WO1993017112A1

WO1993017112A1 - Biosynthesis of methionine using a reduced source of sulfur

Info

Publication number: WO1993017112A1
Application number: PCT/US1993/001351
Authority: WO
Inventors: Jefferson Clay Lievense
Original assignee: Genencor International, Inc.
Priority date: 1992-02-20
Filing date: 1993-02-16
Publication date: 1993-09-02
Also published as: EP0630406A1; CA2130347A1; JPH07503855A

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 SOURCE OF 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-methionine 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 L-Hcmoserine-resistant mutant of Escheridhia coli. " 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 econcmical fermentation production of methionine is possible.

SUMMfiRY 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 homocysteine synthesis comprising the vise of reduced sulfur compounds instead of sulfate as the fermentation sulfur source and/or comprising redesigning and thereby simplifying 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 modifying and thereby simplifying the biochemical pathway.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure la is the ccranon biosynthetic pathway to Lysine, Methionine and

Threonine in Esdherichia coli.

Figure 1b is the Threonine biosynthetic pathway in Esdherichia coli.

Figure 1c is the Lysine biosynthetic pathway in Esdherichia coli.

Figure 1d is the Methionine biosynthetic pathway in Esdherichia coli.

Figure 2. Variations in the pathways for Msthionine biosynthesis: (1) Transsulfurylation pathway; (2) Sulfhydrylation pathway; (3)

MethylsulfhydryJation pathway.

DETAILED DESCRPTlON OF THE INVENTION

The present invention relates to methods for the fermentation synthesis of methionine and homocysteine. 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 E. 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 E. coli. (K.M. Herrmann and R.L. Scmerville, Chapters 9-13 in "Amino Acids:

Biosynthesis and Genetic Regulation," Addison-Wesley 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 Building 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) from 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. Dixon 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-CoA (EU. coli and S. typhimurium) or acetyl-CoA (fungi, yeast, and bacteria such as Brevibacterium and

Bacillus). These reactions are catalyzed by homoserine

succinyltransferase (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 homoserine kinase (EC 2.7.1.39). The homoserine kinase reaction also occurs in microbes, but the resulting O-phosphohonoserine is an intermediate in threonine, but not methionine, biosynthesis. Thus in plants O-phosphohomoserine is the branchpoint between the methionine and threonine pathways, whereas in microbes the brandhpoint is homoserine.

3) In the microbial trarissulfurylation route to methionine,

acylhomoserine, in reactions catalyzed by O-succinylhomoserine

(thiol)-lyase (EC 4.2.99.9) and cystathionine β-lyase (EC 4.4.1.8), accepts reduced sulfur frcm cysteine to give homocysteine. (O- Succinylhomoserine (thiol)-lyase is also known as cystathionine γ- synthase.)

4) In the microbial sulfhydrylation route, homocysteine is produced

directly from acylhomoserine and sulfide by O-succinylhomoserioe (thiol)-lyase or O-acetylhomoserine (thiol)-lyase (EC 4.2.99.10). O- acetylhomoserine (thiol)-lyase is also known as homocysteine synthase and methionine synthase. 5) In the microbial methylsulfhydrylation route, methionine is produced directly from acylhomoserine and methyl mercaptan by O- succinylhomoserine (thiol)-lyase or O-acetylhomoserine (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 phosphchomoserine as a substrate.

7) Homoserine is a poor substrate of O-acetylhosnoserine (thiol)-lyase, except in the case of the enzyme from Schizosaccharomyoes pombe (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," J. Fermentation and Bioengineering 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-acylhomoserine (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 homoserine directly as a substrate in sulfur incαrpαratiαn. Or the O-acetylhomoserine (thiol)-lyase from S. pombe 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 homoserine 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 metabolism 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 homoserine 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, O-aσetylhomoserine (thiol)-lyase, and homocysteine 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

Methionine

Microbe Sulfur Source Produced*

E. coli parent sulfate -

E. coli parent sulfide -

E. coli transformant sulfate +

E. coli transformant sulfide ++

C. glutamicum parent sulfate -

C. glutamicum parent sulfide -

C. glutamicum transformant sulfate +

C. glutamicum transformant sulfide ++

B. flavum parent sulfate -

B. flavum parent sulfide -

B. flavum transformant sulfate +

B. flavum transformant sulfide ++

* low (-), medium (+), high (++) Example 2

Homocysteine Production via Acylhomoserine

(Sulfhydrylation Route)

The parent strains of Exanple 1 are deleted for homocysteine methylase activity

The microbes are then transformed with plasmid(s) encoding homoserine

acetyltransferase and 0-acetylhomoserine (thiol)-lyase. The homocysteine methylase negative parent and transformed microbes are cultivated as in Example

1. Table II indicates the relative amcuπt of homocysteine that is produced by each strain.

Table II

Homocysteine

Microbe* Sulfur Source Produced**

E. coli parent sulfate -

E. coli parent sulfide -

E. coli transformant sulfate +

E. coli transformant sulfide ++

C. glutamicum parent sulfate -

C. glutamicum parent sulfide -

C. glutamicum transformant sulfate +

C. glutamicum transformant sulfide ++

B. flavum parent sulfate -

B. flavum parent sulfide -

B. flavum transformant sulfate +

B. flavum transformant sulfide ++

*A11 strains lack homocysteine methylase activity

**lσw (-), medium (+), high (++)

Example 3

Methionine Production via Acylhomoserine

(Methylsulfhydrylation Route)

The strains of Exanple 2 are cultivated as in Exanple 1 except that

methylmercaptan is supplied as the supplemental sulfur source. Table III 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. glutamicum parent

-

C. glutamicum transformant ++

B. flavum parent

-

B. flavum transformant ++

*A11 strains lack homocysteine methylase activity

**lσw (-), high (++)

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 homocysteine 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 IV

Methionine

Microbe Sulfur Source Produced*

E. coli parent sulfate -

E. coli parent sulfide -

E. coli transformant sulfate +

E. coli transformant sulfide ++

C. glutamicum parent sulfate -

C. glyrtamicum parent sulfide -

C. glutamicum transformant sulfate +

C. glutamicum transformant sulfide ++

B. flavum parent sulfate -

B. flavum parent sulfide -

B. flavum transformant sulfate +

B. flavum transformant sulfide ++

* low (-), medium (+), high (++) Example 5

Methionine Production via Phosphohomoserine

(Methylsulfhydrylation Route)

The deleted parent strains of Exanple 2 are transformed with plasmid(s) encodi homoserine kinase and plant cystathionine 7-synthase. 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. glutamicum parent -

C. glutamicum transformant ++

B. flavum parent -

B. flavum transformant ++

*All strains lack homocysteine methylase activity and were

supplied with methylmercaptan

**lσw (-), high (++)

Example 6

Methionine Production via Homoserine

(Sulfhydrylation Route)

The parent strains of Example 1 are deleted for their homoserine

acyltransferase activity. The microbes are then transformed with

plasmid(s) encoding O-acetylhσmoserine (thiol)-lyase from S. pombe 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

Methionine

Microbe* Sulfur Source Produced**

E. coli parent sulfate -

E. coli parent sulfide -

E. coli transformant sulfate +

E. coli transformant sulfide ++

C. glutamicum parent sulfate -

C. glvttamicum parent sulfide -

C. glutamicum transformant sulfate +

C. glutamicum transformant sulfide ++

B. flavum parent sulfate -

B. flavum parent sulfide -

B. flavum transformant sulfate +

B. flavum transformant sulfide ++

*A11 strains lack homoserine acyltransferase activity

**lσw (-), medium (+), high (++) 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. pombe. 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. glutamicum parent -

C. glutamicum transformant ++

B. flavum parent -

B. flavum transformant ++

*A11 strains lack homoserine 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 homoserine-activating enzyme gene fragment capable of expressing said homoserine-activating enzyme and a sulfur-incorporating enzyme gene fragment capable of eaqpressing said sulfur 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 methionine to said transformed or transduced cell as the sulfur source for methionine 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 oxidized 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 homoserine succiπyltransferase.

5. The method of claim 27 or 28 wherein said sulfur-incorporating enzyme is selected from the group consisting of O-succinylhomoserine (thiol)-lyase, O-acetylhomoserine (thiol)-lyase and plant cystathionine gamma synthase.

6. The method of claim 27 wherein said sulfur-incorporating enzyme converts homoserine and said hydrogen sulfide or a salt thereof directly to homocysteine.

7. The method of claim 27 wherein said sulfur-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 homoserine directly to homocysteine.

9. A method for enhancing homocysteine 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 homoserine-activating enzyme gene fragment capable of expressing any said homoserine-activating enzyme but not including homocysteine 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 cysteine to the said transformed or transduced cell as the sulfur source for homocysteine production.

10. The method of claim 34 wherein said exogenous sulfur cαπpound is a reduced sulfur compound consisting of hydrogen sulfide, or a salt thereof.

11. The method of claim 34 wherein said exogenous sulfur compound is an oxidized sulfur compound consisting of sulfate, sulfite or thiosulfate.

12. The method of claim 35 or 36 wherein said homoserine-activating enzyme is selected from the group consisting of homoserine kinase, homoserine

acetyltransferase and homoserine succinyltransferase.

13. The method of claim 35 or 36 wherein said sulfur-incorporating enzyme is selected frcm the group consisting of O-succinylhomoserine (thiol)-lyase, O-acetylhomoserine (thiol)-lyase and plant cystathionine gamma synthase.

14. The method of claim 35 wherein said sxilfur-incorporating enzyme converts homoserine and said hydrogen sulfide or a salt thereof directly to homocystein

15. The method of claim 36 wherein said sulfur-incorporating enzyme converts homoserine directly to homocysteine.

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-transformed or transduced cell.

17. The method of claim 26 or 34 vftierein said transformed or transduced cell is selected frcm the group consisting of Corynebacteria, 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 homoserine-activating enzyme gene fragment capable of expressing said homoserine-activating enzyme and a sulfur-inσαrporating 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 enzymes are effected;

iii. recovering a transformed αr transduced cell; and

iv. adding an exogenous sulfur compound other than cysteine αr methionine to said transfarmed ar transduced cell as the sulfur source for methionine production.

2. The method of claim 1 wherein said exogenous sulfur compound is a reduced 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 oxidized sulfur compound consisting of sulfate, sulfite αr thiosulfate.

4. The method of claim 2 αr 3 wherein said homoserine-activating enzyme is selected frcm the group consisting of homoserine kinase, homoserine

acetyltransferase and homoserine 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-acetylhomoserine (thiol)-lyase and plant cystathionine gamma synthase.

6. The method of claim 2 wherein said sulfur-incorporating enzyme converts homoserine and said hydrogen sulfide αr a salt thereof directly to homocysteine.

7. The method of claim 2 vtoerein saad sulfur-incorpαrating enzyme converts homoserine and said methyl mercaptan αr a salt thereof directly to methionine.

8. The method of claim 3 wherein said sulfur-incorporating enzyme converts homoserine directly to homocysteine.

9. A method far enhancing homocysteine production in a fermentation process of a microbial cell by modifying the methionine biosynthetic pathway of said cell ocmprising the steps of:

i. transforming αr transducing a homoserine-activating enzyme gene fragment capable of expressing any said homoserine-activating enzyme but not including homocysteine methylase, and a sulfur-incαrpαrating enzyme gene fragment capable of expressing said sulfur enzyme into said cell;

iii. recovering a transformed αr transduced cell; and

iv. adding an exogenous sulfur ccmpound other than methionine or cysteine to the said transformed αr transduced cell as the sulfur source for homocysteine production.

10. The method of claim 9 wherein said exogenous sulfur ccmpound is a reduced sulfur ccmpound consisting of hydrogen sulfide, αr a salt thereof.

11. The method of claim 9 wherein said exogenous sulfur ccmpound is an oxidize sulfur ccmpound consisting of sulfate, sulfite αr thiosulfate.

12. The method of claim 10 αr 11 wherein said homoserine-activating enzyme is selected from the group consisting of homoserine kinase, homoserine

acetyltransferase and homoserine succinyltransferase.

13. The method of claim 10 or 11 wherein said sulfur-incorporating enzyme is selected from the group consisting of O-succinylhomoserine (thiol)-lyase, O-acetylhomoserine (thiol)-lyase and plant cystathionine gamma synthase.

14. The method of claim 10 wherein said sulfur-incαrpαrating enzyme converts homoserine and said hydrogen sulfide αr a salt thereof directly to homocysteine.

15. The method of claim 11 wherein said sulfur-incorporating enzyme converts homoserine directly to homocysteine.

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 Coryneba cteria, Brevibacteraia αr

Escherichia coli.