MXPA00005478A - Method for the production of glycerol by recombinant organisms - Google Patents

Abstract

Recombinant organisms are provided comprising genes encoding a glycerol-3-phosphate dehydrogenase and/or a glycerol-3-phosphatase activity useful for the production of glycerol from a variety of carbon substrates. The organisms further contain disruptions in the endogenous genes encoding proteins having glycerol kinase and glycerol dehydrogenase activities.

Description

METHOD FOR THE PRODUCTION OF GLYCEROL BY RECOMBINANT ORGANISMS FIELD OF THE INVENTION The present invention concerns the field of molecular biology and the use of recombinant organisms for the production of glycerol and compounds from glycerol biosynthetic processes. More specifically, the invention describes the construction of a recombinant cell for the production of glycerol and compounds derived from a carbon substrate, the cell containing foreign genes that encode proteins having glycerol-3-phosphate dehydrogenase (G3PDH) and glycerol activities. 3-phosphatase (G3P phosphatase) where the endogenous genes that encode glycerol converting glycerol activities hydrogenase and glycerol kinase, have been eliminated.

BACKGROUND Glycerol is a highly demanded compound for use in the cosmetics, liquid soaps, food, pharmaceutical, lubricants, REF. 120688 anti-freeze, and numerous other applications. Glycerol esters are important in the fats and oils industry. Historically, glycerol has been isolated from animal fat and similar sources; however, the process is laborious and inefficient. Microbial production of glycerol is preferred.

Not all organisms have a natural ability to synthesize glycerol. However, the biological production of glycerol is known for some species of bacteria, algae, and yeasts. The bacteria Bacill us licheniformis and Lactobacillus lycopersica synthesize glycerol. The production of glycerol is found in the seaweed Dunali ella sp. and Asteromonas gracilis halotolerantes for protection against high concentration of external salts (Ben-A otz et al., (1982) Experientia 38: 49-52). Similarly, several yeast-tolerant yeasts synthesize glycerol as a protective measure. Mostly Saccharomyces strains produce glycerol during alcoholic fermentation and their production can be increased by the application of osmotic stress (Alberryn et al., (1994) Mol Cell Biol .. 14, 4135-4144). At the beginning of this century glycerol was commercially produced with cultures of Saccharomyces to which reactants were added to induce them such as sulfites or alkalis. Through the formation of an inactive complex, the inducing agents block or inhibit the conversion of acetaldehyde or ethanol; thus, excess reducing equivalents (NADH) are available for or "induced" to dihydroxyacetone phosphate (DHAP) for reduction to produce glycerol. This method is limited by the partial inhibition of yeast development that is due to sulfites. This limitation can be partially overcome by the use of alkalies that create excess NADH equivalents by a different mechanism. In this practice, the alkalis initiate a Cannizarro disproportion to produce ethanol and acetic acid from two equivalents of acetaldehyde. In that way, although glycerol production is possible from organisms as they are naturally found, production is often subject to the need to control the osmotic tension of crops and the production of sulfites. A method free of this limitation is desirable. The production of glycerol by biosynthetic process is a possible way for such a method.

A number of the genes involved in the biosynthetic procedure for glycerol have been isolated. For example, the gene encoding glycero-3-phosphate dehydrogenase (DAR1, GPD1) has been cloned and sequenced from Saccaromyces diastaticus (Wang et al., (1994), J. Bact. 176: 7091-7095). The DAR1 gene was cloned into a traveling vector and used to transform E. coli where the expression produced the active enzyme. Wang et al., Supra, recognizes that DAR1 is regulated by the cellular osmotic environment but does not suggest how the gene should be used to improve the production of glycerol 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. Serevisiae (Larason et al., (1993) Mol.Microbiol., 10: 1101). Albertyn et al., (1994) Mol. Cell. Biol., 14: 4135) discloses the cloning of GPD1 encoding glycerol-3-phosphate dehydrogenase from S. Serevisiae. In the same way as Wang et al., Both Alberthyn et al. And Larason et al. Recognize the osmo-sensitivity of the regulation of this gene but do not suggest how the gene should be used in the production of glycerol in a recombinant organism. As with G3PDH, glycerol 3-phosphatase has been isolated from Saccharomyces cerevisiae and the protein identified as being encoded by GPP1 and GPP2 genes (Norbecck et al., (1996) J. Biol. Chem., 271: 13875). In the same way as the genes that encode G3PDH, it seems that GPP2 is osmotically induced.

Although the genes encoding G3PDH and G3P phosphatase have been isolated, there is no teaching in the art that demonstrates the production of glycerol from recombinant organisms with G3PDH / G3P phosphatase expressed together or separately. Additionally there is no teaching to suggest that efficient production of glycerol desired some type of wild type organism is possible using these two enzymatic activities that does not require any tension (salts or an osmolyte) to the cell. In fact, experience suggests that G3PDH activities may not affect the production of glycerol- For example, Eustace ((11987), Can. J. Microbiol., 33: 112-117)) shows hybridized yeast strains that produce glycerol at higher levels than the parent strain. However, Eustace also shows that the G3PDH activity remained constant or slightly lower in the hybridized strains as opposed to the wild type.

Glycerol is an industrially useful material. However, other compounds may be derivatives of the glycerol biosynthetic process which also have commercial significance. For example, glycerol producing organisms can be designed to produce 1, 3-propanediol (USA 5686276), a monomer that has potential utility in the production of polyester fibers and in the manufacture of polyurethanes and cyclic compounds. It is known for example that in some organisms, glycerol is converted to 3-hydroxypropionaldehyde and then to 1,3-propanediol through the actions of a dehydratase enzyme and an oxidoreductase enzyme, respectively. Bacterial strains capable of producing 1,3-propanediol have been found, for example, in the group ci Trobacter, Clostridium, Enterobacter, Iliobacter, Klebsi ella, Lactobacillus, and Pelobacter. The glycerol dehydratase and dehydratase diol systems are described by Seyfried et al. (1996) J. Bacteriol, 178: 5793-5796 and Tobimatsu et al. (1995) J. Biol. Chem. 270: 7142-7148, respectively. Recombinant organisms, which contain exogenous dehydratase enzyme, which are capable of producing 1,3-propanediol have been described (USA 5686276). Although these organisms produce 1,3-propanediol, it is clear that they would benefit from a system that would minimize the conversion of glycerol.

There are a number of engineering advantages of glycerol producing organisms for the production of 1,3-propanediol where the conversion of glycerol is minimized. A microorganism capable of efficiently producing glycerol under physiological conditions is industrially desirable, especially when the glycerol itself will be used as a substrate in vivo as part of a complex catabolic or biosynthetic process that could be disturbed by omotic tension or the addition of inducing agents (eg. example, production of 1,3-propanediol). Some attempts to create mutants of glycerol kinase and glycerol dehydrogenase have been made. Or example, De Koning et al. (1990) Appl. Microbiol. Biotechnology. 32: 693-698 reports that the methanol-dependent production of dihydroxyacetone and glycerol by mutants of the methylphenic yeast Hansenula polymorpha blocked in dihydroxyacetone kinase and glycerol kinase. Methanol and an additional substrate required to replenish the xylose-5-phosphate, co-substrate of the assimilation reaction, was used to produce glycerol; however, a dihydroxyacetone reductase (glycerol dehydrogenase) is also required. Similarly, Shaw and Cameron, Book of Abstracts, 211ava. ACS National Meeting, Neew Orleans, LA, March 24-28 (1996), BIOT-154 Publisher; American Chemical Society, Washimgton, D. C. investigates the elimination of IdhA (lactate dehydrogenase) gJpK (glycerol kinase), and tpiA (triosephosphate isomerase) for the production optimization of 1,3-propanediol. They do not suggest the expression of genes cloned by G3PDH or G3P phosphatase for the production of glycerol or 1,3-propanediol and they do not discuss the impact of glycerol dehydrogenase.

The problem to be solved, however, in the lack of a process to direct coal flow towards coal production by the addition of enhancers of certain enzymatic activities, especially G3PDH and G3P phosphatase that respectively catalyze the conversion of dihydroxyacetone phosphate ( DHAP) to glycerol-3-phosphate (G3P and then to glycerol.) The problem is complicated by the need to control the flow of carbon away from glycerol by eliminating or decreasing the activities of certain enzymes, especially glycerol kinase and glycerol dehydrogenase which respectively they catalyze the conversion of glycerol plus ATPP to G3P and glycerol to dihydroxyacetone (or glyceraldehyde).

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a method for the production of glycerol from a recombinant organism comprising: transforming a suitable host cell with an expression cartridge comprising either or both of (a) a gene encoding a protein having glycerol-3-phosphate dehydrogenase activity and (b) a gene encoding a protein having glycerol 3-phosphate phosphatase activity, wherein the suitable host cell contains an interruption in either one or both of (a) a gene encoding an endogenous glycerol kinase and (b) a gene encoding an endogenous glycerol dehydrogenase, wherein the disruption prevents the expression of the active gene product; culturing the transformed host cell in the presence of at least one carbon source selected from the group consisting of - monosaccharides, oligosaccharides, polysaccharides, and single carbon substrates, by means of which glycerol is produced; and recovering the glycerol produced.

The present invention further provides a process for the production of 1,3-propanediol from a recombinant organism comprising: transforming a suitable host cell with an expression cartridge comprising either or both of (a) a gene encoding a protein having glycerol 3-phosphate dehydrogenase activity and (b) a gene encoding a protein having glycerol-3-phosphate phosphatase activity, the suitable host cell having at least one gene encoding a protein having a dehydratase activity and having an interruption in either or both of (a) a gene encoding an endogenous glycerol kinase and (b) a gene encoding an endogenous glycerol dehydrogenase, in which the disruption in the genes of (a) or (b) prevent the expression of the active gene product; culturing the transformed host cells in the presence of at least one carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and single carbon substrates by which 1,3-propanediol is produced; and recovering the 1, 3-propanediol produced.

Additionally the invention provides for a process for the production of 1,3-propanediol from a recombinant organism where multiple copies of endogenous genes are introduced.

Additional embodiments of the invention include host cells transformed with heterologous genes for the glycerol method as well as host cells containing endogenous genes for the glycerol process.

Additionally, the invention provides recombinant cells suitable for the production of either glycerol or 1,3-propanediol, the host cells having genes that express either or both of a glycerol-3-phosphate dehydrogenase activity and a glycerol activity. - 3-phosphate phosphatase wherein the cell also has breaks in either or both of a gene encoding a glycerol kinase and a coding for a glycerol dehydrogenase, wherein disruption in the genes prevents the expression of an active gene product .

BRIEF DESCRIPTION OF THE FIGURES, BIOLOGICAL DEPOSITS AND LIST OF SEQUENCES.

Figure 1 illustrates the representative enzymatic procedure involving the metabolism of glycerol.

Applicants have made the following biological deposits under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of Patent Procedures: Reference for Int Designation Date of Depositor's Identification Depositor's Deposit Escherichia coli ATCC 98187 26 from pñH21 / DH5a September (containing the 1996 GPP2 gene) Escherichia coli ATCC 98248 6 of DAR1A / AA200) November containing the 1996 gene DAR1) FM5 Escherichia ATCC 98597 25 of Coli RJFl Om (November Contains a 1997 in terption) Reference for Int Designation Date of Depositor Identification Depositor's Deposit FM5 Escherichia ATCC 98598 25 of Coli MSP33.6 November (containing 1997) an interruption gldA) "ATCC" refers to the American Type Culture Collection international deposit located at 12301 Paarklawn Drive, Rockville, MD 20852 USA. The designation is the access number of the deposited material.

Applicants' has provided 43 sequences in accordance with the Rules for the Standard Representation of Sequences of Nucleotides and Amino Acids in Patent Applications (Annexes I and II of the Decision of the President of the EPO, published in Supplement No. 2 of PJ EPO , 12/1992) and with 37 CFR 1. 821- 1825 and Appendices A and B (Requirements for Requests Exhibits containing Nucleotide and amino acid sequences).

DETAILED DESCRIPTION OF THE INVENTION The present invention solves the problem set forth in providing a method for the biological production of glycerol from a source of fermentable carbon in a recombinant organism. The method provides a fast, cheap and environmentally responsible source of glycerol useful in the cosmetic and pharmaceutical industries. The method uses a microorganism containing cloned homologous and heterologous genes encoding glycerol-3-phosphate dehydrogenase (G3PDH) and / or glycerol-3-phosphatase (G3P phosphatase). These genes are expressed in a recombinant host that has interruptions in genes encoding endogenous glycerol kinase and / or glycerol dehydrogenase enzymes. The method is useful for the production of glycerol, as well as some final products for which glycerol is an intermediate. The recombinant microorganism is contacted with a carbon source and cultured and then the glycerol or some final products derived therefrom are isolated from the conditioned medium. The genes can be incorporated into the host microorganisms separately or together for the production of the glycerol.

The process of the applicants has not previously been described for a recombinant organism and required the isolation of genes encoding the two enzymes and their subsequent expression in a host cell that has disruptions in the endogenous kinase and dehydrogenase genes. It will be appreciated by those familiar with the art that the applicants process can generally be applied for the production of compounds in which glycerol is a key intermediate, for example, 1,3-propanediol.

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

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

The terms "glycerol-3-phosphatase", "sn-glycerol-3-phosphatase" or "d, 1-glycerol phosphatase", and "G3P phosphatase" refer to a polypeptide responsible for an enzymatic activity that catalyzes the conversion of glycerol - 3- phosphate and water to glycerol and inorganic phosphate. G3P phosphatase is encoded for example, by GPP1 (GenBank Z47047x25), or GPP2 (GenBank U18813x11).

The term "glycerol kinase" refers to a polypeptide responsible for an enzymatic activity that catalyzes the conversion of glycerol and ATP to glycerol-3-phosphate and ADP. The high energy of the phosphate donor ATP can be replaced by physiological substitutes (for example phosphoenolpyruvate). Glycerol kinase is encoded, for example, by GUT1 (GenBank U11583xl9) and glpK (GenBank L19201).

The term "glycerol dehydrogenase" refers to a polypeptide responsible for an enzymatic activity that catalyzes the conversion of glycerol to dihydroxyacetone (E.C. 1.1.1.6) or glycerol to glyceraldehyde (E.C.1.1.1.72). A responsible polypeptide - of an enzymatic activity that catalyzes the conversion of glycerol to dihydroxyacetone is also referred to as a "dihydroxyacetone reductase". Glycerol dehydrogenase may be dependent on NADH (E.C.l.1.1.6), NADPH (E .C.1.1.1.72), or other cofactors (e.g., E.C.1.1.99.22). A glycerol dehydrogenase dependent on NADH is encoded, for example, by gldA (GenBank U00006).

The term "enzyme dehydratase" will refer to any enzyme that is capable of isomerizing or converting a glycerol molecule to the product 3-hydroxypropionaldehyde. For the purposes of the present invention the dehydratase enzymes include a glycerol dehydratase (E.C. 4.2.1.30) and a diol dehydratase (E.C..2.1.28) having preferred substrates of glycerol and 1, 2-propanediol, respectively. In Citrobacter freundii, for example, glycerol dehydratase is encoded by three polypeptides whose gene sequences are represented by dhaB, dhaC and dhaE (GenBank U09771: base pairs 8556-10223, 10235-10819, and 10822-11250, respectively). In Klebsiella oxytoca, for example, diol dehydrate is encoded by three polypeptides whose gene sequences are represented by pddA, pddB, and pddC (GenBank D45071): base pairs 121-1785, 1796-2470, and 2485-3006, respectively).

The terms "GPDl", "DAR1", "0SG1", "D2830", and "YDL022W" will be used interchangeably and refer to a gene encoding a cytosolic glycerol-3-phosphate dehydrogenase and is characterized by the base sequence as SEC. ID NO: 1.

The term "GPD2" refers to a gene encoding a cytosolic glycerol-3-phosphate dehydrogenase and is characterized by the sequence given in SEQ ID NO: 2.

The terms "GUT2" and "YIL155C" are used interchangeably and refer to a gene encoding a mitochondrial glycerol-3-phosphate dehydrogenase and is characterized by the base sequence given in SEQ ID NO: 3.

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

The terms "GPP2", "H0R2" and "YER062C" are used interchangeably and refer to a gene encoding a cytosolic glycerol-3-phosphatase and characterized by the base sequence given as SEQ ID NO: 5.

The term "GUT1" refers to a gene encoding a cytosolic glycerol cinase and is characterized by the base sequence given as SEQ ID NO: 6. The term "glpK" refers to another gene encoding a glycerol cinase and is characterized by the base sequence given in GeneBank L19201, base pairs 77347-78855.

The term "gldA" refers to a gene encoding a glycerol dehydrogenase and is characterized by the base sequence given in GeneBank U00006, base pairs 3174-4316. The term "dad" refers to another gene encoding glycerol dehydrogenase and is characterized by the base sequence given in GeneBank U09771, peres bese 2577-3654.

How it is used in the present term "Function" and "Enzyme function" refers to the catalytic ectivity of an enzyme to elterate the energy required to effect a specific chemical reaction. Such an activity can be applied to an equilibrium reaction where the production of both product and substrate can be achieved under suitable conditions.

The terms "polypeptide" and "protein" are used interchangeably. The terms "carbon substrate" and "carbon source" refers to a source capable of being metabolized by guest orgenisms of the present invention and particularly means carbon sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and substrates of a carbon or mixtures of these.

"Conversion" refers to the metabolic process of an organism or cell that by means of a chemical reaction degrades or destroys the complexity of a chemical compound or substrate.

The terms "host cell" and "host organism" refer to a microorganism capable of receiving extirpant or heterologous genes and additional copies of endogenous genes and expressing these genes to produce an active gene product.

The terms "cell production" or "production of orgenisms" refer to a cell designed for the production of glycerol or compounds that can be derived from the glycerol biosynthetic process. Cell production will be recombinant and will contain either or both of a gene encoding a protein having an activity glycerol-3-phosphate dehydrogenase and a gene encoding a protein having a glycerol-3-phosphatase activity. In addition, in the genes of G3PDH and G3P phosphate, the host cell will contain interruptions in one or both of a gene encoding an endogenous glycerol kinase and a gene encoding an endogenous glycerol dehydrogenase. Where cell production is designated to produce 1, 3-propendiol, it will additionally contain a gene encoding a protein having dehydratase activity.

The terms "foreign gene", "DNA extrenjero", "heterologous gene", and "heterologous DNA" all refer to and genetic material net of an organism that has been placed in a different host organism.

The term "endogenous" as used herein with reference to genes or polypeptides expressed by genes, refers to genes or polypeptides that are native to a production of. cells and are not derived from other organisms. In the same way an "endogenous glycerol kinase" and an "endogenous glycerol dehydrogenase" are terms that refer to codified polypeptides by native genes for cell production.

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 phosphate for the production of glycerol from edecuted ceric sub-strains. Additionally, the terms "recombinant orgenisms" and "transformed hosts" refer to any organism transformed with endogenous genes (or homologs) as well as increase the number of copies of the genes.

"Gene" refers to a fragment of nucleic acid that expresses a specific protein, which includes regulatory sequences that precede non-coding) and follow e (3 'non-coding) coding region. The terms "native" and "wild type" refer to the gene as it is found in the netureleze with its own regulatory regimens.

The term "coding" refers to the process by which a gene, through the mechanisms of transcription and transfection, produces an amino acid sequence. The process of coding a specific amino acid sequence is the means to include DNA sequences that may involve base changes that do not cause a change in the encoded amino acid, or. that involves base changes that can alter one or more amino acids, but does not affect the functional properties of the protein encoded by the DNA sequence. For this, the invention aberca more than the specific exemplary sequences. Modifications in the sequence, such as eliminations, insertions, or substitutions in the sequence that produce imperceptible signals that do not substense the remaining functional properties of the resulting protein molecule are also contemplated. For example, alterations in the sequence of the gene that reflect the degeneration of the genetic code, or that result in the production of an amino acid chemically equivalent in a given site, are contemplated; similarly, a codon stops the amino acid alanine, a hydrophobic amino acid, can be replaced by a codon encoding other less hydrophobic residues, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. - Similarly, changes that result in the replacement of a negatively charged residue by another, such as aspartic acid with glutamic acid, or a positively charged residue by another, such as lysine by arginine, can also be expected to produce a biologically equivalent product. . Changes in the nucleotides that result in alteration in the N-terminal and C-terminal portions of the protein molecule would not be expected to alter the protein's activity either. In some cases, it may in fact be desirable to make mutants of the sequence in order to study the effect of the alteration of the biological activity of the protein. One of the proposed changes is in the routines of experts in the field, as is the determination of the retention of biological activity in codified products. In addition, the experts in the field recognize that the sequences encompassed by this invention are also defined by their capecided pare hybridizer, under conditions of severity (0.1X SSC, 0.1 L SDS, 65 ° C), with the sequence exemplified herein. .

The term "expression" refers to the transcription and triage of the product of the gene from a gene coding for the sequence of the gene product.

The terms "plasmid", "vector" and "cartridge" as used herein refer to an extrachromosomal element that often drives genes that are not part of the cell's central metabolism and usually in the form of circular DNA molecules of double filament. Such elements can be autonomously replicating sequences, sequences that integrate the genome, phage or nucleotide sequences, linear or circular, in which a number of nucleotide sequences have been joined or recombined in a unique construction that is capable of introducing a promoter fragment and DNA sequence for a selected gene product together with the appropriate non-translocated 3 'sequence in a cell, "transformation cartridge" refers to a specific vector that contains a foreign gene and that has elements in addition to the foreign gene that they facilitate the transformation of a particular host cell. "Expression cartridge" refers to a specific vector that contains a foreign gene and that has elements in addition to the foreign gene that allows enhanced expression of said gene in a foreign host cell.

The terms "transformation" and "transfection" refer to the acquisition of new genes in a cell after incorporation of nucleic acid. The acquired genes can be integrated into a chromosomal DNA or introduced as extrachromosomal replicating sequences. The term "transfor" refers to the cell resulting from a transformation.

The term "genetically altered" refers to the process of changing materiel herediterio by trensformeción or mutation. The terms "interruption" and "interrupted gene" as applied to genes refer to a method of genetically altering an organism by adding to or eliminating a gene a significant portion of said gene such as the protein e encoded by said gene if not is expressed and is not expressed in an effective form.

Biosynthetic glycerol procedure It is contemplated that glycerol can be produced in recombinant orgenisms by manipulation of the synthetic glycerol process found in most microorganisms. Typically, a carbon substrate such as glucose is converted to glucose 6-phosphatase and hexocinese in the presence of ATP.

The glucose-phosphate isomerase catalyzes the conversion of glucose-6-phosphite and fructose-6-phosphate and then e-fructose-1, 6-diphosphate through the action of 6-phosphofructokinase. The diphosphate is then carried in dihydroxyacetone phosphate (DHAP) via aldolate. Finally G3PDH NADH-dependent converts DHAP to glycerol-3-phosphate which is then dephosphorylated to glycerol by G3P phosphatase. (Agarwel (1990), Adv. Biochem. Eng. 41: 114).

Genes encoding G3PDH, glycerol dehydrogenase, G3P phosphatase and glycerol kinase The present invention provides genes suitable for the expression of the activities of G3PDH and G3P phosphates in a host cell.

Genes encoding G3PDH are known, for example, GPD1 has been isolated from Sacharomyces and has the base sequence given by SEQ ID NO: 1, which encodes the amino acid sequence given in SEQ ID NO: 7 (Wang and coleborers, supra). Similarly, the activity of G3PDH has been isolated from Saccharomyces encoded by GPD2 having the sequence given in SEQ ID NO: 2 coding for the amino acid sequence given in SEQ ID NO: 8 (Erikson et al., 1995). Microbiol., 17: 95).

For the purposes of the present invention, it is contemplated that any gene encoding a polypeptide responsible for the activity of G3PDH is determined to dictate the ability of the cetelizer to convert dihydroxycetone phosphite (DHAP) to glycerol-3-phosphate (G3P). . Additionally it is contemplated that any gene encoding the amino acid sequence of G3PDH as given by SEQ ID NO: 7, 8, 9, 10, 11 and 12 corresponding to the genes GPD1, GPD2, GUT2, gpsA, glpD, and the a subunit of glpABC respectively, be functional in the present invention in which said amino acid sequence can encompass substitutions, deletions or additions, of amino acids that do not alter the function of the enzyme. The experts in le meterie appreciate that genes encoding G3PDH isolated from other sources also be suitable for use in the present invention. For example, genes isolated from prokaryotes include accesses to GeneBank M34393, M20938, L06231, U12567, L45246, L45323, L45324, L45325, U32164, U32689, and U39682. Fungal isolated genes include accesses to GeneBank U30625, U30876 and X56162; genes isolated from insects include accesses to GeneBank X61223 and X14179; and genes isolated from source mammals include eats and GeneBank U12424, M25558 and X78593.

The genes that encode G3P phosphatase are known. For example, GPP2 has been isolated from Saccharomyces servisiae and has the base sequence of 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 phosphite activity is suitable for use in the method in which the activity is capable of catalyzing the conversion of glycerol-3-phosphate and water to glycerol and organic phosphate. Additionally, any gene encoding the amino acid sequence of G3P phosphates as given by the sequence SEQ ID NO: 13 and 14 corresponding to the GPP2 and GPP1 genes respectively, will be functional in the present invention that includes any sequence of amino acids that ebarce substitutions, eliminations or additions of amino acids that do not alter the function of the enzyme G3P phosphatase. The experts in the materie will appreciate that genes encoding G3P phosphatase isolated from other sources will be suitable for use in the present invention. For example, the dephosphorylation of glycerol-3-phosphate to produce glycerol can be achieved with one or more of the following generic or specific phosphorylates: phosphonate elcelin (EC 3.1.3.1.) [GenBank M19159, M29663, U02550 or M33965]; acid phosphate (EC 3.1.3.2) [GenBank U51210, U19789, U28658 or L20566]; glycerol-3-phosphatase (EC 3.3.3.-) [GenBank Z38060 or U18813xll]; glucose-1-phosphatase (EC 3.3.3.10) [GenBank M33807]; glucose-6-phosphatase (EC 3.1.3.9) [GenBank U00445]; fructose-1, 6-bisphosphatase (EC 3.1.3.11) [GenBank X 12545 or J03207] or phosphotidyl glycero phosphate phosphatase (EC 3.3.3.27) [GenBank M23546 and M23628].

Genes encoding glycerol kinase are known. For example, GUT1 coding for glycerol kinase from Saccharomyces has been isolated and sequenced (Pavlik and collaborators (1993), Curr. Genet., 24: 21) and the sequence is from SEQ ID NO: 6, which codes sequence of amino acids dade in SEQ ID NO: 15. alternetically, glpK encodes a glycerol kinase from E. coli and is characterized by the base sequence given in Genebank L19201, peres bese 77347-78855.

Genes that encode glycerol dehydrogenase are known. For example, gldA encodes glycerol dehydrogenase from E. coli and is characterized by the base sequence given in GeneBank U00006, base pairs 3174-4316. Alternatively, ded refers to another gene encoding glycerol dehydrogenase from Ci trobacter freundii and is charac terized by the base sequence given in GeneBank U09771, base pairs 2557-3654.

Host cells deduced for the recombinant production of glycerol by the expression of G3PDH and G3P phosphates can be either prokaryotic or eukaryotic and will be limited only by their ability to express enzymes ectives. Preferred host cells will be bacteria, yeasts, and fungal lesions typically useful for the production of glycerol such as Ci trobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus, apergillus, Saccaharomyces, 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.

Where glycerol is a key intermediate in the production of 1,3-propanediol, the host cell will either have an endogenous gene that codes for a protein that has a dehydrating activity or will produce a tel transforming gene. Host cells particularly followed by the production of 1,3-propanediol are Ci trobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus, and Salmonella, which have endogenous genes that encode enzymes dehydrate. In addition, cell hosts lacking an endogenous tel gene include E. coli.

Vectors and Cartridges of Expression The present invention provides a variety of suitable expression and transformation vectors and transducers for the cloning, transformation and expression of G3PDH and G3P phosphatase in a suitable host cell. Appropriate vectors will be those that are compatible with the bacteria used. Suitable vectors can be derived, for example, from a bacterium, a virus (such as bacteriophage T7 or an M13 derived from phage), a cosmid, a yeast, or a plant. Protocols for obtaining and using such vectors are known to those skilled in the art (Sambrook et al., Molecular Cloning: A Leboretory Menuel- Volumes 1, 2, 3 (Cold Spring Herbor Laboratory: Cold Spring Harbor, NY, 1989)).

Typically, the vector or cartridge contains sequences that direct the transcription or translation of the appropriate gene, a selectable marker, and sequences that autonomously allow replication or chromosomal integration. Suitable vectors comprise a 5 'region of the gene harboring transcriptional initiation controls and a 3' region of the DNA fragment that controls transcriptional termination. It is mostly preferred when both control regions are derived from homologous genes of the transformed host cell. Such control regions do not need to be derived from the net genes of the specific species selected as a host for production.

The initiation control regions, or promoters, which are useful to drive the expression of the G3PDH and G3P phosphatase genes in the desired host cell are numerous and familiar to the experts in the meter.

Virtually any promoter capable of driving these genes is suitable for the present invention including but not limited to CYC1, HIS3, GAL1, GALIO, ADH1, PGK, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, and TPI (useful for expression in Saccharomyces); AOXl (useful for expression in Pichia); and lac, trp,? P,? PR, T7, tac, and trc, (useful for expression in E. coli).

Control regions of the termination can also be derived from true native genes of the preferred hosts. Optionally, a termination site may not be necessary; however, it is mostly preferred if it is included.

For effective expression of the instantaneous enzymes, the DNA encoding the enzymes in linkage operebly and between initiation codons and selected expression control regions such that the expression results in the transformation of the appropriate messenger RNA.

Transformation of Suitable Guests and Expression of G3P Phosphatase for the Production of Glycerol Once the right cartridges are built, they are used to keep the guests in their cells. Introduction of the cartridge containing the genes encoding G3PDH and / or G3P phosphatase into the host cell can be achieved by known methods such as transfection, for example, using cells permeabilized with calcium, electroporation, or by transfection using a phage virus recombinant (Sambrook et al., supra).

In the present invention, cartridges AH21 and DAR1 were used to transform the E. Coli DH5 and FM5 as fully described in the GENERAL METHODS AND EXAMPLES.

Alternately, it was contemplated that host cells comprising genes of endogenous G3PDH and / or G3P phosphatase can be manipulated so that the relevant genes are upregulated for the production of glycerol.

Methods for overregulation of endogenous genes are well known in the art. For example, in order to overregulate the desired genes, a structural gene is generally placed at the end of a promoter region on the DNA that is recognized by the recipient microorganism. In addition to the promoter one may include other regulatory sequences that increase or control expression from heterologous genes. In addition, one can alter the regulatory sequences of endogenous genes by any genetic manipulation for the same purpose. The expression can be controlled by an inducer or a repressor so that the microorganism coordinately expresses the genes necessary to complete the desired metabolic procedure.

At the time of the invention, host cells containing endogenous genes encoding the activities of G3PDH and / or G3P phosphatase could be placed under the control of regulated promoters (e.g., lac or osmy) or constitutive promoters. For example, a certucho can be constructed to contain an inducible or constitutive promoter, flenked by DNA of sufficient extension and homologous with the net genes to allow the objective. The introduction of adequate growth conditions will result in homologous recombinations between the cartridge and the target portion of the gene and the replacement of the relevant native promoter with the regulatable promoter. Such methods can be used to effect the up-regulation of endogenous genes coding for G3PDH and / or G3P phosphatase activities for the production of glycerol.

Specific and random Mutagenesis Sites to Disrupt Enzymatic Activities The enzymatic procedure by the cell and the organisms metabolize glycerol are known in the art. Figure 1. Glycerol is converted to glycerol-3-phosphate (G3P) by an ATP-dependent glycerol kinase; The G3P can then be oxidized to DHAP by G3PDH. In a second procedure, the glycerol is oxidized and dihydroxycetone (DHA) by a glycerol hydrogenase; DHA can then be converted to DHAP by a DHA cinase ATP dependent. In a third procedure, glycerol is oxidized and glycereldehyde by a glycerol dehydrogenase; Glyceraldehyde can be phosphorylated to glyceraldehyde-3-phosphite through an ATP-dependent gland. DHAP and glyceraldehyde-3-phosphate, interconverted by the action of triosephosphate isomerase, can be further metabolized by the central metabolism procedure. These procedures, by the introduction of by-products, are deterioration for the production of glycerol.

One aspect of the present invention is the ability to provide an organism production for the production of glycerol wherein the glycerol kinase and glycerol dehydrogenase activities that convert glycerol have been eliminied. Methods of creating mutants by elimination are common and well known in the art. For example, wild type cells can be exposed to a variety of agents such as radiation or chemical agents and then screened for the desired phenotype. When the creation of mutagens through radiations either ultraviolet (UV) or ionizing radiation can be used. Suitable short UV wavelengths for genetic mutations will fall in the range of 200 nm to 300 nm where 254 is preferred. UV radiation at this wavelength mainly causes changes in the nucleic acid sequence from guanidine and cytosine to adenine and thymidine. Since all cells have DNA repair mechanisms that would repair most of the UV-induced mutations, agents such as caffeine and other inhibitors can be added to interrupt the repairing process and maximize the number of effective mutations. Mutations at long UV wavelengths using rays in the range of 300 n up to 400 nm are also possible but are not generally as effective as short UV wavelengths unless the rays are used in conjunction with several ectivadoree teles as tintures psorelem that interectúen with DNA.

Mutagenesis with chemical agents is also effective in generating mutant and commonly used substances that include chemicals that effect neuroreplication of DNA such as HNO_ and NH ^ OH, as well as agents that affect DNA replication such as acridine tinctures, remarkable for cause mutations of mobile structure. Specific methods to create mutants using radiation or chemical agents are well documented in the materie. See for example Thomas D. Brook in Biotechology: A Textbook Industrial Microbiology, second edition (1989) Sineuer Associetes, Inc., Sunderland, MA., Or Deshpande, Mukund V., api .. Biochem. Biotechnol., 36, 227, 1992), incorporated herein by reference.

After the mutagenesis took place, mutants having the desired phenotype can be selected by a variety of methods. It is the most common screener where the mutagenized cells are selected for the ability to produce the desired product or intermediary. Alternatively, selective isolation of mutants can be effected by growth of a mutagenized population on selective medium where only resistant colonies can develop. Methods of mutate selections are highly developed and well known in the field of industrial microbiology. See Brock, Supra, De Mancilha et al., Food Chem., 14, 313, (1984).

Biological mutagenic agents which target genes randomly are well known in the art. See for example De Bruijn and Rossbach in Methods for General and Molecular Bacteriology (1994) American Society for Microbiology, Washington, DC, alternatively, provided that the gene sequence is known, interruption in chromosomal genes with specific elimination or replacement is achieved by homologous recombination with an appropriate plasmid. See, for example, Hemilton et al. (1989) J. Bacteriol. 171-4622, Balbes y colaboredores (1993) Gene 136: 211-213, Gueldener et coleboradores, (1996) Nucleic Acid. Res. 24: 2519-2524, and Smith and coleboredores (1996) Methods Mol. Cell. Biol .. 5: 270-277.

It is contemplated that any of the above-mentioned methods can be used for the elimination or inactivation of glycerol kinase and glycerol dehydrogenase activities in the production of preferred orgenisms.

Medium and Carbon Substrates The fermentation media of the present invention should contain edecuedos carbon substrates. Suitable substrates may include but are limited to monosaccharides such as glucose and fructose, oligosaccharides such as lectose or secerose, polysaccharides such as starch or cellulose or mixtures thereof and unlabeled mixtures of renewable food stocks such as drained whey from cheese, corn soaked liquor, beet sugar molasses, and barley malt. Additionally, the carbon substrate may also be of one-carbon substrates such as carbon dioxide, or methanol for the conversion of the methobolic conversion in chemical intermediates has been demonstrated.

The production of glycerol from single carbon sources (eg methanol, formaldehyde or formate) has been reported in methyltrophobic yeasts (Yamada and collaborators (1989), Agrie, Biol .. Chem., 53 (2): 541-543) and in Becteries (Unter et al., (1985), Biochemistry, 24: 4148-4155). These organisms can assimilate compounds of a single carbon, which vary in the oxidation state from methane to formate, and produce glycerol. The carbon assimilation process can be through ribulose monophosphate, through serine, or through monophosphate of xylulose (Gontschalk, Bacterial Metabolism, Second Edition, Springer-Verlag, New York (1986)). The ribulose monophosphate process involves the condensation of formate with ribulose-5-phosphate to form an ezurium of 6 cerbons which is transformed into fructose and eventually into a three-carbon product, glyceraldehyde-3-phosphate.

In the same way, the serine procedure mimics compounds of a carbon in the glycolytic process via methylenetetrahydrofolate.

In addition to one and two carbon substrates, methyltrophic orgenisms are well known to utilize a number of other carbon-containing compounds such as methylamine, glucosamine and a variety of amino acids for meteorological activity. For example, methyltrophic yeasts are known to utilize the cerium from methylamine to trehalose or glycerol (Bellion et al. (1993), Microb. Growth Cl Compd., [Int.Symp.], 7th, 415.32.) Editors: Murrell, J. Collin, Kelly, Don P. Publishers: Intercept, Andover, UK). Similarly, several species of Candida etabolizer elenin or oleic acid (Sulter et al., (1990), Arch. Microbiol, 153 (5): 485-9). Accordingly, the carbon source used in the present invention may encompass a variety of sub-strains containing cerbono and will only be limited by the selection of the microorganism.

Although all cerbono substrates are mentioned and mixed -of which they are suitable in the present invention, preferred carbon substrates are monosaccharides, oligosaccharides, polysaccharides, single-celled sub-strands or mixtures thereof. More preferred are sugars such as glucose, fructose, sucrose, maltose, lactose and single carbon substrates such as methanol and carbon dioxide. Although all above-mentioned carbon substrates and mixtures thereof are suitable for the present invention, preferred cerium substrates are monoseccharides, oligssecharides, polysaccharides, single carbon substrates or mixtures thereof. More preferred are sugars such as glucose, fructose, saccharose, meltose, lactose and single carbon substrates such as methanol and carbon dioxide. The most preferred of the carbon substrates is glucose.

In addition to an appropriate carbon source, the fermentation medium must contain minerals, salts, cofactors, regulators and other edecuted components, known to those skilled in the art, suitable for the development of crops and promotion of the enzymatic process necessary for glycerol production.

The cells are typically developed at 30 ° C in appropriate medium. Preferred detergent media are commercially available prepared media such as Luria Bertani broth (LB), Dextrose Saboured broth (SD), or medium-halide cell (YM). Other defined or synthetic means of development can also be used and the appropriate medium for the development of the pertinent micro-genomics will be known to experts in microbiology or fermentation sciences. The use of known agents to modulate the repression of cetebolite directly or indirectly, for example adenosine 3'-5'-cyclic monophosphate, can also be incorporated into the reaction medium. Similarly, the use of known agents to modulate enzymatic activities (eg, sulfites, bisulfites, and alkalis) that lead to improved glycerol production can be used in conjunction with or as an alterneive for genetic manipulations.

Suitable pH ranges for fermentation are between pH 5.0 to pH 9.0 where the pH range of 6.0 to 8.0 is preferred for initial conditions.

The reactions can be carried out under aerobic or anaerobic conditions, where aneerobic or microaerobic conditions are preferred.

Identification of the activities of G3PDH, glycerol dehydrogenase, G3P phosfetase, and glycerol The expression levels of the G3PDH, G3P phosphatase, glycerol dehydrogenase, and glycerol kinase proteins are measured by enzymatic assays. Generally, assays for the activity of G3PDH and the glycerol dehydrogenase activity depend on the spectral properties of the co-substrate, NADH, in the conversion of DHAP to G-3-P and the conversion of DHA and glycerol, respectively. NADH tuwo UV absorption intrinsic / vis and its consumption can be monitored spectrophotometrically and 340 mm. The ectivided G3P phosphinate can be measured by any method of measuring the inorganic phosphate released in the reaction. The most commonly used detection method uses the visible spectroscopic determination of the blue colored ammonium phosphomolybdate complex. The glycerol kinase activity can be measured by the detection of G3P from glycerol and ATP, for example by NMR. The teachings can be targeted with more specific characteristics of individual enzymes if necessary, for example, by the use of alternate cofactors.

Identification and recovery of glycerol and other products (for example 1, 3-propanediol) Glycerol and other products (for example 1,3-propanediol) can be identified and quantified by analysis by liquid chromatogram of this execution (HPLC) and gas chromatography / mass spectroscopy (GC // MS) on the extremities of the free cells. The HPLC is a preferred method in which the fermentation media are analyzed on an analytical column of exchange using mobile phase of sulfuric acid 0.01 N in the isocratic style.

The methods for the recovery of glycerol from the fermentation media are known in the art. For example, glycerol can be obtained from the cell medium by subjecting the reaction mixture to the following sequence of steps: filtration; water removal; extraction of organic solvent; Frection distillation (US Pat. No. 2,986,495).

Description of the Preferred Modalities Glycerol production The present invention describes a method for the production of glycerol from a carbon source edecueda using a recombinant organism. Particularly well-known in the invention is a guest cell becteriene, trensformede with a certucho of expression that carries one or another or embos a gene that encodes a protein that has an activity glycerol-3-phosphide dehydrogenase and a gene that encodes and binds protein which has glycerol-3-phosphatase activity. In addition to the G3PDH and G3P phosphates gens, the host cell will contain interruptions in one or the other of the genes encoding the endogenous glycerol kinase and glycerol dehydrogenase enzymes. The combined effect of the foreign G3PDH and G3P phosphatase genes (which provide a carbon-to-glycerol source procedure) with gene disruptions (which block the conversion of glycerol) results in an orgenism that is efficient and efficient. trustworthy glycerol.

Although the optimal origin for the production of glycerol contains the interruption of the mentioned gene, the production of glycerol with a host cell that contains one or the other or both of the G3PDH and G3P phosphatase genes in the absence of such interruptions. For example, the recombinant E. coli strain AA200 carrying the DAR1 gene (Example 1) was able to produce between 0.38 g / L and 0.48 g / L glycerol depending on the fermentation parameters. Similarly, E. Coli DH5a, which carries and expresses the GPP2 gene (Example 2), was capable of a production of 0.2 g / L. Where both genes are present in conjunction with an elimination of endogenous glycerol cinase activity, a reduction in the conversion of glycerol can be seen (Example 8). In addition, witness the ectivided glycerol dehydrogenase is linked to the conversion of glycerol under limited glucose conditions; thus, it is anticipated that the removal of the glycerol dehydrogenase activity will result in the reduction of the glycerol conversion (Example 8).

Production of 1, 3-propanediol The present invention can also be suitable for the production of 1,3-propanediol by the use of recombinant organisms expressing the G3PDH and / or G3P phosphatese genes that are extirpable and which contain interruptions in the activities of endogenous glycerol kinase and / or glycerol dehydrogenase.

Additionally, the invention is provided for the process for the production of 1,3-propanediol from a recombinant organism wherein multiple copies of endogenous genes are introduced. In addition to these genetic alterations, the production of cells will require the presence of a gene that encodes an active dehydrotic enzyme. The enzyme of the enzyme dehydrate can be either a glycerol dehydrate or a diol dehydratase. The activity of the enzyme dehydretase can result in either the expression of an endogenous gene or the expression of a transfected foreign gene in the host organism. The isolation and expression of genes encoding dehydrated enzymes edecuedes are well known in the art, and are disclosed by applicants in PCT / US 96/06705, filed November 5, 1996 and US 5686276 and US 5633362, so they are incorporated herein by reference. It will be appreciated that, since glycerol is an intermediate in the production of 1,3-propanediol, where the host cell contains a dehydrated ectivided in conjunction with the G3PDH and / or G3P phosphoretic genes and in the absence of activities. glycerol kinase and glycerol dehydrogenase that convert glycerol, the cell will be followed by the production of 1,3-propendiol.

The present invention is further defined in the following Examples, which indicate preferred embodiments of the invention, which are given by way of illustration only. From the foregoing discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and elcence thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.

EXAMPLES GENERAL METHODS Methods for phosphorylations, ligations, and transforms are well known in the art. Techniques suitable for use in the following examples can be found in Sambrook et al., Molecular Cloning: a Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press (1989).

Suitable materials and methods for the maintenance and development of bacterial cultures are well known in the materie. Suitable techniques for use in the following examples can be found in Manuel of Methods for Generel Becteriology (Phillipp Gerherdt, R. G. Murrie, Relph N. Cosstilow, Eugene W. Nester, Willis A. Wood, Moel R. Krieg and G. Briggs Phillips, eds), Americen Society for Microbiology, Washington, DC. (1994) or in Biotechnology: A Textbook of Industrial Microbiology (Thomas D. Brock, Second Edition (1989) Sinauer Associares, Inc., Sunderland, MA). All reagents and materials for the development and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, Wl), DIFCO Laboretories (Detroit, MI), GIBCO / BRL (Gaithersburg, MD), or Sigma Chemical Company (St. Louis , MO) unless otherwise specified.

The meaning of the abbreviations are as follows: "h" means time (s), "min" means minute (s), "sec" means second (s), "d" means day (s), "L" means milliliters, "L" means liters.

"Cellular strains" The following strains of E. coli were used for transformation and expression of G3PDH and G3P phosphate. Strains were obtained from the E. Coli Genetic Stock Center, ATCC. Ó Life Technology (Gaithersburg, MD).

AA200 (garN10fhuA22 ompF627 fadL701 relay pit-10 spoTl tpi-1 phoM510 mcrBl) (Anderson and coleboradores, (1970), J. Gen. Microbiol, 62: 329).

BB20 (tonA22 AphoA8 fadL701 reJAI g! PR2 gJpD3 pi t-10 gpsA20 spoTl T2R) (Croman et al., J. bact., 118: 598).

DH5a (deoR endAl gyrA96 hsdR17 recAl relAl supE4 thi-1? DacZYA- argFV169) phl801acZ? M115 F-) (Woodcock et al., (1989), Nuci Acids Res., 17: 3469).

FM5 Escherichia coli (ATCC 53911) Identification of Glycerol The glucose conversion was monitored by HPLC and / or GC. Analyzes were carried out using standard and materiel techniques available to an expert in the chromatography material. An edecuedo method used a system Weters Maximum 820 HPLC using UV (210 nm) and IR detection. The samples were injected onto a Shodex SH-1011P precolumn (6 mm x 50 mm), temperature controlled at 50 ° C, using H SO ^ 0.01 N as the mobile phase at a flow of 0.69 ml / min. When the quantitative analysis was desired, the samples were prepared with a known amount of trimethylacetic acid as an external standard. Typically, the retention times of 1,3-propanediol (IR detection), glycerol (IR detection) and glucose (IR detection) were 21.39 min, 17.03 min and 12.66 min, respectively.

The gliocerol was also added by GC / MS. The detections by gas chromatography with masse spectrometry, seperation and glycerol cuentification were performed using DB-WAX column (30 m, 0.32 mm ID, 0.25 μm film thickness, J &W Scientific, Folsom, CA) the following conditions: Injector: portion, 1: 15; Sample volume: 1 μL; tempereture profile: 150 ° C initial temperature with 30 seconds of waiting, 40 ° C / min at 180 ° C, 20 ° C up to 240 ° C, wait for 2.5 min. Detection: Mass Spectrometry (Hewlett Packard 5971, San Fernando, CA), quantitative SIM using 61 m / z ions and 64 m / z target ions for glycerol and glycerol-d8, and 43 m / z ion qualifying ion for glycerol. Glycerol-d8 was used as an internal standard.

Assay for glycerol-3-phosphatase, G3P phosphatase The assay for enzyme activation was carried out by incubation of the extract with a subset of organic phosphatide in a bis-Tris or MES and megnesium regulator, pH 6.5. the sub-residue used was either 1-a-glycerol phosphate, or d, 1-a-glycerol phosphate. The final concentrations of the reagents in the assay were: regulator (20 mM bis-Tris or 50 M MES); MgCl2 (10 mM); and substrate (20 mM). If the total protein in the sample was low and no visible precipitation occurs with an acid extinguisher, the sample was conveniently assayed in the cell. This method involves incubation of an enzyme sample in a cell that contained 20 mM of regulatory substrate (50 μL, 200 mM) of MES, 10 M of MgCl_, pH 6.5. The assay volume of the phosphatese finel was 0.5 mL. show you that the enzyme contained was added to the reaction mixture; the contents of the cell were mixed and then the cell was placed in a normal water bath at T = 37 ° C for 5 e 120 min, the time period elapsed depending on whether the phosphatase ectivided in the enzyme sample varied from 2 to 0.2 U / mL. The enzymatic reaction was quenched by the addition of acidic molybdate reagent (0.4 mL). After the Fiske SubbaRow reagent (0.1 mL) and dilute distillate (1.5 mL) were added, the solution was mixed and maintained for further development. After 10 min allow color development, the ebsorbance of the samples was read at 660 nm using a Cery 219 UV / Vis spectrophotometer. The centred of inorganic phosphate liberated was compared with a standard curve that was prepared by using a stock solution of inorganic phosphate (0.65 M) and preparation of 6 standards with fine concentrations of inorganic phosphite in the range of 0.026 hete 0.130 μmoles / mL.

Enseyo Spectrophotometric for the Activity of Glycerol 3-Phosphate Dehydrogenase (G3PDH) The following procedure was used as the modified one subsequently published by Bell and coleboredores (1975), J. Biol .. Chem., 250: 7153- 8. This method involved incubating an enzyme sample in a cell containing 0.2 mM NADH; 2.0 mM dihydroxyacetone phosphate (DHAP), and enzyme in 0.1 M Tris / HCl, buffer pH 7.5 with 5 mM DTT, in a total volume of 1.0 ml at 30 ° C. The spectrophotometer was placed in monitor of absorbance changes at the fixed wavelength of 340 nm. The instrument was adjusted with a target in a cell containing regulator only. After the enzyme was added to the cell, an absorbency reading was made. The first substrate, NADH (50 μL 4 mM NADH, "the absorbance will increase approximately 1.25 AU), was added to determine the background ratio, the ratio would continue for at least 3 minutes, the second substrate, DHAP (50 μL 40 mM). of DHAP), was then determined and the ebsorbency monosite against time was monitored for at least 3 minutes to determine the important proportion.The activity of G3PDH was defined by subtracting the background proportion from the important proportion.

Assay of Activity 13, C-NMR for Glycerol Kinase An appropriate amount of enzyme, typically a cell-free crude extract, was added to a reaction mixture containing 40 mM ATP, 20 mM MgSO4, 21 mM glycerol uniformly labeled 1OC (99, Cambridge Isotope Laboretories), and Tris -HCl 0.1 M, pH 9 for 75 min at 25 ° C. the conversion of glycerol to glycerol 3-phosphate was detected by: ~ 'C-NMR (125 MHz); - glycerol (63.11 ppm, d, J = 41 Hz and 72.66 ppm, t, J = 41 Hz); glycerol 3-phosphate (62.93 ppm, d, J = 41 Hz, 65.31 ppm, br d, J = 43 Hz, and 72.66 ppm, dt, J = 6.41 Hz).

Glycerol Dehydrogenase Assay Linked to NADH The activity of glycerol dehydrogenase linked to N7? DH in cepes of. E. Coli (gldA) was determined after separation of the protein by electrophoresis on undenatured polyacrilemide gel. The conversion of glycerol plus NAD + e dihydroxyacetone plus NADH was echopleted with the conversion of 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl tetrazolium bromide (PMS) as a mediator. (Tang et al (1997) J. Bacteriol.140: 182).

Electrophoresis was performed in duplicate by standard procedures using native gels (8-16 i TG, 1.5 mm, 15 fringes of Novex gel, San diego, CA). The residual glycerol was removed from the gels by washing 3x with 50 mM Tris or potassium carbonate regulator, pH 9 for 10 minutes. The duplicate of gels was developed, with and without glycerol (eXexclusively finel concentration of 0.16 M), in 15 ml of test solution containing 50 mM Tris or potassium carbonate, pH 9, 60 mg of ammonium sulfate, 75 mg of NAD +, 1.5 mg of MTT, and 0.5 mg of PMS. The presence or absence of glycerol dehydrogenase activity in the form of NADH in strains of E. coli (gldA) was also determined, following electrophoresis on polyacrylamide gel, by reaction with polyclonal antibodies produced for K. pneumoniae glycerol dehydrogenase (ded).

CONSTRUCTION OF PLASMIDS AND CONSTRUCTION OF STRAINS Cloning and Expression of Glycerol 3- Phosphatase for Increase in Glycerol Production in E. Coli DH5a and FM5 The clone 6592 of chromosome V lembda of Saccharomyces cerevisiae (GeneBank, accession No. U18813 x 11) was obtained from ATCC. The glycerol 3-phosphite phospholipid (GPP2) gene was cloned by cloning from the lambde clone as the target DNA using synthetic primers (SEQ ID NO: 16 with SEQ ID NO: 17) incorporating a BamHI-RBS-XbaI site at the 5 end. 'and a Smal site at the 3' end. The product was subcloned in pCR-Script (Stratagene, medison, Wl) on the SrfT site to generate plasmids pAH15 containing GPP2. Plasmid pAH15 contains the GPP2 gene in the inective direction for expression from the promoter reads in pCR-Script SK +. The BamHI-Smal fragment from pAH15 containing the GPP2 gene was inserted into pBlueScriptII SK + to generate the plasmid pAH19. PAH19 contains the GPP2 gene in the run expression orientation from the lac promoter. The Xbal-Pstl fragment of pAH19 containing the GPP2 gene was inserted into pPHOX2 in the created pAH21 plasmid. The pAH21 / DH5a is the plasmid expression.

Plasmids for Overexpression of PARÍ in E. coli DAR1 was isolated by PCR cloning of S. cerevisiae genomic DNA using synthetic primers (SEQ ID NO: 18 with SEQ ID NO: 19) Successful cloning by PCR places a Ncol site at the 5 'end and from DAR1 where the ATG in Ncol is the methionineinitiator of DARl. at the 3 'end of DAR1 a BamHl site is introduced following the translational terminator. The PCR fragments were designated with Ncol + BamHl and cloned in the same sites in the expression plasmid pTrc99A (Phermecie, Piscawey, NJ) to give pDARl.

In order to create a better ribosome binding site at the 5 'end of DAR1, a Spel-RBS-NcoI stele obtained by chilling followed by cooling of synthetic primers (SEQ ID NO: 20 with SEQ ID NO: 21) was inserted into the Ncol site of pDARlA to create pAH40. Plasmid pAH40 contains the new RBS and the DAR1 gene in the correct orientation for expression from the trc promoter of pTrc99A (Pharmecia, Piscatewey, NJ). The NcoI-BamHI fragment of pDARlA and a second Spel-RBS-Ncol slender group obtained by heating and cooling the synthetic primers (SEQ ID NO: 22 with SEQ ID NO: 23) was inserted into the SpelI-BamHI site of pBC- SK + (Stretegene, medison, Wl) to create the pAH42 plasmid. Plasmid pAH42 contains a chloramphenicol resistant gene.

Construction of Expression Cartridges for PARÍ and GPP2 Expression cartridges for DAR1 and GPP2 were conjugated from the individual subclones DAR1 and GPP2 described above using standard methods of molecular biology. The BamHI-PstI fragment of pAH19 containing the ribosome linker site (RBS) and the GPP2 gene was inserted into pAH40 to create pAH43. The BamHI-Pstl fragment of pAH19 containing the RBS and the GPP2 was inserted into pAH42 to believe pAH45.

The ribosome site appearing at the 5 'end of GPP2 was modified as follows. A BamHI-RBS-Spel link, obtained by heating and then cooling of synthetic primers (GATCCAGGAAACAG (SEQ ID NO: 24) with CTAGTCTGTTTCCTG (SEQ ID NO: 25) in the X al-Pstl fragment from pAH19 containing the GPP2 gene, was inserted into the BamHI-PstI site of pAH40 to create pAH448. Plasmid pAH48 c contains the DAR1 gene, the modified RBS, and the GPP2 gene in the correct orientation for expression from the trc promoter of pTrc99A (Pharmacie, Piscetaway, NJ).

Transformation of E. Coli All plasmids described herein were transformed into E. Coli DH5a or FM5 using standard molecular biology techniques. The trensformentes were verified by their petrón DNA RFLP.

EXAMPLE 1 PRODUCTION OF GLYCEROL DESPE E. COLI TRANSFORMATES WITH THE G3PPH GENE Medium Synthetic medium was used for anaerobic or aerobic glycerol production using E. Cold cells transformed with pDARlA. The medium contained per liter 6.0 g of Na ^ HPO ^, 3.0 g of KH: P0, 1.0 g of NH4C1, 0.5 g of NaCl, 1 mL of Mg.SO; .7H_0 to 20?, 8.0 g of glucose, 40 mg of casemino acids, 0.5 ml of thiamine hydrochloride at 1; , 100 mg of empiciline.

Development Conditions Cepe AA200 protected in pDARl or the vector pTrc99A was developed under aerobic conditions in 50 ml of medium shaking at 250 rpm in a 250 ml flask at 37 ° C. A 6JJ 0.2-0.3 isoprohio-β-galactoside was added to a finel concentration of 1 mM and continuous incubation for 48 hours. Stop samples of aneerbic development of induced cells were used to fill Falcon tubes No. 2054 that were capped and mixed gently by rotation at 37 ° C for 48 hours. The glycerol production was determined by HPLC analysis of the supernatant cultures. The cepes pDARlA / AA200 yielded 0.38 g / L of glycerol after 48 hours, under anaerobic conditions, and 0.48 g / L of eerobic conditions.

EXAMPLE 2 PRODUCTION OF GLYCEROL PESPE E. COLI TRANSFORMAPO CO GENE G3PFOSFATASA Medium Synthetic medium phoA was used in agitation units to demonstrate the increase of glycerol by the expression of GPP2 in E. coli. The medium phoA, contained per liter: amisoy, 12 g; amino sulfate, 0.62 g; MOPS, 10.5 g; sodium citrate, 1.2 g; NaOH (IT), 10 mL; MgSO 1 M, 12 mL; trace elements 100X, 12 mL; 50% glucose, 10 L; 1% thiamine, 10 mL; 100 mg / mL of L-proline, 10 mL; 2.5 mM FeCl_, 5 L; Regulator with phosphate mixture, 2 ml (5 ml of 0.2M NaH2P04 + 9 ml of 0.2 M K_HP0), and pH 7.0. The trace elements 100X for the medium phoA / L contained: ZnS04. H "0., 0.58 g; MnS0 .HO, 0.34 g; CuS0 .5H_0, 0.49 g; CoC1..6H20, 0.47 g; H3B03, 0.12 g, NaMo04.2H.O, 0.48 g.

Agitation of Experiment flasks Strains p? H21 / DH5a (containing the GPP2 gene) and pPH0X2 / DH5a (control were developed in 45 ml of medium (phoA medium, 50 μg / mL of carbeciline, and 1 μg / mL of vitamin B12) in a matre of agitation of 250 mL at 37 ° C. The cultures were developed under erogenous conditions (shaking at 250 rpm) for 24 hours.The glycerol production was determined by HPLC analysis of the supernatant culture PAH21 / DH5a produced 0.2 g / L of glycerol after 24 hours.

EXAMPLE 3 PROPUCCION PE GLICEROL PESPE P-GLUCOSE USING RECOMBINANT E. COLI CONTAINING BOTH GPP2 AND PARI.

The development of the increased glycerol production by E. Coli DH5a containing pAH43 proceeds aerobically at 37 ° C in shake flask cultures (erlen meyer flasks, liquid volume / 5 totel volume).

Crops in minimal medium / 1% glucose shake flasks are initiated by inoculation of overnight LB / culture in glucose at 11 with antibiotic selection. The minimum medium is: defined medium in sterilized filter, final pH 6.8 (HCl), medium content Minimum: 12.6 g of (NH) .S0, 13.7 g of KHP04, 0.2 g of levedura extract (Difco), 1 g of NaHC03, 5 mg of vitemine B12, 5 L of Solution of elements in Trezes de Balch Modified (the composition of the stem can be found in Methods for General and Molecular Bacteriology (P. Gerhardt et al., Eds. P. 158, American Society for Microbiology, Washington, DC (1994).) Methods of egitization are incubated at 37 ° C with vigorous all-night aeration, after which they are sampled for GC analysis of the supernatant.The glycerol production shown by pAH43 / DH5a 3.8 g / L after 24 hours.

EXAMPLE 4 PRODUCTION OF GLYCEROL PESPE P-GLUCOSE USANPO E. COLI RECOMBINANT THAT CONTAINS BOTH GPP2 AND PARI Example 4 illustrates the production of glucose from recombinant E. coli DH5a / pAH48 which contains both GPP2 and DAR1 genes.

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

Pre-cultivation DH5a / pAH48 were precultured by seeding in a fermentation run. The components and protocol for pre-cultivation are enlisted and continued.

Pre-culture medium KH2P04 30.0 g / L Citric acid 2.0 g / L MgSO0 .7H O 2.0 g / L H S04 98 2.0 mL / L Ammonium ferric citrate 0.3 g / L CaCl- .2H O 0.2 g / L Extract of Levadure 5.0 g / L Trazes of metals 5.0 mL / L Glucose 10.0 g / L Carbecillin 100.0 mg / L.

The components of the previous medium were mixed together and the pH was adjusted to 6.8 with NH4OH. The medium was then sterilized by filtrate.

Trace metals were used according to the following recipe: Citric acid, monohydrate 4.0 g / L MgSO4-7H ^ 0 3.0 g / L MnS0 .H_0 0.5 g / L NaCl 1.0 g / L FeS0 .7H-0 0.1 g / L CoCl .6H O 0.1 g / L CeCl 0.1 g / L ZnS0 .7H O 0.1 g / L A1K (S0) ^. 12H.0 10.0 mg / L H-: BOJ 10 mg / L Ne2Mo04.2H20 10 mg / L NiS04.6H20 10 mg / L Na "Se03 10 mg / L Na-W04.2H-0 10 mg / L The cultures were initiated from inoculated culture sowings from 50 μL of frozen reserve (glycerol at 15 μl as a freezing protector) in 600 mL of medium in a 2 L Erlen Meyer matre. The cultures were developed at 30 ° C in an incubator and 250 rpm for approximately 12 hours and then used to sow the fermenter.

Development of Fermentation Container Agitated tank of 15 L Medium KH P0 6.8 g / L Citric acid 2.0 g / L MgSO4.7H-O 2.0 g / L H S0 98 2.0 mL / L Ammonium ferric citrate 0.3 g / L CaCl-.2H-0 0.2 g / L Defoamer Mazu DF204 1.0 ml / L The above components were sterilized together in the fermentation tank. The pH was raised to 6.7 with NH4OH. Extract of levadure (5 g / L and solution of trace minerals (5 ml / L were aseptically added from the reserve solutions sterilized by filtration, Glucose was added from the feeding of 60 r: to give a final concentration of 10 g / L. Carbeciline was added to 100 mg / L. The volume after inoculation was 6 L.

Environmental Conditions for Fermentation The temperature was controlled up to 36 ° C and the air flow was controlled up to 6 liters per minute. The return pressure was controlede e 1 ber. The indexer was set at 350 rpm. Aqueous ammonium was used for pH control at 6.7. The proportion glucose ali entede (60% glucose monohydrate) was controlled to maintain excess glucose.

Results The results of the run fermentation are given in Table 1 Table 1 6 4.7 4.0 2.0 49 14 8 5.4 0 3.6 71 25 10 6.7 0.0 4.7 116 33 12 7.4 2.1 7.0 157 49 14.2 10.4 0.3 10.0 230 70 16.2 18.1 9.7 15.5 259 106 18.2 12.4 14.5 305 20.2 11.8 17.4 17.7 353 119 22.2 11.0 12.6 382 24.2 10.8 6.5 26.6 404 178 26.2 10.9 6.8 442 28.2 10.4 10.3 31.5 463 216 30.2 10.2 13.1 30.4 493 213 32.2 10.1 8.1 28.2 512 196 34.2 10.2 3.5 33.4 530 223 36.2 10.1 5.8 548 38.2 9.8 5.1 36.1 512 233 EXAMPLE 5 ENGINEERING PE MUTANTS PE GLYCEROL CINASA PE E. COLI FM5 FOR THE PROPUCCION PE GLICEROL PESPE GLUCOSE Construction of integration plasmids for glycerol kinase gene replacement in E. coli EM5 Genomic DNA from E. coli FM5 was prepared using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN). A 1 kb DNA fragment containing the gens glpF percial and glycerol kinase (gJpK) was amplified by PCR (Mullis and Faloone, Methods of Enzymol., 155: 335-350, 1987) from FM5 genomic DNA using primers SEQ ID NO: 26 and SEQ ID NO: 27. A 1.1 kb DNA fragment containing the partial glpK and glp genes was amplified by PCR from FM5 genomic DNA using primers SEQ ID NO: 28 and SEQ ID NO: 29. One site Muñí was incorporated into the primer SEQ ID NO: 28. The primer at the 5 'end of SEQ ID NO: 28 was the reverse complement of the SEPARATOR SEQ ID NO: 27 to facilitate the subsequent overlap of the extension by PCR. The union of the gene by the technique of superposition of the extension (Horton and coleboredores, BioTecniques, 8: 528-535, 1990) was used to generate a 2.1 kb fragment by PCR using the two above fragments as primers and primers SEQ ID NO: 26 and SEQ ID NO: 29. these fragments represented a 0.8 deletion kb from the core region of the 1.5 kb glpK gene. Globelly, this fragment had 1.0 kb and 1.1 kb in lateral regions on either side of the Muñí cloning site (in the partial glpK) to allow the replacement of the chromosomal gene by homologous recombination.

The above 2.1 kb PCR fragment was blunt ended and cloned into the smooth vector PCR using the "Zero Blunt PCR Cloning Kit (Invitrogen, San diego, CA) to produce the 5.6 kb pRNIOO plasmid containing genes resistant to kanemicine and Zeocin.The 1.2 kb HincII fragment of pLoxCetl (unpublished results), which contains a chloramphenicol resistant gene flanked by becteriofego sites - Pl loxP (Sneith T. collaborators, Gene, 166: 173-174, (1995), was used to interrupt the glpK fragment in plasmid pRNIOO by binding to Muñí (and with a smooth end) - pRNI O plasmid digested to produce plasmid pRNIQI-l of 6.9 kb. A 376 kb fragment containing the R6K origin was amplified by PCR from vector pGP704 (Miller and Mekalanon, J. Bacteriol, 170: 2575-2583, 1988) using primers SEQ ID NO: 30 and SEQ ID NO: 31, and bound to the 5.3 kb Asp718-AatII fragment of pRN101-l to produce the 5.7 kb pRN102-l plasmid containing genes resistant to kanemicin and chlormephenicol. Read substitution of the ColEl region of origin in pRNlOl-l with the origin R6K to generate pRN102-1 also involved elimination of most of the genes resistant to Zeocin. The host for the replication of pRN102-l was E. coli SY327 (Miller and Mekalanon, J. Bacteriol., 170: 2575-2583, 1988) which contains the pir gene necessary for the function of the R6K origin.

Engineering Glycerol Mutant Kinase RJFllOm with Chloramphenicol Resistance with Gene Break E. coli FM5 was electrotransformed with the non-replicable integration plasmid pRN102-l and transformants that were resistant to chloramphenicol (12.5 μg / mL) and sensitive to kanemicine (30 μg / mL) were further screened for non-glycerol use on M9 medium minimum containing 1 mM glycerol. An EcoRI digester of genomic DNA from a tel mutein, RJFlOm, when tested with the intact gipK gene and Southern analysis (Southern, J. Mol. Biol., 98: 503-507, 1975) indicated that it was a member double-crossing (replacement of the gipK gene) since the two expected bands of 7.9 kb and 2.0 kb were observed, which is characteristic of the presence of an additional EcoRI site in the gene with resistance to chloramphenicol. Wild type control produced the expected single band of 9.4 kb.

An analysis by 13C NMR of the mutant RJFlOm confirmed that it was incapez to convert glycerol 13C-market and ATP glycerol-3-phosphite. This mutein glpK was further amplified by genomic PCR utilizing co-binations of primers SEQ ID NO: 32, SEQ ID NO: 34 and S? C ID NO: 35, and SEQ ID NO: 32 and SEQ ID NO: 35 that produced the cleavage by PCR, 2.3 kb, 2.4 kb and 4.0 kb respectively. Wild type control produced the expected 3.5 kb band with the primers SEQ ID NO: 32 and SEQ ID NO: 35. The glpK mutant, RJFlOm, was electrotransformed with the pAH48 plasmid to allow the production of glycerol from glucose. The mutant of glpK E. coli RJFlOm has been deposited with ATCC under the terms of the Budapest Tretedo on November 20, 1997 ..

Engineering Glycerol Kinase Mutant RJFlOm with Chloramphenicol Resistance with Removal of Gene Removal After developing all night over medium YENB (yeast extract at 0.75 é, broth nutrient al 0. 8 í. ) at 37 ° C, e. Coli RJFlOm in an aqueous suspension was electrotransformed with the plasmid pJW168 (resultsss not published), which contained the bacteriophage gene Pl Cre recombinase under the control of the indiccble-IPTG lacUVS promoter, a temperature-sensitive pSClOl replicon, and a resistant ampicillin gene. In overdevelopment in SOC medium at 30 ° CC, transformants were selected at 30 ° C (permissible temperature for replicate of pJW168) on eger medium LB supplemented with carbecillin (50 μg / mL) and IPTG (lmM). Two series of overnight transfers of groups of colonies were performed at 30 ° C on freshly prepared LB medium supplemented with carbecillin and IPTG in order to allow cleavage of chromosomal gene resistance to chloremfenicol via recombination at ioxP sites. involved in Cre recombinase (Hoess and Abremski, J. Mol. Biol., 181: 351-362, 1985). The resulting colonies were replicated on medium ager LB supplemented with carbecillin and IPTG and LB agar supplemented with chloramphenicol (12.5 μg / mL) to identify colonies that were resistant to carbecillin and sensitive to chloramphenicol indicating the removal of the marker gene. An overnight culture at 30 ° C of such a colony was used to inoculate 10 mL of LB medium. In development at 30 ° C to OD (600 n) of 0.6, the culture was incubated - at 37 ° C overnight. Several dilutions were plated on preheated LB agar medium and the pieces incubated overnight at 42 ° C (the temperature does not allow for replication of pJW168). The resulting scolonies were plaque-replica on the eger medium LB and medium eger LB supplemented with carbecillin (75 μg / mL) to identify colonies that were sensitive to carbecillin indicating loss of plasmid pJW168. Such a mutant of glpK, RJF10, was further analyzed by genomic PCR using the primers SEQ ID NO: 32 and SEQ ID NO: 35 and yielded the expected band of 3.0 kb which confirms the cleavage of the mercer gene. The non-utilization of the glycerol by the RJF10 mutant was confirmed by lack of growth on minimal M9 medium containing 1 mM glycerol. The RJF10 mutant of glpK was electrotransformed with the pAH48 plasmid to allow the production of glycerol from glucose.

EXAMPLE 6 CONSTRUCTION OF PE PE COL. WITH THE AGOTAPO GLPA GENE The gldA gene was isolated from E. coli by PCR (KB Mullis and FA Faloona (1987) Meth. Enzymol 155: 335-350) using the primers SEQ ID NO: 36 and SEV ID NO: 37, which incorporate the Sphl sites and Xbal, repectively, and cloned (T. Menietis 1982 Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor, NY) between the Sphl and Xbal sites in pUCld, pare generer pKP8. pKP8 was cut in the only Sail and Ncol sites in the gldA gene, the ends smoothed with Klenow and religated resulted in an elimination of 109 bp in half of gldA and the regeneration of a single Sail site, to generate pKP9. A 1.4 kb DNA fragment containing the gene that confers resistance to kanemicine (ken), and includes 400 bps of DNA eproximidately at the start of the transletional initiation codon and approximately 100 bps of DNA towards the end of the stop codon traslecionel, was isolated from pET-28A (+) (Novagen, Madison, Wis.) by PCR using the primers SEQ ID NO: 38 and SEQ ID NO: 39, incorporating the Sail terminals, and subclone in the Sali unique site of KP9, pere generer pKP13. A DNA fragment of 2.1 KB that begins 204 bps towards the end of 1 codon of initiation traslecionel of gldA and that finishes 178 bps to the beginning of the stop codon translational of gldA, and that -contains the insert kan, was isolated from pKP13 by PCR using the primers SEQ ID NO: 40 and SEQ ID NO: 41, which incorporate the terminal sites Sphl and Xbal, respectively, were subcloned between the Sphl and Xbal sites, in pMAK705 (Genencor International, Palo Alto, Calif.), to generate pMP33. E. coli FM5 was transformed with pM33 and selected over 20 μg / mL of ken at 30 ° C, which is the permit tempereture for replication of pMAK705. One colony was expanded to overnight at 30 ° C in culture medium supplemented with 20 μg / mL ken. Approximately 32,000 cells were plated on 20 μg / mL ken and incubated for 16 hours and 44 ° C, which is the restricted temperature for the replication of pMAK705. Transformants that grew at 44 ° C had plasmids integrated into the chromosome, which occur at a frequency of approximately 0.0001. Analysis by PCR and Menchedo Southern (E.M. Southern 1975 J. Mol. Biol .. 98: 503-517) were used to determine the nature of the events of chromosomal integration in the transformants. Western blot analysis (H. Torbin et al. (1979) Proc. Nati. Acad. Sci. 76: 4350) was used to determine whether the glycerol dehydrogenase protein, the gldA product, is produced in the transformants. An activity assay was used to determine whether the glycerol dehydrogenase activity remained in the transformants. The activity in the glycerol dehydrogenase bendes on native gels was determined by coupling the conversion of glycerol + NAD (+) - dihydroxyacetone + NADH for the conversion of a tetureolium tinture, MTT [3- (4,5-dimethylthiol) bromide - 2- il) -2,5-diphenyltetrazoliu] for a deep colored formazan, with phenazine methosulafate as an intermediate. Glycerol dehydrogenase also requires the presence of 30 mM of ammonium sulfate and 100 mM of Tris, pH 9 (C.-T. Tang, and collaborators (1997) J. Becteriol, 140: 182). Out of 8 three non-standardized forms, 6 were determined to be agonal. E. coli MSP33.6 has been deposited with ATCC under the conditions of the Budepest Treaty on November 20, 1997.

EXAMPLE 7 CONSTRUCTION OF THE CEPA E. COLI WITH GLPK AND THE GONE SOLD OUT GLDA Ur fragment of 1.6 kb DNA containing the gene gldA and including 2? bps of DNA towards the start of the rlaslacicpal start coder and 220 bps of DNA at the end of the translational stop codon was isolated from E. coll by PCR using the primers SEQ ID NO: 42 and SEQ ID NO: 43, which incorporate the Sphl and Xbal sites, repec iva ep e, and cloned between the sites Sphl and Xbal ae pUCló, to generate p N2, pQN2 was cut in the unique Sail and ccl sites in the gidA gene, the smooth end with Klenow and religated, resulting in a half-elimination of gldA and regeneration of a single SalI site, to generate pQN4 , A 1.2 kb DNA fragment containing the gene that confers resistance to kanemycin (kan), and flanked by l oxP sites was isolated from pLoxKan2 (Genencor International, Palo Alto, Calif.) As a Stul / XhoI fragment, and the ends smoothed with Klenow, and subcloned into the pQN4 site after isolation with Klenow, to generate pQN8, A 0.4 kb DNA fragment containing the replica R6K origin was isolated from pGP704 (Miller and Mekalenos, J. Baetericl, 170: 2575-2583, 1988) by PCR using the primers SEQ ID NO: 44 and SEQ ID NO: 45, which incorporate the Spñl and Xabl sites, respectively, and ligated to the Sphl / Xbal DNA fragment of 2.8 kb containing the gene that confers resistance to chloramphenicol (cam), and flanks by sites- icxP was isolated from pLoxCat2 Genencor International, Palo Alto, Calif.) as a fragment Xba l, and sucked in pK22 on the Xbal site, to generate pKP23. The RJF1C strain of E. coli (see EXAMPLE-5, which is glpK, was transformed with pKP23 and transformants with the kanRcamS phenotype was isolated, indicating double cross-integration, which was confirmed by Southern blot analysis. Glyceryl dehydrogenase activity (as described in EXAMPLE 61 demonstrated that glycerol dehydrogenase ective is not present in these transformants.) The kan marker was removed from the chromosome using the plasmid pJW168 that produces Cre, as described in EXAMPLE 5, to produce KLP23 cepes, several isolates with the kanS phenotype demonstrated no glycerol dehydrogenase activity, and Southern blot analysis confirmed the loss of the kan marker.

SEC ID NO: 44 CACGCATGCAGTTCAACCTGTTGATAGTAC SEC ID NO: 45: GCGTCTAGATCCTTTTAAATTAAAAATG EXAMPLE 8 CONSUMPTION OF GLYCEROL PROPUCIPO PESPE P-GLUCOSE BY E.

RECOMBINANT COLI CONTAINING BOTH GPP2 AND PARÍ CCON AND WITHOUT ACTIVIPAP PE GLYCEROL KINASE (GLPK) The £ example illustrates glycerol consumption by E. coli FM5 / pAH48 recc binante and RJF10 / pAH48. Strains FM5 / pAH48 and RJF10 / pAH48 were constructed as described previously in GENERAL METHODS.

Pre-cultivation FM5 / pAH48 and RJF10 / pAH48 were pre-cultured by sowing in a fermentor in the same medium used for fermentation, or in LB supplemented with 1 l of glucose. Either carbeciin or ampicillin were used (100 mg / L) for plasmid maintenance. The means for fermentation is as described in EXAMPLE 4.

The cultures were initiated from frozen stocks (15 ae glyceroi as cryoprotectant) in 600 ml medium in a 2 L Erlen Meyer flask, developed at 30 ° C on a shaker at 250 rpm for approximately 12 h, and used to seed the Development of Fermentation A 15 L fermenting tank with 5-7 liters of initial volume was prepared as described in EXAMPLE 4. Either carbecillin or ampicillin were used (100 mg / L! For maintenance of the plasmid.

Environmental Conditions for Evaluating Glycerol Kinase Activity (glpK) The temperature was controlled at 30 ° C and the airflow controlled at 6 liters per minute standard. The return pressure was controlled at 0.5 bar. The tension # of dissolved oxygen was controlled 10 i by agitation. Aqueous ammonium was used to control the pH to 6.7. The glucose feed ratio (60% glucose) was controlled by maintaining excess glucose until the glycerol was accumulated to at least 25 g / L. The glucose was depleted, resulting in the net metabolism of glycerol. 2 shows the resulting conversion of glyceroi.

Table 2 Conversion of glycerol by FM5 / pAH48 (weight) and RJF110 / pAH48 8glpK) Dinner Nc. of consumption ratio? j glycerol syrups (g / OD / hr) FM5 / pAH48 0.095 ± 0.015 Table 2 (continued) Strain No. of consumption ratio Examples glycerol (g / OP / hr) RJF110 / pAH48 0.021 ± 0.011 As can be seen in the data in Table 2, the glycerol consumption ratio decreases approximately 4-5 times where the endogenous activity of glyceroi kinase is eliminated.

Environmental conditions to evaluate the Glycerol Dehydrogenase Activity (GldA) The temperature was controlled at 30 ° C and the air flow controlled at the standard of 6 liters per minute. The return pressure was controlled up to 0.5 bar. The dissolved oxygen tension was controlled at 10% by egitetion. Aqueous ammonium was used for pH control at €. ~? . In the first fermentation, the glucose was conserved in excess for the duration of the fermentation. The second fermentation was operated with non-residual glucose after the first 25 hours. Samples against the time of the two fermentations were taken to evaluate the activities of GlpK and GldA. The Teble 3 concludes that the fermentations of RJFI0 / pA48 show the effects of gldA on the selectivity for glycerol.

Table 3 Activities of GldA and GlpK from two Fermentations of RJF110 / pAH 8 Fermentation Time GldA GlpK Selectivity (hours) Total (g / g) 42 49 c _ 54 41 4 c '14 DI 12 As can be seen from the data in Table 3, the presence of glycerol dehydrogenase activity is linked to the conversion of glycerol under limited glucose conditions; thus, it is anticipated that eliminating the glycerol dehydrogenase ectivided will reduce the conversion of glycerol.

EXAMPLE 9 PRODUCTION PE GLYCEROL PESDE P-GLUCOSE USEFUL RECOMBINANT E. BLATTAE CONTAINING BOTH GPP2 AND DAR1 Example 9 illustrates the production of glycerol from D-glucose from recombinant E. blattae containing 15 amino acids senes GP? 2 v DARl.

E. bla z zae, obtained from the ATCC and that has no. AT access 33429, was developed at 30 ° C until the cultivation reached a Ct of approximately 0.6 AU to 600 np .. The culture was then transformed with pAH48, a plasmid cosing genes GPP2 and DAR1 (described in WO 95/21341, using electroporation techniques) The transformants were confirmed by the RFLP DNA standard and antibiotic resistance. mL of carbicillin).

- ^ Transformed E. biattae was developed aerobically at 35 ° C in shake flask cultures. The cultures were developed in a defined medium plus 2 z of glucose with selection of entibiotic and were initiated by inoculum from an overnight culture developed in LB plus 1 glucose with antibiotic selection. The defined medium contained per liter: 27.2 g of KH_P0, 2 g of citric acid, 2 g of MgSO, .7H 0, 1.2 ml of H_S04 to 98 i, 0.3 g of ferric ammonium citrate, 0.2 g of CaC1 ^ .2H0 , 10 g. of yeast extract (Difco), 5 L of Trace solution of Modified Blach elements (the composition of the stem can be found in Methods for General and Molecular Beeteriology (P. Gerhardt and coleboredores, eds P. 158, American Society for Microbiology, Washington, DC (1994.) The defined medium was sterilized by filtration and adjusted to a final p.sub.E of .8 with NH.sub.0 H. The agitation contents were then subjected to HPLC analysis for the presence of glycerci After the overnight incubation, E. bi ate tae containing pAH48 produced 7.63 g / L glyceroi The control, which was E. bia t tae wild type (ATCC 33429) developed under the same conditions, produced = 0.2 g / L glycerol.

EXAMPLE 10 PRODUCTION OF GLYCEROL FROM D-GLUCOSE USING RECOMBINANT COLI DEFICIENT IN GLPA AND GLPK AND CONTAINING BOTH GPP2 AND DAR1 INTEGRATED IN THE CHROMOSOME This example illustrates the production of glycerol from D glucose from recombinant E. coli with the gldA and the gene egotedo glpK and containing embos GPP2 and DAR1 coding loe genes integrated into the. chromosome of the host cell.

The KLP23 strain of E. coli, prepared as described in EXAMPLE 7, is deficient in both activities giicerol kinase (product of glpK) and glyceryl dehydrogenase (product of gldA). KLP23 containing DAR1, and a chloramphenicol-resistant GPP2 gene flanked by io > : P integrated into the chromosome at the ampZ location was prepared and is mentioned as AH76RJcm.

The integration plasmids were designed and constructed on the basis of er. an integration system cre-lox íHoes, supra). In order to create the integration plasmids, a Hindi-Smal fragment of pLoxCatl was inserted into Hind III and linearized Smal pAH48 to create pAH48cm2. Plasmid pAH48 contains the DAR1 and GPP2 genes expressed under the control of the promoter. The 3.5 kb ApaL I fragment from pAH48cm2 was finalized in smooth with T4 DNA polymerase (Boehringer Mannheim Biochemiccel) and dNTPs and inserted into linearized NruI plnt-amp C (Genencor International, CA), using E. coli SY327 (Miller and collaborators, J Bacteriol 170: 2575-2583, 1998) as a host to create pAH76 and pAH76R. The "R" stands for reverse orientation of the integration cartridge. Both plasmids, pAH76 and pAH76R contain a replica R6K origin and are not capable of replicating in KLP23. Plasmids pAH76 and pAH76R were used to transform KLP23 by integration into the ampC location of the E. coli chromosome. Transformants were selected on 10 μg / ml of chloramphenicol and were sensitive kanamycin, producing double cross-over integration. These transformants of E. coli are called AH76Icm and AH76Ricm.

The cultures of AH7óRicm were developed in shake flasks in defined medium (described in Example 9) plus 2.5 glucose initiated by inoculation from an overnight culture of LB having 1:. of glucose and aptibiotic selection. The shake flasks (Erlen Meyer flasks, 1/5 volume volume volume) were incubated at 37 ° C with vigorous shaking overnight, after the supernatant was sampled for glycerol using a colorimetric enzyme (Sigma, Procedure No. 337) On a Monarch 2000 instrument (Instrumentacttion Laboratory Co., Lexington, MA). AH77Ricm showed glycerol production of 6.7 g / L after 25 hours.

E. coli pAH76RI had the chloramphenicol gene removed from AH76Ricm. The chloramphenicol gene was removed from the chromosome using the Cre-producing plasmid, pJW168, as described in Example 5. Transformants were selected for resistance to carbecillin and sensitivity to chloramphenicol under 1 mM and IPTG induction at 30 ° C. After removal of the cioramfeniccl gene, AH76RI was developed on LB medium without any antibiotic to cure pJW168. The final version of AH76RI nc is capable of developing on the selection of chloramphenicol or carbecillin.

The crops of AH76RI were developed in shake flasks in a defined medium plus 2 ° of glucose initiated by inoculation from overnight LB / 1 glucose culture. The shake flasks were vigorously agitated overnight, after the supernatant was sampled for glycerol using a colorimetric assay (Sigma, procedure No. 337) on a Monarch 2000 instrument.

(Instrumentation Laboratory Co. Lexington, MA). AH77RI showed glycerol production of 4.6 g / L after 24 hours.

All the plasmids described in this example were transformed into E. coli KLP23 using standard techniques of molecular biology. The transtormins were verified by DNA RFLP patterns, antibiotic resistance, amplification by. PCR, or phosphatise G3P assay.

LISTING PE SEQUENCES GENERAL INFORMATION: (i! APPLICANT: (A) NAME: E.l. DU PONT DE NEMOURS AND COMPANY (B) STREET: 1007 MARKET STREET (C) CITY: ILMINGTON J; STATE: DELAWARE (E) COUNTRY: E.U.A. (F) POSTAL CODE: 19898 (G) TELEPHONE: 302- 892- 8112 (H) TELEFAX: 302-773- 0164 (I? TELEx: 6717325 (il) TITLE D? THE INVENTION: METHOD FOR THE GLYCLECL CLINIC BY RECOMBINANT ORGANISMS l li) NUMBER D? SEQUENCES: 3 Jv) READING COMPUTER: (A HALF TYPE: Z,: SQUET, 3.5 INCHES 3 'COMPUTER: PC COMPATIBLE WITH IBM (C) OPERATING SYSTEM: MICROSOFT WINDOWS 95 (D) PROGRAM: MICROSOFT WORD VERSION 7.0a (v) NORMAL APPLICATION DATES: (A) APPLICATION NUMBER: (B) REGISTRATION DATE: (C) CLASSIFICATION: (v) PRIORITY DATA OF THE APPLICATION: (A) APPLICATION NUMBER: 08 / 982,783 (B) REGISTRATION DATE: DECEMBER 2, 1997 (C) CLASSIFICATION: 1 (vile, INFORMATION OF OFFICER / APPORTER: (A1 NAME: FL0YO, LINDA AXAMETRY (B) REGISTRATION NUMBER: 33, 692 20 (C) REFERENCE / POWER NUMBER: CR- 9981-C .2) INFORMATION OF THE ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: - (A) EXTENSION: 1380 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: one only (D) TOPOLOGY: linear (ii) MOLECULA TYPE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: i CTTTAATTTT CTTTTATCTT ACTCTCCTAC ATAAGACATC AAGAAACAAT TGTATATTGT 60 ACACCCCCCC CCTCCACAAA CACAAATATT GATAATATAA AGATGTCTGC TGCTGC GAT 120 AGATTAAACT TAACTTCCGG CCACTTGAAT GCTGGTAGAA AGAGAAGTTC CTCTTCTGTT 1S0 TCTTTGAAG3 CTGCCGAAAA GCCTTTCAAG GTTACTGTGA TTGGATCTGG TAACTGGGGT 24C W rtílOjTuU i. TGTAAGGGAT ACCCAGAAGT TTTCGCTCCA 300 ATAGTACAAA CGAAGAAGAG ATCAATGGTG AAAAATTGAC TGAAATCATA 360 AATACTAGAC ATCAAAACGT GAAATACTTG CCTGGCATCA CTCTACCCGA CAATTTGGTT 420 GCTAATCCAG TTCAGTCAAG GATGTCGACA TCATCGTTTT CAACATTCCA 480 CATCAATTTT TGCCCCGTAG CTG AGCCAA TTGAAAGGTC ATGTTGATTC ACACGTCAGA 540 TTTTGAAGT GGTGCTAAAG GTGTCCAATT GCTATCCTCT 600 TACATCACTG AGGAACTAGG GGTGCTCTAT CTGGTGCTAA CATTGCCACC 660 GAAGTCGCTC AAGAACACTC GTC * GAAACA ACAGTTGCTT ACCACATTCC AAAGGATTTC 720 AVJAVJLJL, .unuu Uv ??? neither.- . ? -U? L. r.4? rtÜ u * * Ti va Cu ..G ..? ~ C CAGACCTTAC 7S0 TTCCACGTTA GTGTCATCGA GGTATCTCCA TCTGTGGTGC TTTGAAGAAC 840 GTTGTTGCCT TAGGTTGTGG TTTCGTCGAA GGTCTAGGCT GGGGTAACAA CGCTTCTGCT 900 GCCATCCAAA GAGTCGGTTT GGGTGAGATC ATCAGATTCG GTCAAATGTT TTTCCCAGAA 960 TCTAGAGAAG AAACATACTA CCAAGAGTCT GCTGGTGTTG CTGATTTGAT CACCACCTGC 1020 GCTGGTGGTA GAAACGTCAA GGTTGCTAGG CTAATGGCTA CTTCTGGTAA GGACGCCTGG 1080 GAATGTGAAA AGGAGTTGTT GAATGGCCAA TCCGCTCAAG GTTTAATTAC CTGCAAAGAA 1140 GTTCACGAAT GGTTGGAAAC ATGTGGCTCT GTCGAAGACT TCCCATTATT TGAAGCCGTA 1200 TACCAAATCG TTTACAACAA CTACCCAATG AAGAACCTGC CGGACATGAT TGAAGAATTA 1260 GATCTACATG AAGATTAGAT TTATTGGAGA AAGATAACAT ATCATACTTC CCCCACTTTT 1320 TTCGAGGCTC TTCTATATCA TATTCATAAA TTAGCATTAT GTCATTTCTC ATAACTACTT 1380 (2) INFORMATION OF THE ID NO: (i) CHARACTERISTICS OF THE SEQUENCE: (A) EXTENSION: 2946 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: one only (D) TOPOLOGY: linear (ii) MOLECULES TYPE: DN: genomic) (xi; DESCRIPTION D? THE SEQUENCE: SEQ ID NO: 2 GAATTCGAGC CTGAAGTGCT GATTACCTTC AGGTAGACTT CATCTTGACC CATCAACCCC 60 AGCGTCAATC CTGCAAATAC ACCACCCAGC AGCACTAGGA TGATAGAGAT AATAGAGTAC 120 GTGGTAACGC TTGCCTCATC ACCTACGCTA TGGCCGGAAT CGGCAACATC CCTAGAATTG 180 AGTACGTGTG ATCCGGATAA CAACGGCAGT GAATATATCT TCGGTATCGT AAAGATGTGA 240 TATAAGATGA TGTATACCCA ATGAGGAGCG CCTGATCGTG ACCTAGACCT TAGTGGCAAA 300 AACGACATAT ?? -. Ihift? 1 GGGGAGAGTT TCGTGCAAAT AACAGACGCA GCAGCAAGTA 360 ACTGTGACG TATCAACTCT TTTTTTATTA TGTAATAAGC AAACAAGCAC GAATGGGGAA 420 n u.? * U ?? CAATCACCAA GGTCGTCCCT TTTTTCCCAT TTGCTAATTT AGAATTTAAA 480 GAAACCAAAA GAATGAAGAA AGAAAACAAA TACTAGCCCT AACCCTGACT TCGTTTCTAT 540 GATAATACCC TGCTTTAATG AACGGTATGC CCTAGGGTAT ATCTCACTCT GTACGTTACA 600 AACTCCGGTT ATTTTATCGG AACATCCGAG CACCCGCGCC TTCCTCAACC CAGGCACCGC 660 CCCAGGTAA CG GCGCGAT GAGCTAATCC TGAGCCATCA CCCACCCCAC CCGTTGATGA 720 CAG A * T1 AA AAAACTG GAGCAAGGAA TTACCATCAC CGTCACCATC 780 ACCATCATAT CGCCTTAGCC TCTAGCCATA GCCATCATGC AAGCGTGTAT CTTCTAAGAT 840 TCAGTCATCA TCATTACCGA GTTTGTTTTC CTTCACATGA TGAAGAAGGT TTGAGTATGC 900 TCGAAACAAT AAGAC CGA TGGCTCTGCC ATTGGTTATA TTACGCTTTT GCGGCGAGGT 960 GCCGATGGGT TGCTGAGGG AAGAGTGTTT AGCTTACGGA CCTATTGCCA TTGTTATTCC 1020 GATTAATCTA TTGTTCAGCA GCTCTTCTCT ACCCTGTCAT TCTAGTATTT TTTTTTTTTT 1080 TTT * TGGTTT * a * • * • ** T *** T "» • * •• ** TCTTCTTGCC TTTTTTTCTT GTTACTTTTT TTCTAGTTTT 1140 TTTTCCTTCC ACTAAGCTTT TTCCTTGATT TATCCTTGGG TCTTCTTTC TACTCCTTTA 1200 TTATATATTA ATTTTTAAGT TTATGTATTT TGGTAGATTC AATTCTCTTT 1260 CCCTTTCCTT TTCCTTCGCT CCCCTTCCTT ATCAATGCTT GCTGTCAGAA GATTAACAAG 1320 ATACACATTC CTTAAGCGAA CGCATCCGGT GTTATATACT CGTCGTGCAT ATAAAATTTT 1380 - > Á GCCTTCAAGA TCTACTTTCC TAASAAGATC ATTATTACAA ACACAACTGC ACTCAAAGAT 144 C GACTGCTCAT ACTAATATCA AACAGCACAA ACACTGTCAT GAGGACCATC CTATCAGAAG 1500 ATCGGACTCT GCCGTGTCAA TTGTACATTT GAAACGTGCG CCCTTCAAGG TTACAGTGAT 156C TGGTTCTGGT AACTG3GGGA CCACCATCGC CAAAGTCATT GCGGAAAACA CAGAATTGCA 1620 TTCCCATATC TTCGAGCCAG AGGTGAGAAT GTGGGTTTTT GATGAAAAGA TCGGCGACGA 1680 AAATCTGACG GATATCATAA ATACAAGACA CCAGAACGTT AAATATCTAC CCAATATTGA 1740 CCTGCCCCAT AATCTAGTGG CCGATCCTGA TCTTTTACAC TCCATCAAGG GTGCTGACAT 1800 CCTTGTTTTC AACATCCCTC ATCAATTTTT ACCAAACATA GTCAAACAAT TGCAAGGCCA 1860 CGTGGCCCCT CATGTAAGGG CCATCTCGTG TCTAAAAGGG TTCGAGTTGG GCTCCAAGGG 1920 TGTGCAATTG CTATCCTCCT ATGTTACTGA TGAGTTAGGA ATCCAATGTG GCGCACTATC 1980 GCTGCAAA TTGGCACCGG AAGTGGCCAA GGAGCATTGG TCCGAAACCA CCGTGGCTTA 2040 CCAACTACCA AAGGATTATC AAGGTGATGG CAAGGATGTA GATCATAAGA TTTTGAAATT 2100 GCTGTTCCAC AGACCTTACT TCCACGTCAA TGTCATCGAT GATGTTGCTG GTATATCCAT 2160 TGCCGGTGCC TTGAAGAACG TCGTGGCACT TGCATGTGGT TTCGTAGAAG GTATGGGATG 2220 GGG TAACAAT GCCTCCGCAG CCATTCAAAG GCTGGGTTTA GGTGAAATTA TCAAGTTCGG 2280 TAGAATGTTT TTCCCAGAAT CCAAAGTCGA GACCTACTAT CAAGAATCCG CTGGTGTTGC 2340 AGATCTGATC ACCACCTGCT CAGGCGGTAG AAACGTCAAG GTTGCCACAT ACATGGCCAA 2400 G "w? J» JtJ-v < -7 TCAGCC7TGG AAGCAGAAAA GGAATTGCTT AACGGTCAAT CCGCCCAAGG 2460 G ATC CA TGCAGAGAA3 T7CACGAGTG GCTACAAACA TGTGAGTTGA CCCAAGAATT 2520 CCCAATTATT CGAGGCAGTC TACCAGATAG TCTACAACAA CGTCCGCATG GAAGACCTAC 2580 CGGAGATGAT TGAAGAGCTA GACATCGATG ACGAATAGAC ACTCTCCCCC CCCCTCCCCC 2640 TCTGATCTTT CCTGTTGCCT CTTTTTCCCC CAACCAATTT ATCATTATAC ACAAGTTCTA 2700 CAACTACTAC TAGTAACATT ACTACAGTTA TTATAATTTT CTATTCTCTT TTTCTTTAAG 2760 AATCTATCAT TAACGTTAAT TTCTATATAT ACATAACTAC CATTATACAC GCTATTATCG 2820 TTTACATATC ACATCACCGT TAATGAAAGA TACGACACCC TGTACACTAA CACAATTAAA 2880 TAATCGCCAT AACCTTTTCT GTTATCTATA GCCCTTAAAG CTGTTTCTTC GAGCTTTTCA CTGCAG 2940 2946 (2) INFORMATION SE ID NO: 3:E CHARACTERISTICS: (A) EXTENSION: 3178 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: one only (D) TOPOLOGY: linear (li) MOLECULE TYPE: DN (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3 CTGCAGAACT TCGTCTGCTC TGTGCCCA7C CTCGCGGTTA GAAAGAAGCT GAATTGTTTC 60 ATGCGCAAGG GCATCAGCGA GTGACCAATA ATCACTGCAC TAATTCCTTT TTAGCAACAC 120 ATACTTATAT ACAGCACCAG ACCT7ATGTC TTTTCTCTGC TCCGATACGT TATCCCACCC 180 AACTTTTATT TCAGTTTTGG CAGGGGAAAT TTCACAACCC CGCACGCTAA AAATCGTATT 240 TAAACTTAAA rt? N nn? . CACAAATAGG GAACt ^? G < j? CTAAACGAAG GACTCTCCCT 300 CCCTTATCTT GACCGTGCTA TTGCCATCAC TGCTACAAGA CTAAATACGT ACTAATATAT 360 GTTTTCGGTA ACGAGAAGAA GAGCTGCCGG TGCAGCTGCT GCCATGGCCA CAGCCACGGG 420 GACGCTGTAC TGGATGACTA GCCAAGGTGA TAGGCCGTTA GTGCACAATG ACCCGAGCTA 480 hi or * A? «. N TTCCCCACCG CCGCTCCACC GGCAGuTCTC TAGACGAGAC CTGCTGGACC 540 GTCTGGACAA * Ji-W w ?? ^ .n_-v TTCG? CGTGT TGATCATCGG TGGCGGGGCC ACGGGGACAG 600 GATGTGC CT? UnTuv. «GCJ TCAATGTGGC CCTTGTTGAA AAGGGGGATT 660 TTGCCTCGGG AACG CC C AAATCTACCA AGATGATTCA CGGTACTTAG_720_Go *. ? Gnu 780 AGCGTAAACA TCTTATCAAC ACTGCCCCTC ACCTGTGCAC GGTGCTACCA ATTCTGATCC 840 CCATCTACAG CACCTGGCAG GTCCCGTACA TCTATATGGG CTGTAAATTC TACGATTTCT 900 TTGGCGGTTC CCAAAACTTG AAAAAATCAT ACCTACTGTC CAAATCCGCC ACCGTGGAGA 960 AGGCTCCCAT GCTTACCACA GACAATTTAA AGGCCTCGCT TGTGTACCAT GATGGGTCCT 1020 TTAACGACTC GCGTTTGAAC GCCACTTTAG CCATCACGGG TGTGGAGAAC GGCGCTACCG 1080 TCTTGATCTA TGTCGAGGTA CAAAAATTGA TCAAAGACCC AACTTCTGGT AAGGTTATCG 1140 GTGCCGAGGC CCGGGACGTT GAGACTAATG AGCTTGTCAG AATCAACGCT AAATGTGTGG 1200 TCAATGCCAC GGGCCCATAC AGTGACGCCA TTTTGCAAAT GGACCGCAAC CCATCCGGTC 1260 TGCCGGACTC CCCGCTAAAC GACAACTCCA AGATCAAGTC GACTTTCAAT CAAATCTCCG 1320 TCATGGACCC GAAAATGGTC ATCCCATCTA TTGGCGTTCA CATCGTATTG CCCTCTTTTT 1380 ACTCCCCGAA GGATATGGGT TTGTTGGACG TCAGAACCTC TGATGGCAGA GTGATGTTCT 144C T7TTACCTTG GCAGGGCAAA GTCCTTGCCG GCACCACAGA CATCCCACTA AAGCAAGTCC 1500 CAGAAAACCC TATGCCTACA GAGGCTGATA TTCAAGATAT CTTG? AAGAA CTACAGCACT 1560 ATATCGAA7T CCCCGTGAAA AGAGAAGACG TGCTAAGTGC ATGGGCTGGT GTCAGACCTT 162C TGGTCAGAGA TCCACGTACA ATCCCCGCAG ACGGGAAGAA GGGCTCTGCC ACTCAGGGCG 166C TGGTAAGATC CCACTTCTTG TTCACTTCGG ATAATGGCCT AATTACTATT GCAGGTGGTA 1740 AATGGACTA TTACAGACAA ATGGCTGAGG AAACAGTCGA CAAAGTTGTC GAAGTTGGCG 1800 GATTCCACAA CCTGAAACCT TGTCACACAA GAGATATTAA GCTTGCTGGT GCAGAAGAAT 1860 GGACGCAAAA CTATGTGGCT TTATTGGCTC AAAACTACCA TTTATCATCA AAAATGTCCA 1920 ACTACTTGGT TCAAAACTAC GGAACCCGTT CCTCTATCAT TTGCGAATTT TTCAAAGAAT 1980 CCATGGAAAA TAAACTGCCT TTGTCCTTAG CCGACAAGGA AAATAACGTA ATCTACTCTA 2040 GCGAGGAGAA CAACTTGGTC AATTTTGATA CTTTCAGATA TCCATTCACA ATCGGTGAGT 2100 TAAAGTATTC CATGCAGTAC GAATATTGTA GAACTCCCTT GGACTTCCTT TTAAGAAGAA 2160 CAAGATTCGC CTTCTTGGAC GCCAAGGAAG CTTTGAATGC CGTGCATGCC ACCGTCAAAG 2220 TTATGGGTGA TGAGTTCAAT TGGTCGGAGA AAAAGAGGCA GTGGGAACTT GAAAAAACTG 2280 TGAACTTCAT CCAAGGACGT TTCGGTGTCT AAATCGATCA TGATAGTTAA GGGTGACAAA 2340 GATAACATTC ACAAGAGTAA TAATAATGGT AATGATGATA ATAATAATAA TGATAGTAAT 2400 AACAATAATA ATAATGG7G0 TAATGGCAAT GAAATCGCTA TTATTACCTA TTTTCCTTAA 2460 TGGAA3AGTT AAAG7AAAC? AAAAAAAC7A CAAAAATATA TGAAGAAAAA AAAAAAAAAA 2520 GGTAATAGAC TCTACTACTA CAATTGATCT TCAAATTATG ACCTTCCTAG TGTTTATATT 2580 CTATTTCCAA TACATAATAT AATCTATATA ATCATTGCTG GTAGACTTCC GTTTTAATAT 2640 CGTTTTAATT ATCCCCTTTA TCTCTAGTCT AGTTTTATCA TAAAATATAG AAACACTAAA 2700 TAATATTCTT CAAACGGTCC TGGTGCATAC GCAATACATA TTTATGGTGC AAAAAAAAAA .2760 ATGGAAAATT TTGCTAGTCA TAAACCCTTT CATAAAACAA TACGTAGACA TCGCTACTTG 2820 AAATTTTCAA GTTTTTATCA GATCCATGTT TCCTATCTGC CTTGACAACC TCATCGTCGA 2880 AATAGTACCA TTTAGAACGC CCAATATTCA CATTGTGTTC AAGGTCTTTA TTCACCAGTG 2940 ACGTGTAATG GCCATGATTA ATGTGCCTGT ATGGTTAACC ACTCCAAATA GCTTATATTT 3000 CATAGTGTCA TTGTTTTTCA ATATAATGTT TAGTATCAAT GGATATGTTA CGACGGTGTT 3060 ATTTTTCTTG GTCAAATCGT AATAAAATCT CGATAAATGG ATGACTAAGA TTTTTGGTAA 3120 AGTTACAAAA TTTATCGTTT TCACTGTTGT CAATTTTTTG TTCTTGTAAT CACTCGAC L76 (2) INFORMATION D? THE? ID NO: (i) CHARACTERISTICS OF THE SEQUENCE: (A) EXTENSION: 616 base pairs (B) TYPE.-nucleic acid (C) FILAMENTO: unites only (D) TOPOLOGY: linear (ii) MOLECULUS TYPE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4 ATGAAACGTT TCAATGTTTT AAAATATATC AGAACAACAA AAGCAAATAT ACAAACCATC 60 GCAATGCCTT TGACCACAAA ACCTTTATCT TTGAAAATCA ACGCCGCTCT ATTCGATGTT 12C GACGGTACCA TCATCATCTC TCAACCAGCC ATTGCTGCTT TCTGGAGAGA TTTCGGTAAA 180 GACAAGCCTT ACTTCGATG ATTCACATCT CTCACGGTTG GAGAACTTAC 240 GATGCCATTG CCAAGTTCGC TCCAGA * - A GCTGATGAAG AATACGTTAA CAAGCTAGAA 300 GGTGAAATCC CAGAAAAGTA CGGTGAACAC TCCATCGAAG TTCCAGGTGC TGTCAAGTTG 360 TGTAATGCTT TGAACGCCTT GCCAAAGGAA AAATGGGCTG TCGCCACCTC TGGTACCCGT 420 GACATGGCCA AU? A TUU * T CGACATT .. AAGATCAAGA GACCAGAATA 480 GCCAATGATG TCAAGCAAGG TAAGCCTCAC CCAGAACCAT ACTTAAAGGG TAGAAACGGT 540 -Tui-iu * - n .._ AAu.rtCCCA TCCAAATCTA AGGTTGTTGT CTTTGAAGAC 600 GCACCAGCTG GTATTGC7GC TGGTAAGGCT GCTGGCTGTA AAATCGTTGG TATTGCTACC 660 ACTTTCGATT * oGACTTCT G AuG AAAG GGTTGTGACA TCATTGTCAA GAACCACGAA 720 TCTATCAGAG TCGGTGAA CAACGCTGAA ACCGATGAAG TCGAATTGAT CTTTGATGAC 780 TACTTATACG CTAAGGATG CTTGTTGAAA TGGTAA 816 (2) INFORMATION OF THE ID NO: 5: CHARACTERISTICS D? SEQUENCE: A) EXTENSION: 53 base pairs [B? TYPE: nucleic acid (C) FILAMENTO: one only (D) TOPOLOGY: linear (ii) MOLECULA TYPE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5 ATGGGATTGA CTACTAAACC TCTATCTTTG AAAGTTAACG CCGCTTTGTT CGACGTCGAC 60 GGTACCATTA TCATCTCTCA ACCAGCCATT GCTGCATTCT GGAGGGATTT CGGTAAGGAC 120 AAACCTTATT TCu? I VJ Tu / ". ACACGT ATC - rt? L ¿C v- ATGGTTGGAG AACGTTTGAT 180 GCCATTGCTA AGACTTTGCC AATGAAGAGT ATGTTAACAA ATTAGAAGCT 24C GAAATTCCGG TCAAGTACGG TGAAAAATCC ATTGAAGTCC CAGGTGCAGT TAAGCTGTGC 300 AACGCTTTGA AAAAGAGAAA TGGGCTGTGG CAACTTCCGG 36C ATG? Cnn? A? * «UTTI-? ATCAGGAGAC CAAAGTACTT CATTACCGCT 420 AATGATGTCA AACAGGGTAA GCCTCATCCA GAACCATATC TGAAGGGCAG GAATGGCTTA 480 TCAATGAGCA AGACCCTTCC AAATCTAAGG TAGTAGTATT TGAAGACGCT 54C CCAGCAGG A *** - "r * r * **** -" • GGTTGTAAGA TCATTGGTAT TGCCACTACT 600 TTCGACTTGG ACTTCCTAAA GGAAAAAGGC TGTGACATCA TTGTCAAAAA CCACGAATCC 660 ATCAGAGTTG? UhTnv. ?? TGCCGAAACA GACGAAGTTG AATTCATTTT TGACGACTAC 720 TTATATGCTA AGGACGATCT GTTGAAATGG TAA 753 twenty (2) INFORMATION FROM THE SE IL NC: 6: CHARACTERISTICS LE LA SECUENC: IA: - > (A) EXTENSION: 2520 pairs bas (B) TYPE: nucleic acid (C) FILAMENTO: one only (D) TOPOLOGY: linear (ii) MOLECULA TYPE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6 TGTATTGGCC ACGATAACCA CCCTTTGTAT ACTGTTTTTG TTTTTCACAT GGTAAATAAC 60 GACTTTTATT AAACAACGTA TGTAAAAACA TAACAAGAAT CTACCCATAC AGGCCATTTC 120 GTAATTCTTC TCTTCTAATT GGAGTAAAAC CATCAATTAA AGGGTGTGGA GTAGCATAGT 180 GAGGGGCTGA CTGCATTGAC AAAAAAATTG AAAAAAAAAA AGGAAAAGGA AAGGAAAAAA 240 AGACAGCCAA GACTTTTAGA ACGGATAAGG TGTAATAAAA TGTGGGGGGA TGCCTGTTCT 300 CGAACCATAT AAAATATACC ATGTGGTTTG AGTTGTGGCC GG? ACTATAC AAATAGTTAT 360 ATGTTTTCCCCT CTCTCTTCCG ACTTGTAGTA TTCTCCAAAC GTTACATATT CCGATCAAGC 420 CAGCGCCTTT ACACTAGTTT AAAACAAGAA CAGAGCCGTA TGTCCAAAAT AATGGAAGAT 480 TTACGAAGTG ACTACGTCCC GCTTATCGCC AGTATTGATG TAGGAACGAC CTCATCCAGA 540 TGCATTCTGT TCAACAGATG GGGCCAGGAC GTTTCAAAAC ACCAAATTGA ATATTCAACT 600 TCAGCATCGA AGGGCAAGAT "" TGGGGTGTCT GGCCTAAGGA GACCCTCTAC AGCCCCAGCT 660 CGTGAAACAC CAAACGCCGG TGACATCAAA ACCAGCGGAA AGCCCATCTT TTCTGCAGAA 720 * -.- b n * * k-rtrtunh? hnni .Í? \ ?. -.- «- nvju A_ * J. \ J? - * a* . * - ^, rw or * - AACGAACCCA Cu.TGAAGTT CCCCAAAC G GGTTGGGTTG AGTGCCATCC GCAGAAATTA 6 * íC CTGGTGAACG TCGTCCAA G CCTTGCCTCA AGTTTGCTCT CTCTGCAGAC TATCAACAG 900 GAACGTGTAG CAAACGGTCT CCCACCTTAC AAGGTAATAT GCATGGGTAT AGCAAACATG 560 AGAGAAACCA CAATTCTGTG GTZ CZZZGZ ACAGGAAAAC CAATTGTTAA CTACGGTATT 102C GTTTGGAACG ACACCAGAAC GATCAAAATC GTTAGAGACA AATGGCAAAA CACTAGCGTC 1 C80 GATAGGCAAC TGCAGCTTAG ACAGAAGACT GGATTGCCAT TGCTCTCCAC GTATTTCTCC 1140 TGTTCCAAGC TGCGCTGGTT CCTCGACAAT GAGCCTCTGT GTACCAAGGC GTATGAGGAG 1200 AACGACCTGA TGTGGACACA TGGCTGATTT ACCAATTAAC TAAACAAAAG 126C GCGTTCGTTT CTGACGTAAC CAACGCTTCC AGAACTGGAT TTATGAACCT CTCCACTTTA 1320 AAGTACGACA ACGAGTTGCT GGAATTTTGG GGTATTGACA AGAACCTGAT TCACATGCCC 1380 GAAATTGTGT CCTCATCTCA ATACTACGGT GACTTTGGCA TTCCTGATTG GATAATGGAA 1440 AAGCTACACG ATTCGCCAAA AACAGTACTG CGAGATCTAG TCAAGAGAAA CCTGCCCATA 1500 CAGGGCTGTC TGGGCGACCA AAGCGCATCC ATGGTGGGGC AACTCGCTTA CAAACCCGGT 1560 GCTGCAAAAT GTACTTATGG TACCGGTTGC TTTTTACTGT ACAATACGGG GACCAAAAAA 1620 TTGATCTCCC AACATGGCGC ACTGACGACT CTAGCATTTT GGTTCCCACA TTTGCAAGAG 1680 0 TACGGTGGCC AAAAACCAGA ATTGAGCAAG CCACATTTTG CATTAGAGGG TTCCGTCGCT 1740 GTGGCTGGTG CTGTGGTCCA ATGGCTACGT GATAATTTAC GATTGATCGA TAAATCAGAG 1800 GG-. TW JG? C L? R .. * or -? * TACGOTTCCT GATTCTGGTG GCGTAGTTTT CGTCCCCGCA 1660 TTTAGTGGCC TATTCGCTCC CTATTGGGAC CCAGATGCCA GAGCCACCAT AATGGGGATG 1920 TCTCAATTCA CTACTGCCTC CCACATCGCC AGAGCTGCCG TGGAAGGTGT TTGCTTTCAA 1980 GCCAGGGCTA TCTTGAAGGC AATGAGTTCT GACGCGTTTG GTGAAGGTTC CAAAGACAGG 2040 5 GACTTTTTAG AGGAAATTTC CGACGTCACA TATGAAAAGT CGCCCCTGTC GGTTCTGGCA 2100 GTGGATGGCG GGATGTCGAG GTCTAATGAA GTCATGCAAA TTCAAGCCGA TATCCTAGGT 2160 CCCTGTGTCA AAGTCAGAAG GTCTCCGACA GCGGAATGTA CCGCATTGGG GGCAGCCATT 2220 GCAGCCAATA TGGCTTTCAA GGATGTGAAC GAGCGCCCAT TATGGAAGGA CCTACACGAT 2280 GTTAAGAAAT GGGTCTTTTA CAATGGAATG GAGAAAAACG AACAAATATC ACCAGAGGCT 2340 0 CATCCAAACC TTAAGATATT CAGAAGTGAA TCCGACGATG CTGAAAGGAG AAAGCATTGG 2400 AAGTATTGGG AAGTTGCCGT GGAAAGATCC AAAGGTTGGC TGAAGGACAT AGAAGGTGAA 2460 CA.-GAACAGG TT: CTTCCAATAA CAACATAAAT AATT7CTATT AACAATGTAA 2520 - > «; Phe Leu Prc Ar? He Cys Ser Gln Leu Lys Gly His Val Asp Ser HAS 130 135 140 Val Arg Wing He Ser Cys Leu Lys Gly Phe Glu Val Gly Wing Lys Gly 145 150 155 160 Val Gln Leu Leu Be Ser Tyr He Thr Glu Glu Leu Gly He Gln Cys 165 170 175 Gly Ala Leu Ser Gly Wing Asn He Wing Thr Glu Val Wing Gln Glu HIS 180 185 190 Trp Ser Glu Thr Thr Val Wing Tyr HIS He Pro Lys Asp Phe Arg Gly 195 200 205 Glu Gly Lys Asp Val Asp HIS Lys Val Leu Lys Wing Leu Phe His Arg 210 215 220 Pro Tyr Phe Hxs Val Ser Val He Glu Asp Val Wing Gly He Ser He 225 230 235 240 Cys Gly Ale Leu Lys Asr. Val Val Ala Leu Gly Cys Gly Phe Val Glu 245 250"" 255 Gly Leu Gly Trp Gly Asn Asn Wing Wing Wing He Gln Arg Val Gly 260 265 270 Gly Glu He He Arg Phe Gly Gln Met Phe Phe Prc Glu Ser Arg 275 280 • 285 Glu Glu Tnr Tyr Tyr Glr. Glu Be Wing Gly Val Wing Asp Leu He Thr 29C 295 300 Thr Cys Wing Giy Gly Arg Asn Val Lys Val Wing Arg Leu Met Wing Thr 305 310 315 320 Ser Gly Lys Asp Wing Trp Glu Cys Glu Lys Glu Leu Leu Asn Gly Gln 325 330 335 Be Ala Glr. Gly Leu He Thr Cys Ly. «= Glu Val Hs Glu Trp Leu Glu 340 345 350 Thr Cys Gly Ser Val Glu Asp Phe Pro Leu Phe Glu Ala Val Tyr Gln 355 360 365 le Val Tyr Asr. Asr. Tyr Pro Met Lys Asn Leu Pro Asp Met He Glu 370 375 380 Glu Leu Asp Leu His Glu Asp 3E5 39C (2) INFORMATION SEE ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) EXTENSION: 384 amino acids (B) TYPE: amino acid (C) FILAMENTO: unknown (D) TOPOLOGY: unknown ; ii) MOLECULA TYPE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8 Met Thr Ala HIS Thr Asn He Lys Gln HIS Lys His Cys HIS Glu Asp 1 5 10 15 Kis Pro He Arg Arg Being Asp Being Wing Val Ser He Val HIS Leu Lys 20 25 30 Arg Wing Pro Phe Lys Val Thr Val He Gly Ser Gly Asn Trp Gly Thr 35 40 45 Thr He Wing Lys Val He Wing Glu Asn Thr Glu Leu HIS Be Hxs He 50 55 60 Phe Glu Pro Glu Val Arg Met Trp Val Phe Asp Glu Lys He Gly Asp 65 70 75 80 Glu Asn Le. Thr Asp He He Asn Tr.r Are HÍ S G n Asn Val Lys Tyr 85 90 95 Leu Prc Asn He Asp Leu Pro His Asn Leu Val Wing Asp Pro Asp Leu 100 105 i; 0 Leu HIS Be He Lys Giy Wing Asp He Leu Val Phe Asn He Pro H S 115 120 125 Gln Phe Leu Prc Asn He Vai Lys Gln Leu Gln Gly His Val Aia Prc 130 135 140 His Val Arg Ala He Ser Cys Leu Lys Gly Phe Glu Leu Gly Ser Lys 145 150 155 160 Gly Val Glp Leu Leu Ser Ser Tyr Val Thr Asp Glu Leu Gly He Glr. 165 1 0 175 Cys Gly Aia Leu Ser Gly Wing Asn Leu Aia Pro Giu Val Aia Lys Glu 180 185 190 His Trp Ser Glu Thr Thr Val Wing Tyr Gln Leu Prc Lys Asp Tyr Gln 195 200 205 Gly Asp Gly Lys Asp Val Asp His Lys He Leu Lys Leu Leu Phe His 210 215 220 Ar? Pro Tyr Phe His Val Asn Val lie Asp Asp Vai Aia Gly He Ser 225 230 235 240 'He Ala Gly Ala Leu Lys Asn Vai Val Ala Leu Ala Cys Gly Phe Val 245 250 255 Glu Gly Met Gly Trp Giy Asr. Asn Ala Be Ala Ala He Gln Arg Leu 260 * 265 270 Gly Leu Gly Glu lie He Lys Phe Gly Ar? Met Phe Phe Prc Giu Ser 275 280 285 Lys Val Giu Thr Tyr Tyr Glr. Glu Be Wing Gly Val Wing Asp Leu He 290 295 300 Thr Thr Cys Ser Gly Gly Arg Asn Val Lys Val Wing Thr Tyr Met Wing 305 310 315 320 Lys Thr Gly Lys Ser Ala Leu Glu Ala Glu Lys Glu Leu Leu Asn Gly 325 '330 335 Gln Ser Wing Gln Gly He He Thr Cys Arg Glu Val His Glu Trp Leu 340 345 350 Gln Thr Cys Glu Leu Thr Gln Glu Phe Pro He He Arg Gly Ser Leu 355 '• 360 365 Pro Asp Ser Leu Gln Gln Arg Pro His Gly Arg Pro Thr Gly Asp A = p 370 375 380 (2) INFORMATION D? LA SE ID NO: 9: CHARACTERISTICS OF THE SEQUENCE: (Ai EXTENSION: 614 amino acids (B) TYPE: amino acid (C) FILAMENTO: unknown (D) TOPOLOGY: unknown (ii) MOLECULA TYPE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9 Met Thr Arg Wing Thr Trp Cys Asn Pro Pro Pro Leu His Arg Glr. 1 5 10 15 ' Vai Ser Arg Arg Asp Leu Leu Asp Arg Leu Asp Lys Thr His Gin Phe 20 25 30 Asp Vai Leu He He Gly Gly Gly Wing Thr Gly Thr Gly Cys Ala Leu 35 40 45 Asp Ala Ala Thr Arg Giy Leu Asn Val Ala Leu Val Glu Lys Gly Asp 50 55 60 Pne Wing Ser Giy Thr Ser Ser Lys Ser Thr Lys Met He His Gly Gly 65 70 75 80 Val Arg Tyr Leu Giu Lys Wing Phe Trp Giu Phe Ser Lys Wing Gin Leu is 90 95 Asp Leu Val He Glu Ala Leu Asn Giu Arg Lys His Leu He Asn Thr 10C 105 HO Pro Wing His Leu Cys Tnr Val Leu Pro He Leu He Pro He Tyr Ser 115 120 125 Thr Trp Gir. V¿; Prc Tyr He Tyr Met Giy Cys Lys Phe Tyr Asp Phe 130 135 I Q Phe Giy Giy Ser Glr. Asn Leu Lys Lvs Ser Tyr Leu Leu Ser Lys Ser 145 15C 155 160 Wing Thr Vai Giu Lys Wing Pro Met Leu Thr Thr Asp Asn Leu Lys Wing 165 l 0? 75 Ser Leu Vai Tyr Hs Asp Gly Ser Phe Asn Asp Ser Arg Leu Asn Wing 180 185 190 Thr Leu Wing He Thr Gly Val Glu Asn Gly Wing Thr Val Leu He Tyr 195 200 205 Val Glu Val Gln Lys Leu He Lys Asp Pro Thr Ser Gly Lys Val He 210 215 220 Gly Wing Glu Wing Ar? Asp Val Glu Thr Asn Glu Leu Val Ar? He Asn 225 230 235 240 Wing Lvs Cys Val Vai Asn Wing Tnr Gly Prc Tyr Ser Asp Aia He Leu 245 250 255 Gln Met Asp Arg Asn Pro Ser Giy Leu Pro Asp Ser Pro Leu Asr. Asp 260 265 27C Asn Ser Lys He Lys Ser Thr Pne Asn Gln He Ser Val Met Asp Prc 2 5 '280 285 Lys Met Val He Pro Ser He Gly Val His He Val Leu Pro Ser Phe 290' 295 300 Tyr Ser Pro Lys Asp Met Gly Leu Leu Asp Val Arg Thr Ser Asp Gly 305 310 315 320 Arg Val Met Phe Phe Leu Pro Trp Glr. Gly Lys Val Leu Aia Gly Thr 325 330 335 Thr Asp He Pro Leu Lys Gin Val Pro Glu Asn Pro Met Pro Thr Glu 340 345 350 Wing Asp He 'Glp Asp He Leu Lys Glu Leu Gln KIS Tyr He Glu Phe 355 360 '365 Pro Vai Lys Arg Giu Asp Val Leu Ser Ala Trp Ala Giy Val Ar? Pro 37C 375 380 Leu Vil Arg Asp Pro Arg Thr He Pro Wing Asp Gly Lys Lys Gly Ser 385 390 395 400 Wing T.. R Gln Gly Val Val Are Ser His Phe Leu Phe Thr Ser Asp Asn 405 410 415 Gly Leu He Thr He Wing Gly Gly Lys Trp Thr Thr Tyr Arg Gln Met 420 425 430 Ala Glu Glu T.r Val Asp Ly-. Val Val Giu Val Gly Gly Phe His Asn 435 44C 445 Leu Lys Pro Cys Kis Thr Are Asp He Lys Leu Wing Gly Wing Glu Glu 450 455 460 Trp Tr.r Gln Asr, Tyr Val Aia Leu Leu Wing Gln Asn Tyr His Leu Ser 465 470 475 480 2 ^ Ser Lys Met Ser Asr. Tyr Leu Val Gln Asn Tyr Gly Thr Arg Ser Ser 485 490 495 He He Cys Glu Phe Phe Lys Giu Ser Met Glu Asn Lys Leu Pro Lßu 500 505 510 Ser Leu Wing Asp Lys Glu Asn Asn Val He Tyr Ser Ser Glu Glu Asn 515 520 525 Asn Leu Val Asn Phe Asp Thr Phe Arg Tyr Pro Phe Thr He Gly Glu 530 535 540 Leu Lys Tyr Ser Met Gin Tyr Glu Tyr Cys Arg Thr Pro Leu Asp Pne 545 550 555 560 Leu Leu Ara Arg Thr Arg Phe Wing Phe Leu Asp Wing Lys Giu Aia Leu 565 570 575 Asr. Wing Vai His Aia Thr Val Lys Val Met Gly Asp Glu Phe Asn Trp 580 585 590 Ser Glu Lys Arg Gln Trp Glu Leu Glu Lys Thr Val Asn Phe He 595"600 605 Gin Gly Arg Phe Gly Val 610 INFORMATION OF THE ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: l EXTENSION: 339 amino acids (Bj TYPE: amino acid (C) FILAMENTO: unknown? -: 'TOPOLOGY: unknown MOLECULE TYPE: 'protein ixi 'DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10 Met Asn Gin Arg Asn Wing Being Met Thr Val He Gly Wing Gly Ser Tyr 1 5 10 15 Gly Thr Ala Leu Ala He Thr Leu Ala Arg Asn Gly His Glu Val Val 20 25 30 Leu Trp Giy His Asp Pro Glu His He Wing Thr Leu Glu Arg Asp Arg 3s "40 45 Cys Asn Aia Aia Pne Leu Pro Asp Val Pro Phe Pro Asp Thr Leu His 50 55 '60 Leu Giu Ser Asp Leu Ala Thr Ala Leu Ala Ala Ser Arg Asn He Leu 65 7C 75 80 Val Val Val Pro Ser His Val Phe Giy Glu Val Leu Arg Gln He Lys 85 90 95 Pro Leu Met Arg Pro Asp Wing Arg Leu Val Trp Wing Thr Lys Gly Leu 100 105 110 Glu Wing Glu Thr Gly Arg Leu Leu Gln Asp Val Wing Arg Glu Wing Leu 115 120 125 Gly Asp Gln He Pro Leu Wing Val He Ser Gly Pro Thr Phe Wing Lys 130 135 140 Glu Leu Wing Wing Gly Leu Pro Thr Wing Be Ser Leu Wing Ser Thr Asp 145 150 155 160 Gin Tr.r Fne Wing Asp Asp Leu Gir. Gir. Leu Leu H¿s Cys Giy Lys Ser 165 17C 175 Pne Arg Val Tyr Ser Asr. Pro Asp Phe He Gly Val Gir. Leu Gly Gly 180 185 190 Wing Val Lys Asn Vai He Wing He Gly Wing Gly Met Ser Asp Gly lie 195 200 205 twenty Gly Phe Giy Wing Asn Wing Arg Thr Wing Leu He Thr Arg Gly Leu Wing 210 215 220 Glu Met Ser Arg Leu Gly Aia Wing Leu Gly Wing Asp Pro Aia Thr Phe 225 230 235 240 Met Gly Met Ala Giy Leu Gly Asp Leu Val Leu Thr Cys Thr Asp Aer. 245 250 255 Glp Ser Arg Asn Arg Arg Phe Gly Met Met Leu Gly Gln Giy Met Asp 260 265 270 Val Gir. Be Wing Gin Glu Lys He Gly Gin Vai Vai Glu Gly Tyr Arg 275 280 285 Asn Thr Lys Glu Val Arg Giu Leu Wing HAS Arg Phe Gly Val Glu Met 290 295 300 Pro He Thr Glu Giu He Tyr Gln Val Leu Tyr Cys Gly Lys Asp Ala 305 310 315 320 Arg Glu Ala Aia Leu Tnr Leu Leu Giy Arg Ala Ar? Lys Asp Glu Ar? 325 330 335 Ser Ser Kis .'2) INFORMATION OF THE ID NO: 11. li) CHARACTERISTICS OF THE SEQUENCE: (A) EXTENSION: 501 amino acids (Bj TYPE: amino acid (C) FILAMENTO: unknown (D) TOPOLOGY: unknown (ii) MOLECULE crctema; xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11 Met Glu Thr Lys Asp Leu He Val He Gly Gly Gly He Asn Gly Ala 1 5 10 15 Gly He Wing Wing Asp Wing Wing Gly Arg Gly Leu Ser Val Leu Met Leu 20 25 30 Glu Wing Gln Asp Leu Wing Cys Wing Thr Ser Wing Being Ser Lys Leu 35 40 45 He His Gly Gly Leu Arg Tyr Leu Glu HAS Tyx Glu Phe Arg Leu Val 50 55 60 Ser Giu Aia Leu Aia Giu Arg Giu Val Leu Leu Lys Met Aia Prc His 65 7C 75 80 He Wing Pne Pro Met Arg Phe Arg Leu Pro HAS Arg Pro His Leu Arg 85 90 95 Pro Wing Trp Met He Arg He Gly Leu Phe Met Tyr Asp HAS Leu Giy 100 105 - 110 Lys Arg Tr.r Ser Leu Pro Gly Ser Thr Gly Leu Arg Phe Gly Wing Asn 115 120 '125 Ser Val Leu Lys Pro Glu He Lys Arg Gly Phe Glu Tyr Ser Asp Cys 130 135 140 Trp Vai Asp Asp Ala Arg Leu Val Leu Ala Asn Ala Gln Met Val Vai 145 150 155 160 Ar? Lys Giy Gly Gl ^ Vai Leu Thr Arg Thr Arg Wing Thr Ser Wing Arg 165 170 175 Arg Giu Asr Giy Leu He Val Glu Ala. Glu Asp He Asp Thr G.y 180 185 190 Lys Lys Tyr Ser Trp Glr. Aia Arg Gly Leu Val Asn Wing Thr Giy Pro 195 200 2C5 Trp Val Lys Gln Phe Phe Asp Asp Gly Met HAS Leu Pro Ser Pro ly- 210 215 220 Gly He Arg Leu He Lys Gly Ser KAS He Val Val Pro Aro Vai HAS 225 230 235"2 * 40 Thr Glr. Lys Gln Ala Tyr He Leu Gir. Asn Glu Asp Lys Arg He Val 245 250 255 Pne Val lie Prc Trp Met Asp Giu Pne Ser He Gly Thr Thr Asp 26C 265 27Q Val Glu Tyr Lys Giy Asp Pro Lys Wing Val Lys He Glu Glu Ser Glu 275 280 285 He Asn Tyr Leu Leu Asn Val Tyr Asn Thr His Phe Lys Lys Gln Leu 290 295 300 Ssx Arg Asp Asp He Val Trp Thr Tyr Ser Gly Val Arg Pro Leu Cys 305 * 310 315 320 A = p Asp Glu Ser Asp Ser Pro Gln Wing He Thr Arg Asp Tyr Thr Leu 325 330 335 Asp He HAS Asp Glu Asn Gly Lys Wing Pro Leu Leu Ser Val Phe Gly 340 345 350 Gly Lys Leu Thr Thr Tyr Arg Lys Leu Wing Glu HAS Wing Leu Glu Lys 355 360 365 Leu Tr.r Pro Tyr Tyr Gln Giy He Giy Pro Wing Trp Thr Lys Glu Se: 370 375 380 Val Leu Pro Gly Giy Aia He Giu Gly Asp Arg Asp Asp Tyr Aia Wing 385 390 395 400 Are Leu Arg Arg Arg Tyr Pro Phe Leu Thr Glu Ser Leu Aia Arg HAS 405 410 415 Tyr Ala Ar? Thr Tyr Gly Ser Asn Ser Glu Leu Leu Leu Gly Asr. Aia 420 425 430 Giy Thr Vai Ser Asp Leu Gly Glu Asp Phe Gly HAS Glu Phe Tyr Glu 435 440 445 Wing Giu Leu -ys Tyr Leu Val Asp HAS Glu Trp Val Arg Are Wing Asp 450 455 460 Asp Aia Leu Trp Arg? Rg Thr Lys Gln Gly Met Trp Leu Asn Wing Asp 465 470 475 B0 Glr. Gln Ser Are Vai Ser Gin Trp Leu Val Glu Tyr Thr Gln Gln Arg 485 490 95 Leu Ser Leu Ala Ser 50C (2) INFORMATION OF THE ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) EXTENSION: 542 amino acids (B) TYPE: amino acid (C) FILAMENTO: unknown (D) TOPOLOGY: unknown ii) MOLECULA TYPE: proteine (xi) DESCRIPTION OF THE. SEQUENCE: SEQ ID NO: 12 Met Lys Thr Arg Asp Ser Gln Ser Ser Asp Val He He He Giy Gly 1 5 10 15 Gly Aia Thr Gly Wing Gly He Wing Arg Asp Cys Wing Leu Arg Gly Leu 20 25 30 -Arg Val He Leu Val Glu Arg HAS Asp He Wing Thr Gly Wing Thr Gly 35 40 45 Arg Asn HAS Gly Leu Leu HAS Ser Gly Wing Arg Tyr Ala Val Thr Asp 50 55 60 Wing Glu Ser Wing Arg Glu Cys He Ser Glu Asn Gln He Leu Lys Arg 65 7 ° 75 80 He Wing Arg His Cys Val Giu Pro Thr Asn Giy Leu Phe He Thr Leu 85 90 95 Pro Giu Asp A = p Leu Ser Phe Gln Wing Thr Phe II 100 e Arg Wing C \ 105 110 Glu Ala Gly lle Be Ala Ala Giu Ala He Asp Pr 115 or Gln Gln AJ 120 to Arg Zlt 12. He Giu Pro Ala Vai 130 Asr. Pro Ala Leu He Gly Ala Vai Lys Val 135 140 A 1s4p5 Gly Thr Val Asp Pro Phe Arg Leu Thr Ala Ala Asn Met Leu Asi 150 155 160 Aia Lys Glu HAS Gl Gly Ala Val He Leu Thr Ala HAS Glu Val Thr GH- 1i7 / DU 2-, 5 - * Le lie A: g Giu Gly Wing Thr Val Cys Gly Val Ar 180 g Val Are Asr. HAI 185 190 Leu Thr Gly Giu Tr.r Gln Ala Leu His Al a Pro Val Val Vai Asn Wing 200 205 to Giy He Trp Gly Gln HAS He Wing Glu Tyr Wing Asp Leu Arg H 210 215 220 Arg Met Pne Pro Wing Lys Gly Being Leu Leu He Met Asp HAS Arg He 225 230 235 240 Asn Gln HAS Val He Asn Arg Cys Arg Lys Pro As Asp Aia Asp He 245 250 255 Leu Val Prro Gly, .A. ^ Spry T .n..r- H ..- e S • er Lte-u H "• e Gi¡.y.}. Thr Thr Ser Leu Arg 26C 265 270 He Asp Tyr Asn Glu He Asp Asp Asr. Arg Vai Thr Wing Glu Glu Val 275"2", ° 8"C 285 He leu Ar? Gi. Giy Glu 290 Leu Ala Pro Val Met Ala Lys 295 300 Thr Arg lle Leu Arg Ala Tyr Ser Gly Val Arg Pro Leu Val Ala Ser 305 210 315 320 Asp Asp Asp Pro Be Gly Arg Asn Leu Be Arg Gly He Val Leu Leu 325 3"3n0 335 Asp HAS Ala Glu Aro itr, r. . ^ Arg Asp Gly Leu Asp Gly Phe Ilß Thr? Thr J45 ,,. Gly 350 Gly V *: C.vs Arg Lys Leu Gly Asn Thr Arg prQ Cy = ^ 375 Thr Wing Asp Leu 380 Wing Leu Prc Giy Ser Gin Glu Pro Wing Glu Val Thr Leu Arg Lys Val 385 3-9"0- 3 , 9n5c 400 He Be Pro Leu Pro Pro Leu Arg Gly Ser Aia Val Tyr Arg HAS Glv 405 410 415 Asp Arg Thr Pro Wing Trp Leu Ser Giu Giy Arg Leu His Are Ser Leu 420 425 430 Val Cys Glu Cys Glu Ala Val Thr Ala Giy Giu Val Gin Tyr Ala Val 435 440 445 Giu Asn Leu Asn Val Asp Ser Leu Leu Asp Leu Arg Arg Arg Thr Arg 450 455 460 Val Gly Met .Giy Thr Cys Gln Gly Glu Leu Cys Wing Cys Ring Wing Wing 465 470 475 '480 Gly Leu Leu Gin Arg Pne Asn Val Thr Thr Ser Wing Gln Ser lie Glu 485 490 95 Gin Leu Ser Thr Phe Leu Asn Glu Arg Trp Lys Giy Vai Gln Pro He 50C 505 sio Wing Trp Gly Asp Wing Leu Arg Glu Ser Glu Phe Thr Arg Trp Val Ty- 515 520 '525 Glr. Gly Leu Cys Giy Leu Glu Lys Glu Gln Lys Asp Aia Leu 530 535 54th INFORMATION OF THE ID NO: 13 CHARACTERISTICS OF THE SEQUENCE: (A; EXTENSION: 250 amino acids (B) TYPE: amino acid (C) FILAMENTO: unknown (D) TOPOLOGY: unknown ü) MOLECULA TYPE: protein [xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13 Met Gly Leu Thr Thr Lys Pro Leu Ser Leu Lys Val Asn Ala Ala Leu 1 5 10 15 Phe Asp Vai Asp Gly Thr He He He Ser Gln Pro Wing He Wing Wing 20 25 30 Phe Trp Arg Asp Phe Giy Lys Asp Lys Pro Tyr Phe Asp Wing Glu HAS. 35 40 4s Val He Gln Val Ser HAS Gly Trp Arg Thr Phe Asp Wing Wing Lys 50 55 60 Phe Wing Pro Asp Phe Wing Asn Glu Glu Tyr Val Asn Lys Leu Glu Wing 65 70 75 80 Glu He Pro Vai Lys Tyr Gly Glu Lys Ser He Glu Val Pro Gly Ala 85 90 95 Val Lys Leu Cys Asp Ala Leu Asn Aia Leu Pro Lys Giu Lys rp n_ = .. 100 105 110 Val Ala Tr.r Ser Giy Thr Arg Asp Met Ala Gir. Lys Trp Phe Glu KAS 115 '120 125 Leu Giy He Arg Arg Pro Lys Tyr Pne He Thr Aia Asn Asp Val Lys 130 135 140 Glp Gly Lys Pro HAS Pro Glu Prc Tyr Leu Lys Gly Arg Asn Giy Leu 145 150 155 160 Gly Tyr Pro He Asn Glu Gln Asp Pro Ser Lys Ser Lys Val Val Val 165 170 175 Phe Glu Asp Aia Pro Wing Giy He Wing Aia Gly Lys Aia Wing Gly Cys 180 r 185 190 Lys He He Giy He Wing Thr Thr Phe Asp Leu Asp Phe Leu Lys Glu 195 200 205 Lys Gly Cys Asp He He Val Lys Asr. HAS Glu Ser He Arg Val Gly 210 215 220 Gly Tyr Asn Aia Glu Thr Asp Giu Val Glu Phe He Phe Asp Asp Tyr 225 230 235 240 Leu Tyr Aia Lys Asp Asp Leu Leu Lys Trp 245 250 [2) INFORMATION OF SEE ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) EXTENSION: 271 amino acids (B) TYPE: amino acid (C) FILAMENTO: unknown (D) TOPOLOGY: unknown (ii) MOLECULA TYPE: protein (xi ^ DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14 Met Lys Arg Pne Asn Val Leu Lys Tyr He Arg Thr Thr Lys Wing Asn x 5 10 5 He Gin Thr He Wing Met Pro Leu Thr Thr Lys Pro Leu Ser Leu Lys 20 25 30 He Asn Wing Aia Leu Phe Asp Val Asp Gly Thr He He Ser Ser Gln 35 40 45 Pro Wing Wing Wing Wing Phe Trp Arg Asp Phe Gly Lys Asp Lys Pro Tyr 50 55 60 Phe Asp Wing Glu HAS Val He HAS HAVE BE Gly Trp Arg Thr Tyr 65 70 75 80 Asp Wing Wing Lvs Pne Aia Pro Asp Phe Wing Asp Giu Glu Tyr Vai 85 90 95 Asn Lvs Leu Glu Gly Glu Lie Prc Giu Lys Tyr Gly Giu His Ser He 100 1C5 11C Giu Vai Pro Giy Wing Val Lys Leu Cys Asn Wing Leu Asn Wing Leu Pro 115 120 125 Lys Glu Lys Trp Wing Val Wing Thr Ser Gly Thr Ar ? Asp Ket Aia Lys 130 135 140 Lys Trp Phe Asp He Leu Lys He Lys Arg Prc Giu Tyr Phe He Thr 145 150 155 160 Wing Asn Asp Val Lys Gin Gly Lys Pro His Pro Glu Prc Tyr Leu Lys 165 170 175 Gly Arg Asr. Gly Leu Giy Phe Pro He Asn Glu Gln Asp Pro Ser Lys 130 185 190 Ser Lys Vai Val Val Phe Glu Asp Ala Pro Wing Gly He Wing Aia Glv 195 200 205 Lys Wing Aia Gly Cys Lys He Val Gly He Wing Thr Thr Phe Asp Leu 210 215 220 A = p Phe e Lys Glu Lys Giy Cys Asp He He Val Lys Asn His Glu 225 230 235 240 Ser He Arg Val Giy Glu Tyr Asn Aia Glu Thr Asp Glu Val Glu Leu 245 250 255 lle Fhe Asp Asp Tyr Leu Tyr Aia Lys Asp Asp Leu Leu Lys Trp 260 265 270 (2! INFORMATION FROM SE ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) EXTENSION: 709 amino acids (B) TYPE: amino acid id FILAMENT:. Unknown ÍD) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15 Met Phe Pro Ser Leu Phe Ar? Leu Val Val Phe Ser Lys Arg Tyr He 1 5 10 15 Phe Arg Ser Ser Gln Arg Leu Tyr Thr Ser Leu Lys Gln Glu Gln Ser 20 25 30 Arg Met Ser Lys He Met Glu Asp Leu Arg Ser Asp Tyr Val Pro Leu 35 40 45 Ali Se: He Asp Val Giy Thr Tr.r Ser Se: Are Cys He Phe 5C 60 Asn Are Trp Gly Gln Asp Val Ser Lys HAS Gln He Giu Tyr Ser Thr 65 70 75 80 Be Ala Be Lys Giy Lys He Giy Val Ser Gly Leu Arg Arg Prc Ser 85 90 95 Thr Ala Prc Wing Arg Glu Thr Pro Asn Aia Gly Asp He Lys Thr Ser 100 Í05 11C Gly Lys Pro He Phe Be Wing Glu Gly Tyr Aia He Gln Glu Thr Lys 115 120 125 Phe Le-_ Lys He Glu Glu Leu Asp Leu Asp Phe HAS Asr. Glu Pro Thr 130 125 140 Leu Lys Pne Prc Lys Pro Gly Trp Val Giu Cys His Pro Gln Lys Leu 145 150 '55 '160 Leu Val Asr. Val Vai Gln Cys Leu Ala Ser Ser Leu Leu Ser Leu Gir. 165 170 175 Thr He Asr. Ser Glu Arg Val Aia Asr. Gly Leu Pro Pro Tyr Lys Vai 180 185 190 He Cys Met Giy He Wing Asn Met Arg Glu Thr Thr He Leu Trp Ser 195 200 205 Arg Vai Trp Asn A = p Thr Are Tr.r He Lys He Val Arg Asp Lys Trp Glr. Asn Thr Ser Val 225 230 235 240 Asp Are Gir. Leu Gip Leu Arg Gin Lye Tr.r Gly Leu Prc Leu Leu Ser 245 250 255 Thr Tyr Phe Ser Cys Ser Lys Leu Ar? Trp Phe Leu Asp Asn Glu Prc 260 265 270 Leu Cys Thr Lys Wing Tyr Giu Glu Asn Asp Leu Met Phe Gly Thr Val 275 280 285 Asp Thr Trp Leu He Tyr Gln Leu Thr Lys Gln Lys Wing Phe Val Ser 290 295 300 Asp Vai Thr Asn Ala Ser Ar? Thr Gly Phe Met Asn Leu Ser Thr Leu 305 310 315 320 Lys Tyr Asp Asn Glu Leu Leu Glu Phe Trp Gly He Asp Lys Asn Leu 325 330 335 He His Met Pro Glu He Val Being Ser Gln Tyr Tyr Gly Asp Phe 340 345 350 Gly He Pro Asp Trp He Met Giu Lys Leu HAS Asp Ser Pro Lys Thr 355 360 365 Vai Leu Arg ASD Leu Val Lys Arg Asr. Leu Pro He Glp Giy Cys Leu 370 '375 380 Giy Asr; Gir. Ser Aia Ser Met Val Gly Gln Leu Wing Tyr Lys Pro Gly 3E5"390 395 400 Wing Aia Lys Cvs Thr Tyr Gly Thr Giy Cys Phe Leu Leu Tyr Asr. Thr 405 410 415 Gly Thr Lys Lys Leu He Ser Gln HAS Giy Ala Leu Thr Thr Leu Wing 420 425 430 15 Pne Trp Phe Pro HAS Leu Gln Glu Tyr Gly Gly Gln Lys Pro Giu Leu 435 440 445 Ser Lys Pro His Phe Aia Leu Glu Gly Ser Val Val Wing Ala Gly Wing 450 455 460 Val Val Gir. Leu Arg Asp Asn Leu Arg Leu He Asp Lys Ser Glu 465 470 475 480 Asp Val Gly Prc lle Aia Ser Tnr Val Pro Asp Ser Gly Gly Val Val 485 490 495 Pne Val Prc Wing Phe Ser Gly Leu Phe Wing Pro Tyr Trp Asp Pro Asp 5CC 505 510 Wing Arg Aia Thr He Met Giy Met Ser Gln Phe Thr Thr Ala Ser His 515 520 525 lle Wing Arg Wing Wing Vai Giu Giy Val Cys Phe Gln Ala Ar? Ala He 530 535 540 leu lys Ala Ke Ser Ser Asp Ale Phe Giy Giu Gly Ser Lys Asp Ar? 545 550 555 560 Asp Pne Leu Glu Glu He Ser Asp Val Thr Tyr Glu Lys Ser Pro Leu 565 570 575 Ser Vai Leu Wing Val Asp Gly Gly Met Ser Arg Ser Asn Glu Val Met 580 585 590 Gln He Gln Wing Asp He Leu Gly Pro Cys Val Lys Val Arg Arg Ser 595 600 605 Pro Thr Wing Glu Cys Thr Wing Leu Gly Wing Wing Wing Wing Wing Asn Met 610 615 620 Wing Phe Lys Asp Val A = n Glu Ar? Pro Leu Trp Lys Asp Leu His Asp 625 630 635 640 Val Lys Lys Trp Val Phe Tyr Asn Gly Met Glu Lys Asn Glu Gln He 645 650 655 Ser Prc Giu Ala HAS Prc Asn Leu Lys He Phe Are Glu Ser Asp 660 665 670 Asp Wing Glu Arg Arg Lys HAS Trp Lys Tyr Trp Glu Val Aia Val Giu 675 680 685 Arg Ser Lys Giy Trp Leu Lys Asp He Glu Gly Glu HAS Giu Glr. Vai 690"695 700 Leu Giu Asn Phe Gln 705 : 2 < INFORMATION OF THE ID SE NC: 16: íl) CHARACTERISTICS D? SEQUENCE: (A) EXTENSION: 51 paree base (B) TYPE: nucleic acid (C) FILAMENTO: one only ÍD) TOPOLOGY: linear i? MOLECULUS TYPE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16 GCGCGGATCC AGGAGTCTAG AATTATGGGA TTGACTACTA AACCTCTATC T (2) INFORMATION OF THE ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) EXTENSION: 36 base pairs (B) TYPE: nucleic acid (C; FILAMENTO: one only (D) TOPOLOGY: linear (ii) MOLECULA TYPE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17 GATACGCCCG GGTTACCATT TCAACAGATC GTCCTT (2) INFORMATION OF THE ID NO: 18.

CHARACTERISTICS D? THE SEQUENCE: (A) EXTENSION: 34 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: one only (D) TOPOLOGY: linear (ii) MOLECULA TYPE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1 8 GTGATAATAT AACCATGGCT GCTGCTGCTG ATAG (2) INFORMATION OF THE ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) EXTENSION: 39 base pairs (B) TYPE: nucleic acid ÍC) FILAMENTO: one only (Di TOPOLOGY: linear (ii) MOLECULUS TYPE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 19 GTATGATATG TTATCTTGGA TCCAATAAAT CTAATCTTC (2) INFORMATION OF THE ID NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) EXTENSION: 24 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: one only ÍD) TOPOLOGY: linear (ii) MOLECULA TYPE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 20 CATGACTAGT AAGGAGGACA ATTC ? 2 INFORMATION OF THE ID NO: (i) CHARACTERISTICS OF THE SEQUENCE: (A) EXTENSION: 24 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: one only (D) TOPOLOGY: linear (ii) MOLECULA TYPE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 21 CATGGAATTG TCCTCCTTA TAG Í2) INFORMATION OF THE ID NO: 2: CHARACTERISTICS OF THE SEQUENCE: ÍA; EXTENSION: 19 paree base (B) TYPE: nucleic acid (OR FILAMENT: one selo (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) [xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 22 CTAGTAAGGA GGACAATTC (2) INFORMATION OF THE ID NO: 23 (i) CHARACTERISTICS OF THE SEQUENCE: (A) EXTENSION: 19 base pairs (B) TYPE: nucleic acid IC) FILAMENT: one single TOPICOLOGY: lijieal MOLECULE T ^ PD: DNA (genomic) ixii DESCRIPTION D? THE SEQUENCE: SEQ ID NO: 23 CCTCCTTA (2) INFORMATION OF THE ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) EXTENSION: 15 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: one only (D; TOPOLOGY: linear (nor MOLECULA TYPE: other nucleic acid A) DESCRIPTION: / desc = "primer" (iii1 HYPOTHETICAL: NO [iv > ANTICIPATION: NO (xi SEQUENCE DESCRIPTION:? EC ID NO: 24 GATCCAGGAA ACAGA 2) INFORMATION OF THE ID NO: 25: íl! CHARACTERISTICS L? THE SEQUENCE: (A) EXTENSION: 15 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: one only (D) TOPOLOGY: linear (iii) MOLECULA TYPE: other nucleic acid A) DESCRIPTION: / desc = "primer" mi) HYPOTHETICAL.-NO [iv) ANTICIPATION: NO (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 25 CTAGTCTGTT TCCTG (2) INFORMATION OF THE ID NO: (i) CE CHARACTERISTICS THE SEQUENCE: IA) EXTENSION: 22 base pairs (3 ^ TYPE: nucleic acid (C) FILAMENTO: one only (D) TOPOLOGY: linear (iv) MOLECULA TYPE: other nucleic acid A) DESCRIPTION: / desc = "primer" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 26 GCTTTCTGTG CTGCGGCTTT AG "&! ) CHARACTERISTICS D? SEQUENCE: (A- EXTENSION: 23 base pairs (B TIPG: nucleic acid [OR FILAMENT: one single (D.i TOPOLOGY: linear (I saw MOLECULA ctrc nucleic acid A > DESCRIPTION: / desc = "primer" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 27 TGGTCGAGGA TCCACTTCAC TTT (2) INFORMATION OF THE ID NO: 28: (i) CHARACTERISTICS OF THE SEQUENCE: (A) EXTENSION: 51 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: one only ÍDj TOPOLOGY: linear (vi) MOLECULA TYPE: other nucleic acid A 'DESCRIPTION: / desc = "starter" (xi) DESCRIPTION E? THE SEQUENCE: SEQ ID NO: 28 AAAGTGAAGT GGATCCTCGA CCAATTGGAT GGTGGCGCAG TAGCAAACAA INFORMATION OF THE ID NO: 29: (i) CHARACTERISTICS OF THE SEQUENCE: (A) EXTENSION: 23 pairs kiss (B) TYPE: nucleic acid (C) FILAMENTO: one only (D) TOPOLOGY: linear (vii) MOLECULA TYPE: other nucleic acid ! DESCRIPTION: / desc = "primer" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 26 GGATCACCGC CGCAGAAACT ACG INFORMATION: D? you, CHARACTERISTICS OF THE SEQUENCE: (A'1 EXTENSION: 25 pairs case (3! TYPE: nucleic acid (C) FILAMENTO: one sclc (D: TOPOLOGY: linear (viii) MOLECULE TYPE: other nucleic acid A) DESCRIPTION: / desc = "primer" [xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 30 ÜTGTCAGCCG TTAAGTGTTC CTGTG (2) INFORMATION OF THE ID NO: 31 [i! CHARACTERISTICS OF THE SEQUENCE: EXTENSION: 23 base pairs TYPE: nucleic acid FILAMENTO: one only TOPOLOGY: linear , IX 'MOLÉCULA TIfC: another nucleic acid [ESCPIPTION: / .eec =: ebado , x ?, D? SCRI? .? THE SEQUENCE: SEQ ID NO: 31 CAGTTCAACC TGTTGATAGT ACG [2] INFORMATION OF THE ID NO: 3; (i) SEQUENCE CHARACTERISTICS: (A) EXTENSION: 20 base pairs (SI TIPC: nucleic acid ÍC; FILAMENTO: one only (D) TOPOLOGY: linear (x) MOLECULA TYPE: other nucleic acid A) DESCRIPTION: / desc = "primer" (x: DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3 ATGAGTCAAA CATCAACCTT ! 2l INFORMATION OF THE ID NO: 33: CHARACTERISTICS D? THE SEQUENCE: (A) EXTENSION: 20 peres kiss (B) TYPE: nucleic acid (C) FILAMENTO: one only (D) TOPOLOGY: linear (xi) MOLECULA TYPE: other nucleic acid A) DESCRIPTION: / desc = "primer" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 33 ATGGAGAAAA AAATCACTGG [2! INFORMATION OF THE ID NO: 34 i A CHARACTERISTICS OF THE SEQUENCE: (Ai EXTENSION: 20 base pairs (B) TYPE: nucleic acid (C'i FILAMENTO: one only (D) TOPOLOGY: linear : nucleic acid tro A) DESCRIPTION: / desc = "primer" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 34 TTACGCCCCG CCCTGCCACT [2) INFORMATION OF THE ID NO: 35: ii) 'CHARACTERISTICS OF THE SEQUENCE: and, EXT? NSION: 20 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: one only (D) TOPOLOGY: linear (xiii) MOLECULA TYPE: other nucleic acid TO; DESCRIPTION: / desc = "primer" (xi! DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 35 TCAGAGGATG TGCACCTGCA (2) INFORMATION D? LA ID NO: 36; (i) SEQUENCE CHARACTERISTICS: (A) EXTENSION: 26 peres kiss (B) TYPE: nucleic acid (C) FILAMENTO: one only (D) TOPOLOGY: lineel (xiv) MOLECULA TYPE: other nucleic acid .) DESCRIPTION: / desc = "primer" (xil DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 36 CGAGCATGCC GCATTTGGCA CTACTC 15 (2) INFORMATION OF THE ID NO: 37: (il CHARACTERISTICS OF THE SEQUENCE: 20 ÍA) EXTENSION: 29 paree base Í3 'TIPC: ac nucleic nucleus ÍC) FILAMENT: one selo [D1 TOPOLOGY: linear * <; (xv) MOLECULA TYPE: other nucleic acid A) DESCRIPTION: / desc = "cebedor" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3 ' GCGTCTAGAG TAGGTTATTC CCACTCTTG (2! INFORMATION OF THE ID NO: 38: (i) SEQUENCE CHARACTERISTICS: (AI EXTENSION: 26 base pairs (B) TYPE: nucleic acid (C, FILAMENTO: un solo ÍD) TOPOLOGY: linear [xv: 'other nucleic acid DESCRIPTION: / desc = "primer" DESCRIPTION Z? THE SEQUENCE: SEQ ID NO: 38 GAAGTCGACC GCTGCGCCTT ATCCGG [2) INFORMATION OF SEE ID NO: 39: (i) SEQUENCE CHARACTERISTICS: (A) EXTENSION: 26 base pairs (B) TYPE: nucleic acid (C; FILAMENTO: one only and, TOPOLOGY: linear [xvii) MOLECULA TYPE: other nucleic acid l DESCRIPTION: / desc = "primer" : xi? DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 39 CGCGTCGACG TTTACAATT7 CAGGTGGC .2! INFORMATION OF THE ID NO: 40: CHARACTERISTICS OF THE SEQUENCE: : XTE 3I0N: res case (B) TYPE: nucleic acid (C) FILAMENTO: one only (D) TOPOLOGY: linear (xvi ii) MOLECULA TYPE: other nucleic acid A) DESCRIPTION: / desc = "primer" (xi; DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 40 GCAGCATGCT GGACTGGTAG TAG (2) INFORMATION OF THE ID NO: 41: 1 CHARACTERISTICS OF THE SEQUENCE: (A) EXTENSION: 27 base pairs (B? TIP0: nucleic acid (C? FILAMENTO: une sole 20 (D «TOPOLOGY: linear (xix MOLECULA TYPE: other nucleic acid A, DESCRIPTION: / desc = "primer '-> (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 41 CAGTCTAGAG TTATTGGCAA ACCTACC [2] INFORMATION OF THE ID NO: 42: (i) D CHARACTERISTICS? SEQUENCE: (To the EXTENSION: 25 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: one only (D) TOPOLOGY: linear (xx, another nucleic acid A) DESCRIPTION: / desc = "primer ixi) DESCRIPTION D? THE SEQUENCE: SEQ ID NO: 42 GATGCATGCC CAGGGCGGAG ACGGC [2, INFORMATION 0? THE ?? ID NO: 43 (i) CHARACTERISTICS OF THE SEQUENCE: (A) EXTENSION: 29 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: one only (D) TOPOLOGY: linear (xxi) MOLECULA TYPE: other nucleic acid A- DESCRIPTION: / desc = "primer" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 43 CIAACGATTG TTCTCTAGAG AAAATGTCC Í2 '> INFORMATION OF THE ID NO: 44: CHARACTERISTICS D? SEQUENCE: y: EXTENSION: 30 base pairs ÍB) TYPE: nucleic acid (C FILAMENTO: joins only (D- TOPOLOGY: linear íiii MOLECULA TYPE: nucleic acid chorus (A) DESCRIPTION: / desc = "primer" (iii) HYPOTHETICAL: NO ; ÍV) ANTISENTIDO: NO [xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 44 CACGCATGCA GTTCAACCTG TTGATAGTAC (2) INFORMATION OF SEQ ID NO: 45: (i) SEQUENCE CHARACTERISTICS: ÍA) EXTENSION: 28 base pairs (B> TYPE: nucleic acid IC [FILAMENT: one only (Di TOPOLOGY: linear (ii <MOLECULE TYPE: other nucleic acid 20 (A) D? SCRIPTION: / desc = "primer" iiii! HYPOTHETICAL: C - > iv) ANTIS? NTIDO.-NC (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 45 GCGTCTAGAT CCTTTTAAAT TAAAAATG It is noted that in relation to this date the best method known to the applicant to practice and practice the said invention is that which is clear from the present description of the invention.

Having described the invention as above, it is claimed as property in the following: !. "> 0

Claims (20)

1. A Method for the production of giicerol from a recombinant organism, which is characterized in that it comprises: (i) transforming a cell-guest to < decueda with a cartridge and expression comprising either or both of: (a) a gene encoding a protein having sii-cyano-3-phosphate dehydrogenase activity, and (b> a gene that encodes a protein that has gi gi-3-phosphate phosphatase activity, the host cell that has an i :: -tí: r- i J. either e :. ^; I in both of ?to; an enagenic gene encoding a polypeptide having glyceroi kinase activity, and r an alien gene that encodes a polypeptide that dehydrogenase, where the interruption prevents the expression of the piudu iu of the active gene, " (ii) cultivating the host cell transformed from (i) into ie presence of at least one carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and substrates of a single carbon, by which is produced giiceroi; Y (iii) optionally recover the glycerol produced

2. The method of claim 1 which is characterized in that the expression cartridge comprises a gene coding for autologous and 3-phosphate dehydrogenase.

3. The method of claim 1 is characterized in that the expression cartridge comprises u;: yei; what c? dlíioe u :. glyceiul-3-phosphate rosettase enzyme.

4. The ethoac of claim 1, which is characterized in that the expression cartridge comprises genes encoding a giicerol-3-phosphate phos- phetase enzyme and a giiceroi- 3-phosphate dehydrogenase enzyme.

5. The cell of id claim 1 which is characterized in that the host cell contains an interruption in a gene encoding an endogenous giicerol kinase enzyme in which the interruption prevents the expression of the active gene product.

6. The method of claim 1 which is characterized in that the host cell contains an interruption in a gene encoding an endogenous enzyme giiceroi dehydrogenase in which the interruption prevents the expression of the active gene product.

7. Method of claim 1 which is characterized in that the host cell contains a) an interruption in a gene encoding an endogenous glycerol kinase enzyme and b; an interruption in a gene encoding an endogenous enzyme giicerol dehydrogenase, in which disruption in the respective genes prevents the expression of the active gene product.

8. The method of claim 1 which is characterized in that the suitable host cell is selected from the group consisting of becteria, yeasts, and filamentous fungi.

9. The method of claim 8 which is characterized in that a suitable host cell is selected from the group consisting of Ci trobacter, Enterobacter, Clostridium, Klebsieila, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Pichia, Kl uyveromyces, Candida Hansenula. , Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella, Bacillus, Streptomyces, and Pseudomonas.

10. The method of claim 9 which is characterized in that the appropriate host cell is E. Coli or Sd chai orr.y is sp.

11. r.1 method of claim 1 which is characterized in that the source of cerbono is glucose.

12. The method of claim 1 which is raised because the protein having giicerol-3-phosphate dehydrogenase activity corresponds to amino acid sequences selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 , SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 and in which the amino acid sequences aberce substitutions, 5 deletions or insertions of amino acids that do not alter the functional properties of the enzyme.

13. The method of claim 1 which is characterized because it protects it has activity of 10 giicerol-3-phosphatase corresponds to the amino acid sequences selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 14, and the amino acid sequences may encompass substitutions, deletions or additions of amino acids not surrounding the function from 15 the enzyme.

14. A transformed host cell which is characterized in that it comprises: 20? A) a gene encoding a protein having an alicerol-3-phosphate phosphatase activity. (Or a gene that coats a protein that has aeziviaaa ae gncere_ - r> - resrato rosiatese. - > < (c) an interruption in a gene encoding an endogenous • -glycerol kinase; (d) an interruption in a gene encoding an endogenous ylicerol dehydrogenase; in which the disruption in the genes of (O and (d) prevent the expression of the active gene product, and wherein the host cell converts at least one carbon source selected from the group consisting of monosaccharides, cligosaccharides, polysaccharides, and subtracts of a single carbon to glycerol.

15. A transformed host cell that is "acterized because it comprises: . to; a gene that encodes a protein that has a nviaaa "o o .. - sratc aesnicrogenasa; tb, a gene that coacts a protein that has glycero-3-phosphate phosphatase activity; Y (Or an interruption in a gene encoding an endogenous rehydroxydehydrogenase; in which the interruption in the gene of (c) prevents the expression of the product of the ective gene, and where the guest cell converts at least one cerium source selected from the group consisting of monosaccharides, oiigosaccharides, poiisaccharides and substrates of a single carbon to glycerol.

16. A transformed host cell which is characterized in that it comprises: (a) a gene encoding e binds protein having a glycerol-3-phosphate dehydrogenase activity; ib) a gene encoding a protein having glycerol-3-phosphate phosphate activity; Y (c) an interruption of a gene that encodes e unites roomasoma enaognana, wherein the digestion in the gene of (c) prevents the expression of the product of the ective gene, and wherein the host cell conveys at least one carbon source selected from the group consisting of monosaccharides, ¿, Olxsdcei i ob, and substrates of a single carbon to alicercl.

17. Method for the production of 1,3-propanediol from a recombinant organism which is characterized in that it comprises: (i) transforming a suitable host cell with an expression cartridge comprising either one or both of (a) a gene encoding a protein having glycero-3-phosphate dehydrogenase activity, and (b'- a gene that codes for a protein that has glycerol-3-phosphate phosphatase activity = a, the appropriate host cell having at least one gene encoding a protein having an activity dehydrated and having an interruption in either one or both of; a) an endogenous gene encoding a polypeptide having glyceryl activity, and ib.- an endogenous gene that encodes a polypeptide that has glyceral dehydrogenase activity, in which the interruption in the genes of (a) or (b) prevents the expression of the product of the ective gene; (ii) culturing the transformed host cell of (i) in the presence of at least one carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and single carbon substrates whereby 1,3-propanediol is produced; Y (iii) recover the 1,3-propanediol produced from [ii)

18. The method of claim 17 which is characterized in that the protein having an ectivided dehydrette is selected from the group consisting of a glycerol dehydrease enzyme and a diol dehydratase of the enzyme.

19. The method of claim 18 which is characterized in that the glycerol dehydrate enzyme is encoded by a gene, the gene isolated from a microorganism, the microorganism selected from the group consisting of Klebsiella, Lactobacillus, Enterobacter, Ci trobacter, Pelobacter, Ilyobacter and Clostridi. um.

20. The method of claim 1 which is characterized because the enzyme diol dehydrate is encoded by a gene, the gene from a microorganism, the microorganism selected from the group consisting of Kiebsieila and Seimonelle. METHOD FOR THE PRODUCTION OF GLYCEROL BY RECOMBINANT ORGANISMS SUMMARY OF THE INVENTION Recombinant organisms are provided- ^ an-t¿e- =; which comprise genes encoding glyceral-3-phosphate dehydrogenase activity and / or a glycerol-3-phosphatase useful for the production of slicerol in a variety C- i containing interruptions in the endogenous genes encoding the protein that has actividaaes of glycerol cinase and glycerin dehydrogenase.