. A method according to any one of Claims 20 to 22, wherein the carbon source is glucose. (N
14. 24. A method according to any one of Claims 20 to 23, wherein the gene encoding a glycerol-3-phosphate dehydrogenase enzyme is selected from the group consisting of the OO 0, amino acid sequence given in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID c SEQ ID NO:11 and SEQ ID NO:12 and wherein the amino acid sequence encompasses amino C acid substitutions, deletions or insertions that do not alter the functional properties of the 0 10 enzyme. A method according to any one of Claims 20 to 23, wherein the gene encoding a glycerol-3-phosphatase 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.
15. 26. A method according to Claim 1 substantially as herein described with reference to any one of the Examples.
16. 27. An Escherichia coli pAH21/DH5c 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.
17. 28. An Escherichia coli pDAR1A/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. Dated this 12 th day of July, 2005 E.I. Du Pont De Nemours and Company and Genencor International, Inc. By Their Patent Attorneys: CALLINAN LAWRIE I 4I -24- SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: NAME: E. I. DU PONT DE NEMOURS AND COMPANY STREET: 1007 MARKET STREET 00 CITY: WILMINGTON STATE: DELAWARE COUNTRY: U.S.A. POSTAL CODE 19898 TELEPHONE: 302-892-8112 TELEFAX: 302-773-0164 TELEX: 6717325 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 (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 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:l: SEQUENCE CHARACTERISTICS: LENGTH: 1380 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CTTTAATTTT CTTTTATCTT ACTCTCCTAC ATAAGACATC AAGAAACAAT TGTATATTGT ACACCCCCCC AGATTAAACT TCTTTGAAGG ACTACTATTG ATAGTACAAA AATACTAGAC GCTAATCCAG CATCAATTTT GCTATCTCCT CCTCCACAAA TAACTTCCGG CTGCCGAAAA CCAAGGTGGT TGTGGGTGTT ATCAAAACGT ACTTGATTGA TGCCCCGTAT GTCTAAAGGG CACAAATATT CCACTTGAAT GCCTTTCAAG TGCCGAAAAT CGAAGAAGAG GAAATACTTG TTCAGTCAAG CTGTAGCCAA TTTTGAAGTT GATAATATAA GCTGGTAGAA GTTACTGTGA TGTAAGGGAT ATCAATGGTG CCTGGCATCA GATGTCGACA TTGAAAGGTC GGTGCTAAAG AGATGTCTGC AGAGAAGTTC TTGGATCTGG ACCCAGAAGT AAAAATTGAC CTCTACCCGA TCATCGTTTT ATGTTGATTC GTGTCCAATT TGCTGCTGAT CTCTTCTGTT TAACTGGGGT TTTCGCTCCA TGAAATCATA CAATTTGGTT CAACATTCCA ACACGTCAGA GCTATCCTCT TACATCACTG GAAGTCGCTC AGAGGCGAGG TTCCACGTTA GTTGTTGCCT GCCATCCAAA TCTAGAGAAG GCTGGTGGTA GAATGTGAAA GTTCACGAAT TACCAAATCG GATCTACATG TTCGAGGCTC AGGAACTAGG AACAACACTG GCAAGGACGT GTGTCATCGA TAGGTTGTGG GAGTCGGT TT AAACATACTA GAAACGTCAA AGGAGTTGTT GGTTGGAAAC TTTACAACAA AAGATTAGAT TTCTATATCA TATTCAATGT GTCTCAAACA CGACCATAAG AGATGTTGCT TTTCGTCGAA GGGTGAGATC CCAAGAGTCT GGTTGCTAGG GAATGGCCAA ATGTGGCTCT CTACCCAATG TTATTGGAGA 26 CCTGCTCTAT ACAGTTGCTT GTTCTAAACG GGTATCTCCA GGTCTAGGCT ATCAGATTCG GCTGGTGTTC CTAATGGCTA TCCGCTCAAG GTCGAAGACT AAGAACCTGC AAGATAACAT CTGGTGCTAA. ACCACATTCC CCTTGTTCCA TCTGTGGTGC GGGGTAACAA GTCAAATGTT CTGATTTGAT CTTCTGGTAA GTTTAATTAC TCCCATTATT CGGACATGAT ATCATACTTC CATTGCCACC AAAGGATTTC CAGACCTTAC TTTGAAGAAC CGCTTCTGCT TTTCCCAGAA CACCACCTGC GGACGCCTCG CTGCAAAGAA TGAAGCCGTA TGAAGAATTA CCCCACTTTT 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 R TATTCATAAA TTAGCATTAT GTCATTTCTC ATAACTACTT 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: GAATTCGAGC CTGAAGTGCT GATTACCTTC AGGTAGACTT CATCTTGACC CATCAACCCC AGCGTCAATC CTGCAAATAC ACCACCCAGC AGCACTAGGA TGATAGAGAT AATATAGTAC 27 GTGGTAACGC AGTACGTGTG TATAAGATGA AACGACATAT AOTGTGAOGA AGOOTATGTG GAAACOAAAA GATAATACCC AAOTCCGGTT CCCAGGTAAC CAGCAATTCG ACOATOATAT TCAGTCATCA TOGAAAOAAT GCCGATGGCT GATTAATCTA TTTTTGGTTT TTTTCCT TOO GATTTTTTTT TTGCCTCATC ATCCGGATAA TGTATACA CTATTATAGT TATCAACTCT CAAT OACCAA GAATGAAGAA TGCTTTAATG ATTT TAT CG CGTGCGCGAT GGAGGGCGAA CGCCTTAGCC TCATTACCGA AAGAOGACGA TGCTGACGO TTGTTCAGCA TACT TTTTTT ACTAAGCTTT TTATATATTA ACCTACGCTA CAACGGCAGT ATGAGGAGCG GGGGAGAGTT TTTTTTATTA GOT OGTCCCT AGAAAACAAA AACGGTATGC AACATCCGAG GAGCTAATOC AATAAAACTG TCTAGCCATA GTTTGTTTTC TGGOTOTGCC AAGAGTGTTT GCTCTTCTCT TCTTCTTGCC TTCCTTGATT ATTTTTAAGT TGGCCGGAAT GAATATATCT CCTGATCGTG TCGTGCAAAT TGTAATAAGC TTTTTCCCAT TAO TAG CCC T COTAGGCTAT CACCCGOGOC TGAGCCATOA GAGCAAGGAA GCCATCATGC CTTCAOATGA ATTGGTTATA AGOTTACGGA ACOCTGTOAT TTTTTTTCTT TATCCTTGGG TTATGTATTT CGGCAACATC TCGGTATCGT ACOTAGACOT AACAGACGCA AAACAAGCAC TTGCTAATTT AACCCTGACT ATOTOACTOT TTCCTCAACC COCACCOCAC TTAOOATOAO AAGCGTGTAT TGAAGAAGGT TTACGCTTTT CCTATTGCOA TCTAGTATTT GTTACTTTTT T TO TTOTT TO TGGTAGATTC OOTAGAATTG AAAGATGTGA TAGTGGOAAA GOAGOAAGTA GAATGGGGAA AGAATTTAAA TOGTTTCTAT GTACGTTACA OAGGOAOOGO OOGTTGATGA CGTCACCATO OTTOTAAGAT TTGAGTATGC GCGGOGAGGT TTGTTATTOO TTTTTTTTTT TTCTAGTTTT TACTCCT TTA AATTCTCTTT 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 COTTTOOTT TTOOTTOGOT OOOOTTCCTT ATOAATGOTT GOTGTCAGAA GATTAACAAG ATACACAT TC GCCTTCAAGA CACTGCTCAT ATCCGACTCT TCGTTCTGGT TTCCCATATC AAATCTGACC CCTCCCCCAT CCTTGTTTTC CCTCGCCCCT TGTCCAATTG TCCTGCAAAC CCAACTACCA GCTGTTCCAC TGCCGGTGCC CCC TAACAAT TAGAATCTTT AGATCTGATC CACCGGTAAG GATAATCACA CCCAATTATT CTTAACAA TCTACTTTCC ACTAATATCA GCCCTGTCAA AACTGCCCCA TTCCACCCAG CATATCATAA AATCTAGTG AACATCCCTC CATGTAAGCC CTATCCTCCT TTCCCACCCC AAGGAT TATC AGACCT TACT TTGAAGAACG GCCTCCGCAC TTCCCACAAT ACCACCTCCT TCACCCTTCG TGCACAGAAC CGAGGCAGTC CGCATCCGCT TAAGAAGATC AACACCACAA TTCTACATTT CCACCATCC AGGTGACAAT ATACAAGACA CCGATCCTGA ATCAATTTTT CCATCTCGTC ATCTTACTGA AAGTCGCCAA AAGCTCATGG TCCACGTCAA TCGTGCCACT CCATTCAAAG CCAAACTCCA CAGGCGGTAC AAGCAGAAAA TTCACGACTG TACCACATAG -28- GT TATATACT ATTATTACAA ACACTGTCAT GAAACCTGCG CAAAGTCATT GTGGGTTTTT CCAGAACCTT TCTTTTACAC ACCAAACATA TCTAAAAGGG TGAGTTAGGA GGAGCATTGG CAAGGATGTA TGTCATCGAT TGCATGTGGT GCTGGGTTTA GACCTACTAT AAACGTCAAG GGAAT TCCTT GCTACAAACA TCTACAACAA C GT C CT CAT ACACAACTC CAGGACCATC CCCTTCAAGG CCGGAAAACA GATGAAAAGA AAATATCTAC TCCATCAAGG GTCAAACAAT TTCCACTTGG ATCCAATGTG TCCGAAACCA CAT CATAAGA GATGT TGCTG TTCGTAGAAG GGTGAAATTA CAAGAATCCG GTTGCCACAT AACGGTCAAT TGTGAGTTGA CGTCCGCATG ATAAAATTTT ACTCAAACAT CTATCAGAAG TTACAGTGAT CAGAATTGCA TCGGCGACGA CCAATATTGA GTGCTGACAT TGCAAGGCCA GCTCCAACCC GCGCACTATC CCGTGGCTTA T TT TCAAATT GTATATCCAT GTATGGGATG TCAAGTTCCC CT GGT CTTC ACATGGCCAA CCCCCCAACC CCCAACAATT GAAGACCTAC 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 29 CGGAGATGAT TCTGATCTTT CAACTACTAC AATCTATCAT TTTACATATC TAATCGCCAT CTGCAG TGAAGAGCTA CCTGTTGCCT TAGTAACATT TAACGT TAAT ACATCACCGT AACCT T TTCT GACATCGATG CTTTTTCCCC ACTACAGTTA TTCTATATAT TAATGAAAGA GTTATCTATA ACGAATAGAC CAACCAATTT TTATAATTTT ACATAACTAC TACGACACCC GCCCTTAAAG ACTCTCCCCC ATCATTATAC CTATTCTCTT CAT TATACAC TGTACACTAA CTGTTTCTTC (2) CTGCA( ATGCG( ATACT~ AACTT7 TAAAC CC CTT GTTTT( INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 3178 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear ii) MOLECULE TYPE: DNA (genomic) xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ;AACT TCGTCTGCTC TGTGCCCATC CTCGCGGTTA GAAAGAAGCT 'AAGG GCATCAGCGA GTGACCAATA ATCACTGCAC TAATTCCTTT FATAT ACAGCACCAG ACCTTATGTC TTTTCTCTGC TCCGATACGT ~TATT TCAGTTTTGG CAGGGGAAAT TTCACAACCC CGCACGCTAA LTAAA AGAGAACAGC CACAAATAGG GAACTTTGGT CTAAACGAAG ~TCTT GACCGTGCTA TTGCCATCAC TGCTACAAGA CTAAATACGT IGGTA ACGAGAAGAA GAGCTGCCGG TGCAGCTGCT GCCATGGCCA CCCCT CC CCC ACAAGTTCTA TTTCTTTAAG GCTATTATCG CACAATTAAA GAGCTTTTCA GAATTGTTTC T TAG CAACAC TATCCCACCC AAATCGTATT GACTCTCCCT ACTAATATAT CAGCCACGGG 2640 2700 2760 2820 2880 2940 2946 120 180 240 300 360 420 GACGCTGTAC TGCATGACTA GCCAAGGTGA TACGCCGTTA GTGCACAATG ACCCGAGCTA CATGGTGCAA CTCTGGACAA GATGTGCCCT TTGCCTCGGG AGAAGGCCTT AGCGTAAACA CCATCTACAG TTGCGTTC AGGCTCCCAT TTAACGACTC TCTTGATCTA GTGCCCAGGC TCAATGCCAC TGCCGGACTC TCATGGACCC ACTCCCCGAA TTTTACCTTG CAGAAAACCC ATATCGAATT TTCCCCACCG GACGCATCAA AGATGCTGCG AACGTCGTCC CTGGGAGTTC TCTTATCAAC CACCTGGCAG CCAAAACTTG GCTTACCACA GCGTTTCAAC TGTCGAGGTA CCGGGACGTT GGGCCCATAC CCCGCTAAAC GAAAATGGTC GGATATGGGT GCAGGGCAAA TATGCCTACA CCCCGTGAAA CCGCTCCACC TTCGACGTGT ACCAGGGGAC AAATCTACCA TCCAAGGCAC ACT GC CC C TC GTCCCGTACA AAAAAATCAT GACAATTTAA GCCACTTTAG CAAAAATTGA GAGACTAATG AGTGACGCCA GACAACTCCA ATCCCATCTA TTGTTGGACG GTCCTTGCCG GAGGCTGATA AGAGAAGACG GGCAGGTCTC TGATCATCGG TCAATGTGGC AGATGATTCA AACTGGATCT ACCTGTGCAC TCTATATGGG ACCTACTGTC AGGCCTCGCT CCATCACGCG TCAAAGACCC AGCTTGTCAG TTTTGCAAAT AGATCAAGTC TTGGCGTTCA TCAGAACCTC GCACCACAGA T TCAAGATAT TGCTAAGTGC TACACGAGAC TGGCGGGGCC CCT TGT TGAA CGGTGGGGTG GGTCATCGAG GGTGCTACCA CTGTAAATTC CAAATCCGCC TGTGTACCAT TCTGCACAAC AACTTCTGGT AATCAACGCT GGACCGCAAC GACTTTCAAT CATCGTATTG TGATGGCAGA CATCCCACTA CTTGAAAGAA ATGGGCTGGT CTGCTGGACC ACGGGGACAG AAGGGGGATT CGGTACTTAG GCACTCAACG ATTCTGATCC TACGATTTCT ACCGTGGAGA GATGGGTCCT GCCCTACCC AAGGTTATCG AAATGTGTGG CCATCCGGTC CAAATCTCCG CCCTCTTTTT GTGATGTTCT AAGCAAGTCC CTACAGCACT GTCAGACCTT 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 TGGTCAGAGA TCCACGTACA ATCCCCGCAG ACGGGAAGAA GGGCTCTGCC ACTCAGGGCG -31 TGGTAAGATC AATGGACTAC GATTCCACAA GGACGCAAAA ACTACTTGGT CCATGGAAAA GCGAGGAGAA TAAAGTATTC CAAGATTCGC TTATGGGTGA TGAACTTCAT GATAACATTC AACAATAATA TGGAAGAGTT GGTAATAGAC CTATTTCCAA CGTTTTAATT TAATATTCTT ATGGAAAATT AAATTTTCAA CCACTTCTTG TTACAGACAA CCTGAAACCT CTATGTGGCT TCAAAACTAC TAAACTGCCT CAACTTGGTC CATGCAGTAC CTTCTTGGAC TGAGTTCAAT CCAAGGACGT ACAAGAGTAA ATAATGGTGG AAAGTAAACT TCTACTACTA TACATAATAT ATCCCCTTTA CAAACGGTCC TTGCTAGTCA GTTTTTATCA TTCACTTCGG ATGGCTGACG TGTCACACAA TTATTGGCTC GGAACCCGTT TTGTCCTTAG AATTTTGATA GAATATTGTA GCCAAGGAAG TGGTCGGAGA TTCGGTGTCT TAATAATGGT TAATGGCAAT AAAAAAACTA CAATTGATCT AATCTATATA TCTCTAGTCT TGGTGCATAC TAAACCCTTT GATCCATGTT ATAATGGCCT AAACAGTCGA GAGATATTAA AAAACTACCA CCTCTATCAT CCGACAAGGA CTTTCAGATA CAACTCCCTT CTTTGAATGC AAAAGAGGCA AAATCGATCA AAT CAT GATA GAAATCGCTA CAAAAATATA TCAAATTATG ATCATTGCTG AGTTTTATCA GCAATACATA CATAAAACAA T CC TAT CT GC AATTACTATT CAAAGTTGTC GCTTGCTGGT TTTATCATCA TTGCGAATTT AAATAACGTA TCCATTCACA GGACTTCCTT CGTGCATGCC GTGGGAACTT TGATAGTTAA ATAATAATAA TTATTACCTA TGAAGAAAAA ACCTTCCTAG GTAGACTTCC TAAAATATAG TTTATGGTC TACGTAGACA CTTGACAACC GCAGGTGGTA GAAGTTGGCG GCAGAAGAAT AAAATGTCCA TTCAAAGAAT ATCTACTCTA ATCGCTGAGT TTAAGAAGAA ACCGTCAAAG GAAAAAACTG GGGTGACAAA TGATAGTAAT TTTTCCTTAA AAAAAAJAAGA TGTTTATATT GTTTTAATAT AAACACTAAA AAAAAAAAAA TCGCTACTTG TCATCGTCGA 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 -32- AATAGTACCA TTTAGAACGC CCAATATTCA CATTGTGTTC AAGGTCTTTA TTCACCAGTG ACGTGTAATG GCCATGATTA ATGTGCCTGT ATGGTTAACC ACTCCAAATA GCTTATATTT CATAGTGTCA TTGTTTTTCA ATATAATGTT TAGTATCAAT GGATATGTTA CGACGGTGTT ATTTTTCTTG GTCAAATCGT AATAAAATCT CGATAAATGG ATGACTAAGA TTTTTGGTAA AGTTACAAAA TTTATCGTTT TCACTGTTGT CAATTTTTTG TTCTTGTAAT CACTCGAG INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 816 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 2940 3000 3060 3120 3178 (xi) SEQUENCE DESCRIPTION: ATGAAACGTT TCAATGTTTT AAAATATATC SEQ ID NO:4: GCAATGCCTT GACGGTACCA GACAAGCCTT GATGCCATTG GGTGAAATCC TGTAATGCTT GACATGGCCA GCCAATGATG TTGGGTTTCC TGACCACAAA TCATCATCTC ACTTCGATGC CCAAGTTCGC CAGAAAAGTA TGAACGCCTT AGAAATGGTT TCAAGCAAGG CAATTAATGA ACCTTTATCT TCAACCAGCC CGAACACGTT TCCAGACTTT CGGTGAACAC GCCAAAGGAA CGACATTTTG TAAGCCTCAC ACAAGACCCA AGAACAACAA TTGAAAATCA ATTGCTGCTT ATTCACATCT GCTGATGAAG TCCATCGAAG AAATGGGCTG AAGATCAAGA CCAGAACCAT TCCAAATCTA AAGCAAATAT ACGCCGCTCT TCTGGAGAGA CTCACGGTTG AATACGTTAA TTCCAGGTGC TCGCCACCTC GACCAGAATA ACTTAAAGGG AGGTTGTTGT ACAAACCATC ATTCGATGTT TTTCGGTAAA GAGAACTTAC CAAGCTAGAA TGTCAAGTTG TGGTACCCGT CTTCATCACC TAGAAACGGT CTTTGAAGAC 33 GCACCAGCTG GTATTGCTGC TGGTAAGGCT GCTGGCTGTA AAATCGTTGG TATTGCTACC ACTTTCGATT TGGACTTCTT GAAGGAAAAG GGTTGTGACA TCATTGTCAA GAACCACGAA TCTATCAGAG TCGGTGAATA CAACGCTGAA ACCGATGAAG TCGAATTGAT CTTTGATGAC TACTTATACG CTAAGGATGA CTTGTTGAAA 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 ATGGGATTGA CTACTAAACC TCTATCTTTG AAAGTTAACG CCGCTTTGTT CGACGTCGAC GGTACCATTA AAACCTTATT GCCATTGCTA GAAATTCCGG AACGCTTTGA ATGGCACAAA AATGATGTCA GGATATCCGA CCAGCAGGTA TCATCTCTCA TCGATGCTGA AGT TCGCTCC TCAAGTACGG ACGCTCTACC AATGGTTCGA AACAGGGTAA TCAATGAGCA TTGCCGCCGG ACCAGCCATT ACACGTTATC AGACTTTGCC TGAAAAATCC AAAAGAGAAA G CAT C TGGGA GCCTCATCCA AGACCCTTCC AAAAGCCGCC GCTGCATTCT CAAGTCTCGC AATGAAGAGT ATTGAAGTCC TGGGCTGTGG ATCAGGAGAC GAACCATATC AAATCTAAGG GGTTGTAAGA GGAGGGATTT ATGGTTGGAG ATGTTAACAA CAGGTGCAGT CAACTTCCGG CAAAGTACTT TGAAGGGCAG TAGTAGTATT TCATTGGTAT CGGTAAGGAC AACGTTTGAT ATTAGAAGCT TAAGCTGTGC TACCCGTGAT CATTACCGCT GAATGGCTTA TGAAGACGCT TGCCACTACT 34 TTCGACTTGG ACTTCCTAAA GGAAAAACGC TGTGACATCA TTGTCAAAAA CCACGAATCC ATCAGAGTTG GCGGCTACAA TGCCGAAACA GACGAAGTTG AATTCATTTT TGACGACTAC TTATATGCTA AGGACGATCT GTTCAAATGG TAA INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 2520 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: TGTATTGGCC ACGATAACCA CCCTTTGTAT ACTGTTTTTG TTTTTCACAT GACTTTTATT GTAATTCTTC GAGGGGCTGA AGACAGCCAA CGAACCATAT ATGTTTCCCT CAGCGCCTTT TTACGAAGTG TGCATTCTGT TCAGCATCGA CGTGAAACAC AAACAACGTA TCTTCTAATT CTGCATTGAC GACTTTTAGA AAAATATACC CTCTCTTCCG ACACTAGTTT ACTACGTCCC TCAACAGATG AGGGCAAGAT CAAACGCCGG TGTAAAAACA GGAGTAAAAC AAAAAAATTG ACGGATAAGG ATGTGGTTTG ACT TGTAGTA AAAACAAGAA GCT TAT CGC C GGGCCAGGAC TGGGGTGTCT TGACATCAAA. TAACAAGAAT CAT CAAT TAA AAAAAAAAAA TGTAATAAAA AGTTGTGGCC TTCTCCAAAC CAGAGCCGTA AGTATTGATG GTTTCAAAAC GGCCTAAGGA ACCAGCGGAA CTACCCATAC AGGGTGTGGA AG GAAAAG GA TGTGGGGGGA GGAACTATAC GT TACATAT T TGTCCAAAAT TAGGAACGAC ACCAAATTGA GACCCTCTAC AGCCCATCTT GGTAAATAAC AGGCCATTTC GTAGCATAGT AAGGAAAAAA TGCCTGTTCT AAATAGT TAT CCGATCAAGC AATGGAAGAT CTCATCCAGA ATATTCAACT AGCCCCAGCT TTCTGCAGAA 120 180 240 300 360 420 480 540 600 660 720 35 GGCTATGCCA AACGAACCCA CTGGTGAACG GAACGTGTAG AGAGAAACCA GTTTGGAACG GATAG6CAAC TGTTCCAAC AACGACCTGA GCGTTCGTTT AAGTACGACA GAAATTGTGT AAGCTACACG CAGGGCTGTC GCTGCAAAAT TTGATCTCCC TACGGTGGCC GTGGCTGGTG GATGTCGGAC TTCAAGAAAC CGTTGAAGTT TCGTCCAATG CAAACGGTCT CAATTCTGTG ACACCAGAAC TGCAGCTTAG TGCGCTGGTT TGTTCGGCAC CTGACGTAAC ACGAGTTGCT CCTCATCTCA ATTCGCCAAA TGGGCGACCA GTACTTATGG AACATGGCGC AAAAACCAGA CTGTGGTCCA CGATTGCATC CAAATTCCTA CCCCAAACCG CCTTGCCTCA CCCACCTTAC GTCCCGCCGC GATCAA-AATC ACAGAAGACT CCTCGACAAT TGTGGACACA CAACGCTTCC GGAATTTTGG ATACTACGGT AACAGTACTG AAGCGCATCC TACCGGTTGC ACTGACGACT AT TGAGCAAG ATGGCTACGT TACGGTTCCT AAAATCGAGG GGTTGGGTTG AGTTTGCTCT AAGGTAATAT ACAGGAAAAC GTTAGAGACA GGATTGCCAT GAGCCTCTGT TGGCTGATTT AGAACTGGAT GGTATTGACA GACTTTGGCA CGAGATCTAG ATGGTGGGGC TTTTTACTGT CTAGCATTTT CCACATTTTG GATAATTTAC GATTCTGGTG AATTGGACTT AGTGCCATCC CTCTGCAGAC GCATGGGTAT CAATTGTTAA AATGGCAAAA TGCTCTCCAC GTACCAAGGC ACCAATTAAC TTATGAACCT AGAACCTGAT TTCCTGATTG TCAAGAGAAA AACTCGCTTA ACAATACGGG GGTTCCCACA CATTAGAGGG GATTGATCGA GCGTAGTTTT GGACTTCCAT GCAGAAAT TA TATCAACAGC AGCAAACATG CTACGGTATT CACTAGCGTC GTATTT CT CC CTATGAGGAG TAAACAAAAG CTCCACT TTA TCACATGCCC GATAATGCAA CCTGCCCATA CAAACCCGGT GACCAAAAAA TTTGCAAGAC TTCCGTCGCT TAAATCAGAG CGTCCCCGCA 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 TTTAGTGGCC TATTCGCTCC CTATTGGGAC CCAGATGCCA GAGOCACCAT AATGGGGATG TCTCAATTCA GCCAGGGCTA GACTTTTTAG GTGGATGGCG CCCT CTCT CA GCAGCCAATA GTTAAGAAAT CATCCAAACC AAGTATTGGG CACGAACAGG CTACTGCCTC TCT TGAAGGC AGGAAATTTC GGATGTCGAG, AAGTCAGAAG TGGCTTTCAA GGGTCTTTTA TTAAGATATT AAGTTGCCGT TTCTAGAAAA CCACATCGCC AATGAGTTCT CGACGTCACA GTCTAATGAA GTCTCCGACA GGATGTGAAC CAATGGAATG CAGAAGTGAA GGAAAGATCC 36 AGAGCTGCCG GACGCCTTTG TATGAAAAGT GTCATGCAAA GCGGAATGTA GAGCGCCCAT GAGAAAAACG TCCGACGATG AAAGGTTGGC TGGAAGGTGT GTGAAGGTTC C GCC C CTGTC TTCAAGCCGA CC GOAT T CC TATGGAAGGA AACAAATATC CTGAAAGGAG TGAAGGACAT TTGCTTTCAA. CAAAGACAGG GGTTCTGGCA TATCCTAGGT GOCAGO CAT T CCTACACGAT ACCAGAGGOT AAAGCATTCC AGAAGGTGAA 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 CTTCCAATAA CAACATAAAT 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 Cly 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 37 Ile Ala Lys Val Val Ala Asn Cys Lys Gly Pro Giu Val Phe le Asn Gly Giu Ala 65 Pro Ile Val Gin Met 70 Trp Val Phe Giu Giu Giu Gin Asn Val Lys Leu Thr Giu Ile Asn Thr Arg Lys Tyr Leu Asp Leu Ile 110 Pro His Gin Pro Gly Ile Asp Ser Val 115 Thr 100 Leu Pro Asp Asn Val Ala Asn Pro Lys Asp Val Asp Ile 120 Ile Val Phe Asn Ile 125 Phe Leu 130 Pro Arg Ile Cys Ser 135 Gin Leu Lys Gly Vai Asp Ser His Val1 145 Arg Ala Ile Ser Leu Lys Gly Phe Giu 155 Val Gly Ala Lys Val Gin Leu Leu Ser Tyr Ile Thr Giu Lou Gly Ile Gin Cys 175 Gly Ala Lou Trp Ser Giu 195 Gly Ala Asn Ile Ala 185 Thr Giu Val Ala Gin Giu His 190 Phe Arg Giy Thr Thr Val Ala His Ile Pro Lys Asp 205 Giu Giy 210 Lys Asp Val Asp His 215 Lys Val Leu Lys Ala Lou Phe His 220 Ala Gly Ile Ser Arg Pro 225 Tyr Phe His Val Gly Ala Lou Lys 245 Val Ile Giu Asp Val1 235 Ile 240 Cys Asn Val Val Ala Gly Cys Gly Phe Val Giu 255 Gly Lou Gly Gly Asn Asn Ala Ser 265 Ala Ala Ile Gin Arg Val Gly 270
18. 38- Leu Gly Glu 275 Ile Ile Arg Phe Gly 280 Gin Met Phe Phe Pro 285 Glu Ser Arg Glu Glu 290 Thr Tyr Tyr Gin Ser Ala Gly Val Asp Leu Ile Thr Cys Ala Gly Gly Arg 310 Asn Val Lys Val Ala 315 Arg Leu Met Ala Ser Gly Lys Asp Trp Glu Cys Glu Glu Leu Leu Asn Gly Gin 335 Ser Ala Gin Gly 340 Leu Ile Thr Cys Lys 345 Glu Val His Glu Trp Leu Glu 350 Val Tyr Gin Thr Cys Gly 355 Ser Val Glu Asp Pro Leu Phe Glu Ala 365 Ile Val 370 Tyr Asn Asn Tyr Pro 375 Met Lys Asn Leu Asp Met Ile Glu Glu 385 Leu Asp Leu His Glu Asp 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 39 Arg Ala Pro Phe Lys Val Thr Ile Gly Ser Gly Asn Trp'Gly Thr Thr Ile Ala Lys Val Ile Giu Asn Thr Giu His Ser His Ile Phe 65 Glu Pro Giu Val Arg 70 Met Trp Val Phe Asp Giu Lys Ile Gly Glu Asn Leu Thr Asp Ile Ile Asn Thr His Gin Asn Val Lys Tyr Leu Pro Asn Leu His Ser 115 Asp Leu Pro His Asn 105 Leu Val Ala Asp Pro Asp Leu 110 Ile Pro His Ile Lys Giy Aia Ile Leu Val Phe Asn 125 Gin Phe 130 Leu Pro Asn Ile Val1 135 Lys Gin Leu Gin His Val Ala Pro His 145 Val Arg Ala Ile Cys Leu Lys Gly Phe 155 Glu Leu Gly Ser Lys 160 Gly Val Gin Leu Leu 165 Ser Ser Tyr Val Asp Giu Leu Giy Ile Gin 175 Cys Gly Ala His Trp Ser 195 Ser Gly Ala Asn Leu 185 Ala Pro Giu Val Ala Lys Giu 190 Asp Tyr Gin Giu Thr Thr Val Tyr Gin Leu Pro Gly Asp 210 Gly Lys Asp Val Tyr Phe His Val 230 Asp 215 His Lys Ile Leu Lys 220 Leu Leu Phe His Arg 225 Pro Asn Val Ile Asp Val Ala Gly Ile lie Ala Gly Ala Leu Lys Asn Val Val Ala Leu Ala Cys Gly Phe Val 245 Trp 250 Ser Glu Gly Met Gly Asn Asn Ala 265 Gly Ala Ala Ile 255 Gin Arg Leu 270 Pro Glu Ser Gly Leu Gly 275 Ile Ile Lys Phe 280 Glu Arg Met Phe Lys Val 290 Thr Thr Glu Thr Tyr Tyr Gin 295 Arg Ser Ala Gly Val 300 Ala Asp Leu Ile Cys Ser Gly Asn Val Lys Thr Tyr Met 305 Lys Thr Gly Lys Ser 325 Gly Leu Glu Ala Glu 330 Arg Glu Leu Leu Asn Gly 335 Gin Ser Ala Ile Ile Thr Glu Val His Gin Thr Cys 355 Pro Asp Ser Leu Thr Gin Glu 360 Pro Pro Ile Ile Glu Trp Leu 350 Gly Ser Leu Gly Asp Asp Leu Gin Gin Arg His Gly Arg Pro 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 -41 Val Ser Arg Arg Asp Leu Leu Asp Arg Leu Asp Lys Thr His Gin Phe Asp Val Leu Ile Ile Gly Gly Gly Ala Thr Gly Thr Gly Cys Ala Leu Asp Ala Ala Thr Arg Gly Leu Asn Val Ala Leu Val Glu Lys Gly Asp Phe Ala Ser Gly Thr Ser Ser Lys Ser Thr Lys Met Ile His Gly Gly Val Arg Tyr Leu Glu Lys Ala Phe Trp Glu Phe Ser Lys Ala Gin Leu Asp Leu Val Ile Glu Ala Leu Asn Glu Arg Lys His Leu Ile Asn Thr Ala Pro His Leu Cys Thr Val Leu Pro Ile Leu Ile Pro Ile Tyr Ser Thr Trp Gin Val Pro Tyr Ile Tyr Met Gly Cys Lys Phe Tyr Asp Phe Phe Gly Gly Ser Gin Asn Leu Lys Lys Ser Tyr Leu Leu Ser Lys Ser 145 150 155 160 Ala Thr Val Glu Lys Ala Pro Met Leu Thr Thr Asp Asn Leu Lys Ala Ser Leu Val Tyr His Asp Gly Ser Phe Asn Asp Ser Arg Leu Asn Ala 180 185 190 Thr Leu Ala Ile Thr Gly Val Glu Asn Gly Ala Thr Val Leu Ile Tyr 195 200 205 Val Glu Val Gin Lys Leu Ile Lys Asp Pro Thr Ser Gly Lys Val Ile 210 215 220 Gly Ala Glu Ala Arg Asp Val Glu Thr Asn Glu Leu Val Arg Ile Asn -42- 225 240 Ala Lys Cys Val Val 245 Asn Ala Thr Gly Tyr Ser Asp Ala Ile Leu 255 Gin Met Asp Asn Pro Ser Gly Leu 265 Pro Asp Ser Pro Leu Asn Asp 270 Met Asp Pro Asn Ser Lys 275 Ile Lys Ser Thr Asn Gin Ile Ser Val 285 Lys Met 290 Val Ile Pro Ser Pro Lys Asp Met 310 Ile 295 Gly Val His Ile Val 300 Leu Pro Ser Phe Tyr 305 Ser Gly Leu Leu Asp Arg Thr Ser Asp Gly 320 Arg Val Met Phe Phe 325 Leu Pro Trp Gin Lys Val Leu Ala Gly Thr 335 Thr Asp Ile Leu Lys Gin Val Pro Glu Asn Pro Met 345 Pro Thr Glu 350 Ile Glu Phe Ala Asp Ile 355 Gin Asp Ile Leu Glu Leu Gin His Pro Val 370 Lys Arg Glu Asp Val 375 Leu Ser Ala Trp Ala 380 Gly Val Arg Pro Leu 385 Val Arg Asp Pro Arg 390 Thr Ile Pro Ala Gly Lys Lys Gly Ala Thr Gin Gly Val Arg Ser His Phe 410 Leu Phe Thr Ser Asp Asn 415 Gly Leu Ile Ala Glu Glu 435 Ile Ala Gly Gly Trp Thr Thr Tyr Arg Gin Met 430 Phe His Asn Thr Val Asp Lys Val 440 Val Glu Val Gly -43- Leu Lys 450 Pro Cys His Thr Arg 455 Asp Ile Lys Leu Gly Ala Glu Glu Trp 465 Thr Gin Asn Tyr Ala Leu Leu Ala Gin 475 Asn Tyr His Leu Ser Lys Met Ser Asn 485 Tyr Leu Val Gin Tyr Gly Thr Arg Ser Ser 495 Ile Ile Cys Ser Leu Ala 515 Phe Phe Lys Glu Ser 505 Met Glu Asn Lys Leu Pro Leu 510 Glu Glu Asn Asp Lys Glu Asn Val Ile Tyr Ser Ser 525 Asn Leu 530 Val Asn Phe Asp Thr 535 Phe Arg Tyr Pro Thr Ile Gly Glu Leu 545 Lys Tyr Ser Met Tyr Glu Tyr Cys Arg 555 Thr Pro Leu Asp Phe 560 Leu Leu Arg Arg Thr 565 Arg Phe Ala Phe Asp Ala Lys Glu Ala Leu 575 Asn Ala Val Ala Thr Val Lys Val 585 Met Gly Asp Glu Phe Asn Trp 590 Ser Glu Lys 595 Lys Arg Gin Trp Leu Glu Lys Thr Val 605 Asn Phe Ile Gin Gly 610 Arg Phe Gly Val INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 339 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown 44 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l0: Ala Ser Met Thr Val Met Asn Gin Arg Asn 1 Gly 5 Ala 10 Arg Ile Gly Ala Gly Ser Tyr Asn Gly His Giu Val Val Thr Ala Leu Ile Thr Leu Ala 25 Ile Ala Thr Leu Leu Trp Gly Cys Asn Ala His Asp Pro Glu Giu Asp Arq Asp Arg Thr Leu His Ala Phe Leu Pro Thr Val Pro Phe Leu Val Glu Ser Asp Leu Ala Leu Ala Al a Val1 Arq Asn Ile Val Val Pro Ser Pro Val Phe Gly Glu 90 Val1 Leu Arg Gin Ile Lys Pro Leu Met Asp Ala Arg Leu 105 Gin Trp Ala Thr Glu Ala Glu 115 Gly Asp Gin 130 Gly Arg Leu Asp Val Ala Arg 125 Thr Lys Gly Leu 110 Glu Ala Leu Phe Ala Lys Ile Pro Leu Ala 135 Pro Ile Ser Gly Glu 145 Gin Leu Ala Ala Gly Thr Ala Ile Ser 155 Leu Ala Ser Thr Thr Phe Ala Asp 165 Ser Leu Gin Gin His Cys Gly Lys Ser 175 Gly Gly Phe Arg Val Asn Pro Asp Phe 185 Gly Val Gin Ala Val Lys 195 Asn Val Ile Ala Ile 200 Gly Ala Gly Met Ser 205 Asp Gly Ile Gly Phe 210 Gly Ala Asn Ala Thr Ala Leu Ile Arg Gly Leu Ala Glu 225 Met Ser Arg Leu Gly 230 Ala Ala Leu Gly Ala 235 Asp Pro Ala Thr Met Gly Met Ala Leu Gly Asp Leu Leu Thr Cys Thr Asp Asn 255 Gin Ser Arg Val Gin Ser 275 Asn 260 Arg Arg Phe Gly Met 265 Met Leu Gly Gin Gly Met Asp 270 Gly Tyr Arg Ala Gin Glu Lys Gly Gin Val Val Glu 285 Asn Thr 290 Lys Glu Val Arg Glu 295 Leu Ala His Arg Gly Val Glu Met Ile Thr Glu Glu Tyr Gin Val Leu Tyr 315 Cys Gly Lys Asn Arg Glu Ala Ala Leu Thr Leu Leu Gly 325 Ala Arg Lys Asp Glu Arg 335 Ser Ser His INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 501 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
19. 46- Met 1 Glu Thr Lys Leu Ile Val Ile Gly 10 Gly Gly Ile Asn Gly Ala Gly Ile Ala Ala Asp Ala Ala Gly Gly Leu Ser Val Leu Met Leu Ser Lys Leu Glu Ala Gin Asp Leu Ala Cys Ala 40 Thr Ser Ser Ala Ser Ile His Gly Gly Leu Arg Leu Glu His Tyr Phe Arg Leu Val Glu Ala Leu Ala Glu Arg Glu Val Leu Leu Lys Met Ala Pro lie Ala Phe Pro Arg Phe Arg Leu Pro His Arg Pro His Leu Arg Pro Ala Trp Met 100 Ile Arg Ile Gly Leu 105 Phe Met Tyr Asp His Leu Gly 110 Gly Ala Asn Lys Arg Thr 115 Ser Leu Pro Gly Thr Gly Leu Arg Phe 125 Ser Val 130 Leu Lys Pro Glu Ile 135 Lys Arg Gly Phe Tyr Ser Asp Cys Trp 145 Val Asp Asp Ala Leu Val Leu Ala Asn Ala Gin Met 155 Arg Ala Thr Ser Val Arg Lys Gly Gly Val Leu Thr Arg Ala Arg 175 Arg Glu Asn Gly 180 Leu Trp Ile Val Glu 185 Ala Glu Asp Ile Asp Thr Gly 190 Thr Gly Pro Lys Lys Tyr 195 Ser Trp Gin Ala Gly Leu Val Asn Ala 205 Trp Val 210 Lys Gin Phe Phe Asp 215 Asp Gly Met His Pro Ser Pro Tyr 47 Gly 225 Ile Arg Leu Ile Lys 230 Gly Ser His Ile Val1 235 Val Pro Arg Val Thr Gin Lys Gin Tyr Ile Leu Gin Giu Asp Lys Arg Ile Vai 255 Phe Vai Ile Vai Giu Tyr 275 Pro 260 Trp Met Asp Giu Phe 265 Ser Ile Ile Giy Thr Thr Asp 270 Giu Ser Glu Lys Giy Asp Pro Aia Vai Lys Ile Giu 285 Ile Asn 290 Tyr Leu Leu Asn Vai 295 Tyr Asn Thr His Phe Lys Lys Gin 300 Vai Arq Pro Leu Leu Arg Asp Asp Ile Trp Thr Tyr Ser Giy 315 Cys 320 Thr Leu 335 Asp Asp Giu Ser Asp 325 Ser Pro Gin Ala Thr Arq Asp Tyr Asp Ile His Giy Lys Leu 355 Giu Asn Gly Lys Aia 345 Pro Leu Leu Ser Vai Phe Gly 350 Leu Giu Lys Thr Thr Tyr Arg Leu Ala Glu His Aia 365 Leu Thr 370 Pro Tyr Tyr Gin Giy 375 Ile Gly Pro Aia Thr Lys Giu Ser Val1 385 Leu Pro Giy Gly Leu Arg Arg Arg 405 Ile Giu Giy Asp Arg 395 Asp Asp Tyr Aia Aia 400 Arg Tyr Pro Phe Leu Giu Ser Leu Aia Arg His 415 Tyr Aia Arg Tyr Giy Ser Asn Ser 425 Giu Leu Leu Leu Giy Asn Ala 430 Giy Thr Vai Ser Asp Leu Giy Giu Asp Phe Giy His Giu Phe Tyr Giu -48- 445 Ala Glu Leu Lys Tyr Leu Val Asp His Glu Trp Arg Arg Ala Asp Asp 465 Ala Leu Trp Arg Thr Lys Gin Gly Met 475 Trp Leu Asn Ala Gin Gin Ser Arg Val Ser Gin Trp Leu 485 Glu Tyr Thr Gin Gin Arg 495 Leu Ser Leu Ala Ser 500 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 542 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Lys Thr Arg Asp Ser Gin Ser Ser Val Ile Ile Ile Gly Gly Gly Ala Thr Arg Val Ile Ala Gly Ile Ala Arg Asp Cys Ala Leu Arg Gly Leu Ala Thr Gly Leu Val Glu Arg Asp Ile Ala Thr Gly Arg Asn His Gly Leu Leu His 55 Ser Gly Ala Arg Tyr Ala Val Thr Gin Ile Leu Lys Asp Ala 65 Glu Ser Ala Arg Cys Ile Ser Glu Asn 75 Arg 49 Ile Ala Arg His Val Giu Pro Thr Gly Leu Phe Ile Thr Leu Pro Glu Asp Asp 100 Leu Ser Phe Gin Al a 105 Thr Phe Ile Arq Ala Cys Giu 110 Ala Arg Ile Giu Ala Gly 115 Ile Ser Ala Giu Ile Asp Pro Gin Gin 125 Ile Giu 130 Pro Ala Val Asn Ala Leu Ile Gly Vai Lys Val Pro Gly Thr Val Asp Pro 150 Phe Arg Leu Thr Ala 155 Ala Asn Met Leu Ala Lys Glu His Ala Val Ile Leu Ala His Glu Val Thr Gly 175 Leu Ile Arg Giu 180 Giy Ala Thr Val Cys 185 Gly Val Arg Val Arg Asn His 190 Vai Asn Ala Leu Thr Giy 195 Giu Thr Gin Ala His Ala Pro Val Val1 205 Ala Giy 210 Ile Trp Gly Gin Ile Ala Giu Tyr Asp Leu Ar Ile Arg 225 Met Phe Pro Ala Gly Ser Leu Leu Ile 235 Met Asp His Arg Asn Gin His Vai Ile 245 Asn Arg Cys Arg Pro Ser Asp Ala Asp Ile 255 Leu Val Pro Giy 260 Asp Thr Ile Ser Leu 265 Ile Gly Thr Thr Ser Leu Arg 270 Giu Glu Vai Ile Asp Tyr 275 Asn Giu Ile Asp Asn Arg Val Thr Ala 285 Asp Ile 290 Leu Leu Arg Giu Giy 295 Giu Lys Leu Aia Vai Met Ala Lys Thr 305 Arg Ile Leu Arg Tyr Ser Gly Val Arg Pro Leu Val Ala Ser Asp Asp Asp Pro Ser 325 Gly Arg Asn Leu Arg Gly Ile Val Leu Leu 335 Asp His Ala Gly Lys Leu 355 Val Cys Arg 370 Arg Asp Gly Leu Asp 345 Gly Phe Ile Thr Ile Thr Gly 350 Thr Asp Ala Met Thr Tyr Arg Lys Leu Gly Asn 375 Met Ala Glu Trp Thr Arg Pro Cys Thr Ala Asp Leu Ala 385 Leu Pro Gly Ser Ser Leu Pro Ala 405 Glu Pro Ala Giu Val Thr Leu Arq 395 Ala Val Tyr Arg Lys Val 400 Ile Pro Leu Arg Gly His Gly 415 Asp Arg Thr Val Cys Glu 435 Ala Trp Leu Ser Giu 425 Gly Arg Leu His Arg Ser Leu 430 Tyr Ala Val Cys Glu Ala Val Ala Gly Giu Val Glu Asn 450 Leu Asn Val Asn Ser 455 Leu Leu Asp Leu Arg 460 Arq Arg Thr Arg Val1 465 Gly Met Gly Thr Gin Gly Giu Leu Ala Cys Arg Ala Gly Leu Leu Gin Arg 485 Phe Asn Val Thr Ser Ala Gin Ser Ile Giu 495 Gin Leu Ser Phe Leu Asn Glu Arg 505 Trp Lys Gly Val Gin Pro Ile 510 Ala Trp Gly Asp Ala Leu Arg Glu Ser Giu Phe Thr Arg Trp Val Tyr -51 cqq oo O (N <3 e-4 00 (N 0 -m 0 (N in 515 520 525 Gin Gly Leu Cys Gly Leu Glu Lys Glu Gin Lys Asp Ala Leu 530 535 540 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 250 amino acids 10 TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) (xi) MOLECULE TYPE: protein SEQUENCE Met Gly Leu Thr Thr DESCRIPTION: Lys Pro Leu Thr Ile Ile SEQ ID NO:13: 1 Phe 5 Gly Ser Leu 10 Ile Ser Lys Val Asn Ala Ala Leu Asp Val Asp Trp Arg Asp Gin Pro Ala 25 Lys Phe Phe Gly Lys Pro Tyr Phe Val Ile Gin Asp Ala Ile Ala Ala Ala Glu His Ile Ala Lys Val Ser His Gly Asn Arg Thr Phe Phe Glu Ala Pro Asp Phe Glu Glu Tyr Val 75 Ile Lys Leu Glu Ala Ile Pro Val Lys Asn Gly Glu Lys Glu Val Pro Gly Ala Val Lys Leu Val Ala Thr 115 Cys 100 Ser Ala Leu Asn Ala 105 Met Pro Lys Glu Lys Trp Ala 110 Phe Glu His Gly Thr Arg Ala Gin Lys Trp 125 -52- in oo C) (N O 00 (N 0q m% 0D (N in Leu Gly Ile Arg Arg Pro Lys Tyr Phe Ile Thr Ala Asn Asp Val Lys 130 Gly 135 Glu Gin 5 145 Gly Lys Pro His Pro Tyr Leu Lys 155 Lys Arg Asn Gly Tyr Pro Ile Gin Asp Pro Ser Lys Val Val Val 175 Phe Glu Asp Lys Ile Ile 195 Lys Gly Cys 210 Ala 180 Gly Ala Gly Ile Ala 185 Phe Gly Lys Ala Ile Ala Thr Asp Leu Asp Ala Gly Cys 190 Leu Lys Glu Arg Val Gly Asp Ile Ile Val 215 Asp Asn His Glu Ser 220 Ile Gly 225 Leu Tyr Asn Ala Glu Glu Val Glu Phe 235 Phe Asp Asp Tyr Ala Lys Asp Leu Leu Lys Trp 250 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: Met 1 Ile LENGTH: 271 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Lys Arg Phe Asn Val Leu Lys Tyr Ile Arg Thr Thr Lys Ala Asn 5 10 Gin Thr Ile Ala Met Pro Leu Thr Thr Lys Pro Leu Ser Leu Lys 25
20. 53- Ile Asn Ala Ala Leu Phe Asp Asp Gly Thr Ile Ile Ile Ser Gin Pro Ala Ile Ala Ala Phe Trp Arg Asp Phe Gly Asp Lys Pro Tyr Asp Ala Glu His Ile His Ile Ser His Gly Trp Arg Ala Asp Glu Glu Thr Asp Ala Ile Ala Phe Ala Pro Asp Tyr Val Asn Lys Leu Glu 100 Gly Glu Ile Pro Glu 105 Lys Tyr Gly Glu His Ser Ile 110 Ala Leu Pro Glu Val Pro 115 Gly Ala Val Lys Cys Asn Ala Leu Asn 125 Lys Glu 130 Lys Trp Ala Val Ala 135 Thr Ser Gly Thr Asp Met Ala Lys Lys 145 Trp Phe Asp Ile Lys Ile Lys Arg Pro 155 Glu Tyr Phe Ile Thr 160 Ala Asn Asp Val Lys 165 Gin Gly Lys Pro His 170 Pro Glu Pro Tyr Leu Lys 175 Gly Arg Asn Leu Gly Phe Pro Asn Glu Gin Asp Pro Ser Lys 190 Ala Ala Gly Ser Lys Val 195 Val Val Phe Glu Asp 200 Ala Pro Ala Gly Lys Ala 210 Ala Gly Cys Lys Ile 215 Val Gly Ile Ala Thr 220 Thr Phe Asp Leu Asp 225 Phe Leu Lys Glu Lys 230 Gly Cys Asp Ile lie 235 Val Lys Asn His Ser Ile Arg Val Gly Glu Tyr Asn Ala Glu Thr Asp Glu Val Glu Leu in O o O 1 (N ¢3 O >-1 (N 00 (N 0D 8 (N i! -54- 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 0 TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) (xi) MOLECULE TYPE: protein SEQUENCE DESCRIPTION: SEQ ID Met Phe Pro Ser Leu Phe Arg Leu Val Val Phe Ser Lys Arg 1 Phe Arg Ser Ser Met Ser Lys 5 Gin Arg Leu Ile Met Glu Tyr Ile Tyr Thr 25 Leu Leu Lys Gin Arg Ile Ala Ser Asp 40 Thr Arg Ser Asp Tyr Cys Glu Gin Ser Val Pro Leu Ile Leu Phe Ile Asp Val Thr Ser Ser Arg Asn Ser Trp Gly Gin Asp 70 Lys Ser Lys His Gin Gly Glu Tyr Ser Ala Ser Lys Ile Gly Val Leu Arg Arg Pro Ser Thr Ala Pro Gly Lys Pro 115 Ala 100 Ile Glu Thr Pro Asn 105 Gly Gly Asp Ile Lys Thr Ser 110 Glu Thr Lys Phe Ser Ala Glu 120 Tyr Ala Ile Gin 125 55 Phe Leu Lys Ile Glu Giu Leu Asp Leu Asp Phe 130 135 Asn Giu Pro Thr Leu 145 Lys Phe Pro Lys Giy Trp Val Glu Cys 155 His Pro Gin Lys Leu Val Asn Val Gin Cys Leu Ala Ser Ser Leu Leu 170 Gly Leu Pro Pro Thr Ile Asn Ile Cys Met 195 Ser 180 Glu Arg Val Ala Asn 185 Ser Leu Gin 175 Tyr Lys Val 190 Leu Trp Ser Gly Ile Ala Asn Arg Giu Thr Thr Ile 205 Arg Arq 210 Thr Gly Lys Pro Ile 215 Val Asn Tyr Gly Ile Val Trp Asn Asp 220 Gin Asn Thr Ser Val Thr 225 Arg Thr Ile Lys Arg Gin Leu Gin 245 Vai Arg Asp Lys Trp 235 Asp Leu Arg Gin Lys Gly Leu Pro Leu Leu Ser 255 Thr Tyr Phe Leu Cys Thr 275 Cys Ser Lys Leu Arg 265 Trp Phe Leu Asp Asn Giu Pro 270 Giy Thr Vai Lys Ala Tyr Giu Asn Asp Leu Met Phe 285 Asp Thr 290 Trp Leu Ile Tyr Gin 295 Leu Thr Lys Gin Ala Phe Val Ser Asp 305 Val Thr Asn Ala Arg Thr Gly Phe Met 315 Asn Leu Ser Thr Leu 320 Lys Tyr Asp Asn Giu 325 Leu Leu Giu Phe Gly Ile Asp Lys Asn Leu 335 Ile His Met Giu Ile Val Ser Ser 345 Ser Gin Tyr Tyr Gly Asp Phe 350 56 Gly Ile Pro 355 Asp Trp Ile Met Lys Leu His Asp Ser 365 Pro Lys Thr Val Leu 370 Arg Asp Leu Val Lys 375 Arg Asn Leu Pro Gin Gly Cys Leu Gly 385 Asp Gin Ser Ala Met Val Giy Gin Leu 395 Ala Tyr Lys Pro Ala Ala Lys Cys Tyr Gly Thr Gly Phe Leu Leu Tyr Asn Thr 415 Gly Thr Lys Lys 420 Leu ile Ser Gin His 425 Gly Ala Leu Thr Thr Leu Ala 430 Pro Glu Leu Phe Trp Phe 435 Pro His Leu Gin Tyr Gly Gly Gin Lys 445 Ser Lys 450 Pro His Phe Ala Leu 455 Giu Gly Ser Val Val Ala Gly Ala Val Gin Trp Leu Asp Asn Leu Arg Leu 475 Ile Asp Lys Ser Asp Val Gly Pro Ile 485 Ala Ser Thr Val Asp Ser Giy Gly Val Val 495 Phe Val Pro Phe Ser Giy Leu Phe 505 Ala Pro Tyr Trp Asp Pro Asp 510 Ala Ser His Ala Arg Ala 515 Thr Ile Met Giy Ser Gin Phe Thr Thr 525 Ile Ala 530 Arg Ala Ala Val Giu 535 Gly Val Cys Phe Ala Arg Ala Ile Leu 545 Lys Ala Met Ser Asp Ala Phe Gly Gin 555 Gly Ser Lys Asp Asp Phe Len Giu Giu Ile Ser Asp Val Thr Tyr Glu Lys Ser Pro Leu -57- Ser Val Leu Ala 580 Val Asp Gly Gly Met Ser Arg Ser Asn 585 575 Glu Val Met 590 Arg Arg Ser Gin Ile Gin 595 Ala Asp Ile Leu Gly 600 Pro Cys Val Lys Val 605 Pro Thr 610 Ala Glu Cys Thr Ala 615 Leu Gly Ala Ala Ala Ala Asn Met Ala 625 Phe Lys Asp Val Glu Arg Pro Leu Trp 635 Lys Asp Leu His Val Lys Lys Trp Val 645 Phe Tyr Asn Gly Glu Lys Asn Glu Gin Ile 655 Ser Pro Glu Asp Ala Glu 675 His Pro Asn Leu Lys 665 Ile Phe Arg Ser Glu Ser Asp 670 Ala Val Glu Arg Arg Lys His Lys Tyr Trp Glu Val 685 Arg Ser 690 Lys Gly Trp Leu Lys 695 Asp Ile Glu Gly His Glu Gin Val Leu Glu Asn Phe Gin 705 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: -58- n) GCGCGGATCC AGGAGTCTAG AATTATGGGA TTGACTACTA AACCTCTATC T 51 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid 00 STRANDEDNESS: single O TOPOLOGY: linear 8r (C (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: -59- GTATGATATG TTATCTTGGA TCCAATAAAT CTAATCTTC 39 S(2) INFORMATION FOR SEQ ID Sni e- SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs 00 TYPE: nucleic acid S(C) STRANDEDNESS: single r n 10 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) ln (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: CCTAGTAAGGA GGACAATTC 19 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: 00 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 -61 9I TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "PRIMER" 00 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID CTAGTCTGTT TCCTG -62- SWhere the terms "comprise", "comprises", "comprised" or "comprising" are used in Sthis 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 18854/02 which is itself a divisional application of AU 54307/98, the disclosures of which are incorporated herein by way of reference. 00 (N- 0"- mt' 0"-