BIOSYNTHESIS OF METHIONINE USING A REDUCED SOURCE OF SULFUR

Abstract: There are provided methods for the fermentation synthesis of methionine and homoserine using a reduced source of sulfur such as sulfide or methylmercaptan; and/or by modifying the methionine biosynthetic pathway in a producing microbe. Also provided are methods for the fermentation synthesis of methionine and homoserine using an oxidized sulfur source such as sulfate, sulfite or thiosulfate; and/or by modifying the methionine biosynthetic pathway in a producing microbe.

Description: BIOSYNTHESIS OF MEIHIONINE USING A REDUCED OTIHΠR W SULFUR BACKGROUND OF THE INVENTION Methionine is an essential amino acid in the diet of animals and is used widely as a food and feed supplement. It is conventionally produced by various multi-step chemical syntheses which generally employ acrolein, methyl mercaptan, and cyanide as starting materials. (H.H. Szmant, "Organic Building Blocks of the Chemical Industry," page 182, John Wiley & Sons, New York, 1989.) There are two resulting product forms: D,L-methion±ne and its hydroxy analog. Unlike all other amino acids, D-methionine is converted to the required L-form in vivo. As a result, chemical syntheses, which typically result in the D,L mixture, are feasible and cost-effective in this case. However, fermentation production methods, which are common methods for making many low-cost amino acids, do not exist in the case of methionine. (K. Aida, I. Chibata, K. Nakayama, K. Takinami, and H. Yamada, "Biotechnology of Amino Acid Production," Progress in Industrial Microbiology 24. Elsevier, 1986.) This is surprising given that the biochemically related essential amino acids lysine and threonine are both produced cost-effectively by fermentation methods losing inexpensive raw materials such as molasses, starch hydrolysates, corn steep liquor, and soy hydrolysates. (See for example: P.L. Rogers, R.G. Cail, D.F. Midgley, and C. Fryer, "The Prospects for L-Lysine Production in Australia," Food Technology in Australia 38. pp. 514-518, 1986; and S. Furukawa, A. Ozaki, and T. Nakanishi, "L-Threonine Production by L-Aspartate- and Ir-Hcmoserine- resistant mutant of Escheridhia σoli." Applied Microbiology and Biotechnology 29. pp. 550-553, 1988.) Various microbes have been used to produce L-lysine and L-threonine. These have been developed through classical methods of mutagenesis and selection as well as genetic engineering. (K. Aida, supra.) Greatest success has been realized historically with the Corvnebacteria and Brevibacteria. but it is also clear that other microbes such as Escheridhia coli are viable. There is a need for methods to reduce the metabolic cost and complexity of methionine biosynthesis, ideally making it similar to that for lysine or threαnine, such that an e∞ncmical fermentation production of methionine is possible. SCMMfiRY OF THE INVENTION There are provided feasible fermentation methods for methionine synthesis comprising the use of reduced sulfur compounds instead of sulfate as the fermentation sulfur source and/αr comprising re-designing and thereby simplifying the biochemical pathway. Also provided are fermentation methods for hcmocysteine synthesis ccnαprising the vise of reduced sulfur compounds instead of sulfate as the fermentation sulfur source and/or αxπprising redesigning and thereby siπplifying the biochemical pathway. In a preferred embodiment of the present invention the reduced sulfur source is hydrogen sulfide, methyl mercaptan or salts thereof. In another preferred embodiment of the present invention there are provided improved methods for such fermentation processes comprising re-designing or modi^ing and thereby simplifying the biochemical pathway. BRIEF DESCRIPTION OF THE DRfiWINGS Figure la is the ccranon biosynthetic pathway to Lysine, Methionine and Threαnine in Esdherichia coli. Figure lb is the Threonine biosynthetic pathway in Esdherichia coli. Figure lc is the Lysine biosynthetic pathway in Esdherichia coli. Figure Id is the Methionine biosynthetic pathway in Esdherichia coli. Figure 2. Variations in the pathways for Msthionine biosynthesis: (1) Transsu_]__i_ιrylation pathway; (2) Sulfhydrylation pathway; (3) MethylsulfhydryJation pathway. DEIAILEP n Srτrr TlO OF THE INVENTION The present invention relates to methods for the fermentation synthesis of methionine and hcmocysteine. To understand why a cost-effective fermentation method for methionine synthesis does not exist, whereas such methods are available for lysine and threonine, it is instructive to consider in more detail the differences among the methionine, lysine, and threonine biosynthetic pathways. All three amino acids are biochemically derived from the same intermediate metabolite, aspartic acid (Fig.1) . In fact, threonine and methionine also share additional biochemical steps and the common intermediate homoserine. But the syntheses diverge substantially when their specific pathway branches are considered (Fig. 1) . These are compared in Table I (J.L. Ingraham, O. Maaloe, and F.C. Neidhardt, "Growth of the Bacterial Cell," pp. 122-135, Sinauer Assoc., Inc., Sunderland, Mass., 1983.) The pathways present in _ coli are chosen as a basis of comparison, recognizing however that there is diversity in these pathways among microbes and plants and that this comparison should in no way be interpreted as limiting the present invention to pathways using Ej. coli. (K.M. Herrmann and R.L. Scmerville, Chapters 9-13 in "Amino Acids: Biosynthesis and Genetic Regulation," Addison- esley Publishing Co., 1983; W.B. Jakoby and O.W. Griffith, Section III.D. in Methods in Enzymology 143. Academic Press, New York, 1987.) Table I Biochemical Bui] ing Blocks Needed to Synthesize Lysine. Threonine. and Methionine Amino Acid Aspartate Pyruvate ATP NADFH 1-C S Lysine 1 1 2 3 0 0 Threonine 1 0 2 2 0 0 Methionine 1 0 7 8 1 1 It is evident from Table I that the biochemical energy requirements for methionine biosynthesis, in terms of adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide phosphate (NADFH) , are about three times higher than for lysine and threonine. This is due to the requirements of sulfate assimilation (J.L. Ingraham, supra.) A total of three moles of ATP and four moles of NftDEH are required to biochemically reduce sulfate to sulfide. Two additional moles of ATP are required, one each to transport sulfate into the cell and to incorporate sulfide into cysteine. It is cysteine, finally, that serves as the sulfur donor in the biosynthesis of methionine (Fig. 1) . In addition, methionine biosynthesis uniquely requires the incorporation of a methyl group (Fig. 1, Table I) . This is derived as 5- methyl-tetrahydrofolate (CH3-THF) frcm the conversion of serine to glycine. Clearly considering the foregoing, the metabolic cost and complexity of synthesizing methionine with sulfate as the sulfur source is much greater than that for lysine or threonine. There is natural diversity among microbes and plants in the biosynthesis of methionine. This is represented schematically by Figure 2 and can be summarized as follcws (K.M. Hermann, supra; W.B. Jakαby, supra; F.C. Neidhardt, Chapter 27 in Escherichia coli and Salmonella typhimurium. American Society for Microbiology, Washington, D.C, 1987; M. Dixαn and E.C. Wfebb, "Enzymes," 3rd edition. Academic Press, New York, 1979; S. Yamagata, Biochimie 71 (1989) 1125-1143): 1) In the methionine biosynthetic pathways of all microbes, homoserine is first activated either by succinyl-OoA (EU. coli and ∑ _ tart imuriuitrt or acetyl-CoA (fungi, yeast, and bacteria such as Brevibacterium and Bacillus) . These reactions are catalyzed by homoserine su∞inyltransferase (EC 2.3.1.46) and homoserine acetyltransferase (EC 2.3.1.31) , respectively. 2) In the methionine biosynthetic pathway of plants, homoserine is activated by ATP in a reaction catalyzed by hcmoserine kinase (EC 2.7.1.39) . The hcmoserine kinase reaction also occurs in microbes, but the resulting O-phosphchαnoserine is an intermediate in threonine, but not methionine, biosynthesis. Thus in plants O-phosphαhcmoserine is the branchpoint between the methionine and threonine pathways, whereas in microbes the tα__andhpoint is hcmoserine. 3) In the microbial trarissulfurylation route to methionine, acylhσmoserine, in reactions catalyzed by O-succiπylhcπioserine (thiol)-lyase (EC 4.2.99.9) and cystathionine β-lyase (EC 4.4.1.8), accepts reduced sulfur frcm cysteine to give hcmocysteine. (O- Su∞inylhomoserine (thiol)-lyase is also kncwn as cystathionine y- synthase.) 4) In the microbial sulfhydrylation route, hcmocysteine is produced directly from acylhcmoserine and sulfide by O-su∞iπylhαmoserine (thiol)-lyase or O-acetylhcmoserine (thiol)-lyase (EC 4.2.99.10). O- acetylhσmoserine (thiol)-lyase is also kncwn as hcmocysteine synthase and methionine synthase. 5) In the microbial methylsulfhydrylation route, methionine is produced directly from acylhcmoserine and methyl mercaptan by O- succinyHαmoserine (thiol)-lyase or O-acetylhcmoserine (thiol)-lyase. 6) The transsulfhydrylatiαn and sulfhydrylatiαn routes in plants are catalyzed by cystathionine γ-syπthase. The plant enzyme cystathionine γ-synthase is distinct from EC 4.2.99.9 and is unique in vising O phosphcho oserine as a substrate. 7) Homoserine is a poor substrate of O-acetylhcsnoserine (thiol)-lyase, except in the case of the enzyme frcm Sc zosarr-harπ yoes pgribe (S. Yamagata, supra) . The methionine biosynthetic enzymes above belong to the group of pyridαxal phosphate-containing enzymes. These are flexible catalysts kncwn to carry out various elimination and replacement reactions. (C. Walsh, Chapter 24 in "Enzymatic Reaction Mechanisms," W.H. Freeman & Co., San Francisco (1979) . Another of this group, tryptophan synthase converts serine and sulfide at a very high rate to cysteine (K. Ishiwata, T. Nakamura, M. Shimada, and N. Makigudhi, "Enzymatic Production of L-cysteine with Tryptophan Synthase of Esdherichia coli r " J. Fermentation and Bioenuineering 67: 169-172, 1989). This reaction is analogous with the reaction of homoserine and sulfide. The various reactions relating to sulfur incorporation and methionine biosynthesis have yet to be considered in the design of a viable fermentation method. The use of sulfide or methyl mercaptan instead of sulfate reduces the metabolic cost of methionine synthesis to the levels of lysine and threonine. In the present invention two ATP and three NADPH are required since the active transport of sulfate, reduction of sulfate, arri synthesis of cysteine are all eliminated. Use of sulfide or methyl mercaptan also reduces the metabolic complexity of methionine biosynthesis since the biosynthesis of cysteine and, in the case of methyl mercaptan, CH3-THF are eliminated. Further simplification is possible and may be desirable by adapting the plant biosynthetic pathway to microbes by methods known to those skilled in the art. Since homoserine kinase is already present as an enzyme functioning in the microbial threonine pathway, this modification requires only introduction of plant cystathionine γ-lyase activity. This cculd be accomplished by structurally .modifying microbial O-acylhcmoserine (thiol)-lyase or by expressing plant cystathionine γ-lyase in the producing microbe. Alternatively, structural modifications could be made in these enzymes or other candidate pyridoxal phosphate enzymes such as tryptophan synthase in order to effectively vise hcmoserine directly as a substrate in sulfur incαrpαratiαn. Or the O- acetylhcmoserine (thiol)-lyase frcm J . pcmbe could be used without modification. While reduced forms of sulfur would be preferred to minimize the requirement for biochemical energy, other more oxidized forms of sulfur are also beneficial. As described above, an improvement through metabolic simplification results whenever sulfide, rather than cysteine, is incorporated directly into hcmoserine or an activated derivative. Thus more oxidized forms such as sulfate, sulfite, and thiosulfate may be provided as sulfur sources and biochemically reduced to sulfide. Sulfite and thiosulfate also diminish the need for biochemical energy relative to sulfate since they are more reduced forms, although the energy requirement is greater than for sulfide or methyl mercaptan. Ey reducing the complexity of the methionine biosynthetic pathway, the engagement of microbial metaboli m in methionine over-production is less extensive. This reduces the number of genetic changes that must be introduced into the producing microbe by classical or genetic engineering methods in order to de-regulate methionine biosynthesis and limits the disruption of microbial metabolism, in general. As used herein, "de¬ regulate" means any effect on the self-regulation of the microbial metabolism for example, any effect on microbial self-regulation by feed-back inhibition or repression. Such de-regulation can be achieved through methods known to those skilled in the art such as for example, classical mutagenesis and selection or genetic engineering. The net result is to transform the methionine biosynthetic pathway to one that compares favorably with those for lysine and threαnine in terms of metabolic cost and complexity. In this way, a feasible fermentation method of methionine production can be realized. EXPERIMENTAL The following disclosure is intended to serve as a representation of embodiments herein, and should not be construed as limiting the scope of this application. Example 1 Methionine Production via Acylhoπoserine (Sulfhydrylation Route) E. coli, C. qlutamicum. and B flavum are de-regulated far hcmoserine over¬ production by classical or genetic engineering methods. The sulfhydrylation route to methionine is introduced into these microbes by transfarming them with plasmid(s) encoding homoserine acetyltransferase, < aσetylhcmoserine (thiol)-lyase, and hcmocysteine methylase. The parent and transformed microbes are cultivated individually in a fermentation medium containing glucose, soy hydrolysate, and inorganic nutrients. The medium is supplemented either with sulfate or sulfide as a source of sulfur for methionine production. Table I indicates the relative amount of methionine that is produced by each strain. Table I Microbe E. coli parent E. coli parent E. coli transformant E. coli transformant C. glutamicum parent C. qlutamicum parent C. qlutamicum transformant C. qlutamicum transformant B. flavum parent B. flavum parent B. flavum transformant B. flavum transformant * low (-) , medium (+) , high (-H-) Example 2 Hbroocysteine Production via Acylhcmoserine (Snlfhyrirylation Route) The parent strains of Exanple 1 are deleted for hcmocysteine methylase activity The microbes are then transformed with plasmid(s) encoding hcmoserine acetyltransferase and 0-acetylhcmoserine (thiol)-lyase. The hcmocysteine methylase negative parent and transformed microbes are cultivated as in Exanple 1. Table II indicates the relative amcuπt of hcmocysteine that is produced by each strain. Table II *A11 strains lack hcmocysteine methylase activity **lσw (-), medium (+) , iύ (++) Example 3 Methionine Production via Acylhcmoserine fM j =t.hγisulfhydrylation Route) The strains of Exanple 2 are cultivated as in Exanple 1 except that methylmercaptan is supplied as the supplemental sulfur source. Table HI indicates the relative amount of methionine that is produced by each strain. Methionine production is indicative of a functioning methylsulfhydrylation pathway. Table III Methionine Microbe* Produced** E. coli parent E. coli transformant C. qlutamicum parent C. qlutamicum transformant B. flavum parent B. flavum transformant *A11 strains lack hcmocysteine methylase activity **lσw (-), hi . (++) Example 4 Methionine Production via Phosphohcmoserine (Sulfhydrylation Route) The parent strains of Exanple 1 are transformed with plasmid(s) encoding homoserine kinase, plant cystathionine 7-synthase and hcmocysteine methylase. The parent and transformed microbes are cultivated as in Exanple 1. Table IV indicates the relative amount of methionine that is produced by each strain. Table 3V Microbe E. coli parent E. coli parent E. coli transformant E. coli transformant C. qlutamicum parent C. qlyrtamicum parent C. qlutamicum transformant C. qlutamicum transformant B. flavum parent B. flavum parent B. flavum transformant B. flavum transformant * low (-), medium (+) , hiφi (++) Example 5 Methionine Production via Riosphchcmoserine (Methylsulfhvdrylation Route) The deleted parent strains of Exanple 2 are transformed with plasmid(s) encodi hcmoserine kinase and plant cystathionine 7-syπthase. The parent and transfar microbes are cultivated as in Exanple 3. Table V indicates the relative amoun of methionine that is produced by each strain. Table V Methionine Microbe* Produced** E. coli parent E. coli transformant ++ C. qlutamicum parent - C. qlutamicum transformant ++ B. flavum parent - B. flavum transformant ++ *A11 strains lack hcmocysteine methylase activity and were supplied with methylmercaptan Example 6 Methionine Production via Hcmoserine (Sulfhydrylation Route) The parent strains of Example 1 are deleted for their hcmoserine acyltransferase activity. The microbes are then transformed with plasmid(s) encoding O-acetylhσmoserine (thiol)-lyase frcm S pcmbe and hαnocysteine methylase. The parent and transformed microbes are cultivated as in Exanple 1. Table VI indicates the relative amount of methionine that is produced by each strain. Table VI Microbe* E. coli parent E. coli parent E. coli transformant E. coli transformant C. qlutamicum parent C. qlvttamicum parent C. qlutamicum transformant C. qlutamicum transformant B. flavum parent B. flavum parent B. flavum transformant B. flavum transformant *A11 strains lack hcmoserine acyltransferase activity **lσw (-), medium (+) , hiφ. (44-) . Example 7 Methionine Production via Hcmoserine (Methylsulfhvdrylation Route) The deleted parent strains of Example 6 are transformed with a plasmid encoding O-acetylhomoserine (thiol)-lyase frcm S po be. The parent and transformed microbes are cultivated as in Example 3. Table VII indicates the relative amount of methionine that is produced by each strain. Table VII Methionine Microbe* Produced** E. coli parent E. coli transformant C. qlutamicum parent C. qlutamicum transformant B. flavum parent B. flavum transformant *A11 strains lack hcmoserine acyltransferase activity **low (-), high (++)

Claims: WHAT IS CLAIMED IS: 1. A method for enhancing methionine production in a fermentation process of microbial cell by modifying the methionine biosynthetic pathway of said cell ccmprising the steps of: i. transforming or transducing a hσmoserine-activating enzyme gene fragment capable of expressing said hcmoserine-activating enzyme and a su___^ur-incαrpαrating enzyme gene fragment capable of eaqpressing said sulf enzyme into said cell; ii. growing said cell under such conditions that transformation or transductiαn of both gene enzymes are effected; iii. recovering a transformed or transduced cell; and iv. adding an exogenous sulfur compound other than cysteine or methioni to said transformed or transduced cell as the sulfur source for methionin production. 2. The method of claim 26 wherein said exogenous sulfur compound is a reduced sulfur coπpαund consisting of hydrogen sulfide, methyl mercaptan or a salt thereof. 3. The method of claim 26 wherein said exogenous sulfur compound is an oxidiz sulfur ccmpound consisting of sulfate, sulfite or thiosulfate. 4. The method of claim 27 or 28 wherein said hσmoserine-activating enzyme is selected frcm the group consisting of homoserine kinase, homoserine acetyltransferase and hcmoserine succiπyltransferase. 5. The method of claim 27 or 28 wherein said sulfur-incorporating enzyme is selected frcm the group consisting of O-succinylhomoserine (thiol)-lyase, O- acetylhcmoserine (thiol)-lyase and plant cystathionine gamma synthase. 6. The method of claim 27 viierein said sulfur-incorporating enzyme converts homoserine and said hydrogen sulfide or a salt thereof directly to hcmocysteine 7. The method of claim 27 wherein said svilfur-incorporating enzyme converts homoserine and said methyl mercaptan or a salt thereof directly to methionine. 8. The method of claim 28 wherein said sulfur-incorporating enzyme converts hcmoserine directly to hcmocysteine. 9. A method for enhancing hcmocysteine production in a fermentation process a microbial cell by modifying the methionine biosynthetic pathway of said cell comprising the steps of: i. transforming or transducing a hαmoserine-activating enzyme gene fragment capable of expressing any said hcmoserine-activating enzyme but not including hcmocysteine methylase, and a svαfur-incαrporating enzyme gene fragment capable of expressing said sulfur enzyme into said cell; ii. growing said cell under such conditions that transformation or transduction of both gene enzyme fragments are effected; iii. recovering a transformed or transduced cell; and iv. adding an exogenous sulfur compound other than methionine or cyste to the said transformed or transduced cell as the sulfur source for hcmocysteine production. 10. The method of claim 34 wherein said exogenous sulfur cαπpound is a reduce sulfur compound consisting of hydrogen sulfide, or a salt thereof. 11. The method of claim 34 wherein said exogenous sulfur compound is an oxidi sulfur compound consisting of sulfate, sulfite or thiosulfate. 12. The method of claim 35 or 36 wherein said hαmoserine-activating enzyme is selected frcm the group consisting of homoserine kinase, homoserine acetyltransferase and hcmoserine succinyltransferase. 13. The method of claim 35 or 36 wherein said sulfur-incorporating enzyme is selected frcm the group consisting of O-succinylhαmoserine (thiol)-lyase, o- acetylhcmoserine (thiol)-lyase and plant cystathionine gamma synthase. 14. The method of claim 35 wherein said sxilfur-incorporating enzyme converts hcmoserine and said hydrogen sulfide or a salt thereof directly to hαmocystein 15. The method of claim 36 wherein said sulfur-incorporating enzyme converts hcmoserine directly to hcmocysteine. 16. The method of claim 26 or 34 wherein said transformed or transduced cell produces an amino acid that is greater than said amino acid of a non-transforme or transduced cell. 17. The method of claim 26 or 34 vftierein said transformed or transduced cell i selected frcm the group consisting of CbryneJaσteria, Brevibacteria or Escherichia coll. [received by the International Bureau on 3 August 1993 (03.08.93); original claims 2-8, 10 and 17 amended; other claims unchanged (3 pages)] 1. A method for enhancing methionine production in a fermentation process of microbial cell by modifying the methionine biosynthetic pathway of said cell ocmprising the steps of: i. transforming αr transducing a hcmoserine-activating enzyme gene fragment capable of expressing said hcmoserine-activating enzyme and a sulfur-inσαrporating enzyme gene fragment capable of expressing said sul enzyme into said cell; ii. growing said cell under such conditions that transformation or transduction of both gene enzymes are effected; iii. recovering a transformed αr transduced cell; and iv. adding an exogenous sulfur compound other than cysteine αr methion to said transfarmed ar transduced cell as the sulfur source for methioni production. 2. The method of claim 1 wherein said exogenous sulfur compound is a reduce sulfur compound consisting of hydrogen sulfide, methyl mercaptan or a salt thereof. 3. The method of claim 1 wherein said exogenous sulfur compound is an oxidi sulfur compound consisting of sulfate, sulfite αr thiosulfate. 4. The method of claim 2 αr 3 wherein said hcmoserine-activating enzyme is selected frcm the group consisting of hcmoserine kinase, hcmoserine acetyltransferase and hcmoserine succiπyltransferase. 5. The method of claim 2 or 3 wherein said sulfur-incorporating enzyme is selected frcm the group consisting of O-suσciπylhcπoserine (thiol)-lyase, O- acetylhcπoserine (thiol)-lyase and plant cystathionine gamma synthase. 6. The method of claim 2 wherein said sulfur-incorporating enzyme converts hcmoserine and said hydrogen sulfide αr a salt thereof directly to hcmocystein 7. The method of claim 2 vtoerein said sulfur-incorpαrating enzyme converts hcmoserine and said methyl mercaptan αr a salt thereof directly to methionine. 8. The method of claim 3 wherein said sulfur-incorporating enzyme converts hcmoserine directly to hcmocysteine. 9. A method far enhancing hcmocysteine production in a fermentation process a microbial cell by modifying the methionine biosynthetic pathway of said cell ocmprising the steps of: i. transforming αr transducing a hcmoserine-activating enzyme gene fragment capable of expressing any said hcmoserine-activating enzyme but not including hcmocysteine methylase, and a sulfur-incαrpαrating enzyme gene fragment capable of expressing said sulfur enzyme into said cell; ii. growing said cell under such conditions that transformation αr transduction of both gene enzyme fragments are effected; iii. recovering a transformed αr transduced cell; and iv. adding an exogenous sulfur ccmpound other than methionine or cyste to the said transformed αr transduced cell as the sulfur source for hcmocysteine production. 10. The method of claim 9 wherein said exogenous sulfur ccmpound is a reduce sulfur ccmpound consisting of hydrogen sulfide, αr a salt thereof. 11. The method of claim 9 wherein said exogenous sulfur ccmpound is an oxidi sulfur ccmpound consisting of sulfate, sulfite αr thiosulfate. 12. The method of claim 10 αr 11 wherein said hcmoserine-activating enzyme is selected frcm the group consisting of hcmoserine kinase, hcmoserine acetyltransferase and hcmoserine succinyltransferase. 13. The method of claim10 or11 wherein said sulfur-incorporating enzyme is selected from the group consisting of O-succinyhcmoserine (thiol)-lyase, O- acetylhc oβerine (thiol)-lyase and plant cystathionine gamma synthase. 14. The method of claim 10 wherein said sulfur-incαrpαrating enzyme converts hcmoserine and said hydrogen sulfide αr a salt thereof directly to hcmocystein 15. The method of claim11 wherein said sulfur-incorporating enzyme converts homoserine directly to hcmocysteine. 16. The method of claim 1 αr 9 wherein said transformed or transduced cell produces an amino acid that is greater than said amino acid of a non-transfarm or transduced cell. 17. The method of claim 1 or 9 wherein said transformed αr transduced cell selected from the group consisting of Cttrynefcβc±eria, BreviJbacteria αr ∑scherichia coli.

Inventors: LIEVENSE JEFFERSON CLAY, LIEVENSE, JEFFERSON, CLAY
Applicants: GENENCOR INT, GENENCOR INTERNATIONAL, INC.
Application year: 1993
Main classification: C12P13
IPC codes: C12N9, C12N15, C12P13