JPH07503855A - Methionine biosynthesis using a sulfur reducing source - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] A method characterized by comprising each step.

2. The exogenous sulfur compound is hydrogen sulfide, methyl mercaptan, or The method according to claim 1, which is a reduced sulfur compound consisting of a salt of Law.

3. The exogenous sulfur compound is an acid consisting of sulfate, sulfide or thiosulfate. The method according to claim 1, characterized in that it is a sulfur compound.

4. The homoserine activating enzyme is homoserine kinase, homoserine acetyl A group consisting of transferases and homoserine succinyltransferases The method according to claim 2 or 3, characterized in that the method is selected from:

5. The sulfur uptake enzyme is 0-succinylhomoserine (thiol)-rea lyase, 0-acetylhomoserine (thiol)-lyase and plant cismethionine Claim 2, characterized in that it is selected from the group consisting of gamma synthase. Or the method described in Section 3.

6. The sulfur uptake enzyme homoserine and hydrogen sulfide or their salts The method according to claim 2, characterized in that it is directly converted to cysteine.

7. The sulfur uptake enzyme is homoserine and methyl mercaptan or The method according to claim 2, characterized in that the salt of methionine is directly converted into methionine. .

8. The sulfur uptake enzyme directly converts homoserine to homocysteine. A method according to claim 3, characterized in that:

9. By changing the methionine biosynthesis pathway of microbial cells, A method for increasing the production of homocysteine in a fermentation process, the method comprising: i) homocysteine; Homoserine activating enzymes that can express homoserine activating enzymes other than methylase The gene fragment and the sulfur uptake enzyme gene fragment capable of expressing the sulfur uptake enzyme are ii) transform or transduce into the microbial cell, growing the microbial cell under conditions that permit transformation or transduction of the microorganism; 1ii) Collect the transformed or transduced microbial cells, i. v) Other than cysteine and methionine as a sulfur source for producing homocystine the transformed microbial cells or transduced microorganisms with an exogenous sulfur compound of add to cells, A method characterized by comprising each step.

10. Reductive sulfurization in which the exogenous sulfur compound is hydrogen sulfide or a salt thereof 10. The method according to claim 9, which is a compound.

11. The exogenous sulfur compound is an acid consisting of sulfate, sulfide or thiosulfate. 10. The method according to claim 9, which is a sulfur compound.

12. The homoserine activating enzyme is homoserine kinase, homoserine acetyl A group consisting of transferases and homoserine succinyltransferases The method according to claim 10 or 11, characterized in that the method is selected from .

13. The sulfur uptake enzyme is 0-succinylhomoserine (thiol)-rea lyase, 0-acetylhomoserine (thiol)-lyase and plant cismethionine Claim 10, characterized in that it is selected from the group consisting of gamma synthase. or the method described in paragraph 11.

14. The sulfur uptake enzyme homocytes homoserine and hydrogen sulfide or its salts. 11. A method according to claim 10, characterized in that it is directly converted into tin.

15. The sulfur uptake enzyme directly converts homoserine to homocysteine. 12. The method according to claim 11, characterized in that:

16. The transformed microbial cell or transduced microbial cell is transformed Amino acids greater than those in microbial cells that do not transduce or transduce microbial cells 10. The method according to claim 1 or 9, characterized in that an acid is produced.

17. The transformed microbial cell or transduced microbial cell is selected from the group consisting of Brevibacteria, Brevibacteria or Escherichia coli. 10. A method according to claim 1 or claim 9, characterized in that:

Specification Methionine is an essential amino acid in animal diets and is a food and feed supplement. widely used. Methionine has traditionally been commonly used as acrolein, methyl mer captan, and various multistep chemical syntheses using cyanide as starting materials. (H. H. Zumant, “Organic Building Blocks of the Chemical Industry”) (Building Blocks) J 182 pages, Joan Willie and Sons, New York, 1989). The resulting product has two forms , that is, D,L-methionine and its hydroxy analog Ru. Unlike all other amino acids, D-methionine has a preferred L converted into shape. As a result, chemical synthesis is feasible and cost-effective in this case. Good rate. Note that chemical synthesis usually produces a mixture of the L-type and the L-type.

However, fermentation production, which is a common way to produce many low-cost amino acids, method is not applicable in the case of methionine (K, Aida, ■, Chibata, K, Naka Yama, K. Takinami, and H. Yamada, “Biotechnology of Amino Acid Production. ”, Advances in Industrial Microbiology 24, El Sepia-11986). Biochemistry related essentials Both the amino acids lysine and threonine are found in molasses, starch hydrolysates, and corona. production using cheap raw materials such as tea liquor and soybean hydrolyzate. This is surprising given that it can be produced cost-effectively by fermentation methods. (For example, P, L, Rogers, R, G, Kyle, D, F, Mitsushiri-1 and C. Fryer, “Prospects for lysine production in Australia”, Aus. Tralia Food Technology 38, pp. 514-518, 1986, and Furusawa, S. , A. Ozaki, and T. Nakanishi, “Escherichia coli aspartic acid and L. -Threonine Production by Homoserine-Resistant Mutants”, Applied Microbiology and Biotechnology 29, pp. 550-553, 1988).

Various microorganisms have been used to produce L-lysine and L-threonine.

These microorganisms can be developed using traditional methods using mutagenesis and selection as well as genetic It has been developed through engineering (K., Ida, supra). Historically, Corynebacteria Although great success has been achieved using Brevibacteria and Brevibacteria, large intestine It is clear that other microorganisms such as fungi can also be used. Methioni The complexity and substitution of methionine biosynthesis has been reduce metabolic costs and ideally reduce them to the same extent as for lysine or threonine. A method is needed to do so.

Summary of the invention The present invention proposes the use of reduced sulfur compounds instead of sulfates as fermentation sulfur sources. contain and/or redesign a biochemical pathway, thereby simplifying that biochemical pathway. The present invention provides a viable fermentation method for synthesizing methionine, including Ru. The present invention also utilizes reduced sulfur compounds instead of sulfates as fermentation sulfur sources. and/or redesign a biochemical pathway, thereby improving its biochemical pathway. provides a fermentation method for synthesizing homocystine that be. In a preferred embodiment of the invention, the reduced sulfur source is hydrogen sulfide, methyl sulfide, lucaptan or their salts.

In another preferred embodiment of the invention, redesigning or altering biochemical pathways; thereby providing a method for improving the fermentation process, including simplifying its biochemical pathways. Ru.

Brief description of the drawing Figure 1a shows the normal biosynthesis of lysine, methionine and threonine in E. coli. It is a diagram showing a route.

FIG. 1b is a diagram showing the threonine biosynthesis pathway in E. coli.

FIG. 1c is a diagram showing the ricin biosynthesis pathway in E. coli.

FIG. 1d is a diagram showing the methionine biosynthesis pathway in E. coli.

Figure 2 shows the various pathways of methionine biosynthesis: (1)) Lance sulfurylation Pathway; (2) Sulfhydrylation pathway; (3) Methyl sulfhydryl sheath FIG.

The present invention relates to a method for fermentative synthesis of methionine and homocystine.

While cost-effective fermentation methods for methionine synthesis do not exist, lysine and To understand why there is such a method for methionine and threonine, To take a closer look at the differences in the biosynthetic pathways of nin, lysine, and threonine. is beneficial. Are all three amino acids the same intermediate metabolite (aspartic acid)? (Figure 1). In fact, threonine and methionine are They share a chemical process and a common intermediate, homoserine. But those Synthesis becomes quite useful when considering the branching of a particular pathway (Figure 1). Table these Compare to 1 (J. L. Ingraham, 0. Marlow, and F. C. Nee. Dohard, “Growth of Bacterial Cells J,” pp. 122-135, Massachusetts. Sunderland, Sunderland, Shinara Associates Ltd., 1983). present in E. coli The existing pathways in microorganisms and plants are selected as the basis for comparison. There are differences between the routes, and this comparison limits the invention to the E. coli route. (K, M, Berman and R, L, Summer) Bill, “Amino Acids: Biosynthesis and Genetic Regulation” Chapters 9-13, Ajiso Wesley Publishing, 1983. W, B, Jacopee and O, W, Griffey. Section II1 of Methods in Enzymology 143.　D　　　,Academic Cup Les, New York, 1987).

Ricin 1 12300 Threonine 1 02200 Methionine 1 07811 Adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide Biochemical energy requirements for methionine biosynthesis regarding phosphoric acid (NADPH) It is about 3 times larger than those of Rishin and Threonine. This is a necessary condition for sulfate assimilation. (J.L. Ingraham, supra). 3 moles of ATP and 4 moles of NADPH is required to biochemically reduce sulfate to sulfide.

1 mole each to transport sulfate into the cell and incorporate sulfide into cysteine. ATP, ie 2 more moles of ATP are required. Finally, methionine Cysteine functions as a sulfur donor in the biosynthesis of cystein (Fig. 1).

Furthermore, methionine biosynthesis uniquely requires the incorporation of a methyl group (the Figure 1, Table ■). This methyl group is converted to 5-methyl- by conversion of serine to glycine. Originated as tetrahydrofolic acid (CH3-THF). Considering the above Clearly, the metabolic costs and metabolic costs of synthesizing methionine with sulfate as a sulfur source The complexity is much greater than that of lysine or threonine.

There is natural diversity among microorganisms and plants in methionine biosynthesis. this child is schematically represented in Figure 2 and can be summarized as follows (K, M, barma N, supra; W, B,') Jacobi-1 supra, F, C, Needhard, Escherichia coli and Typhimurium Chapter 27, American Society for Microbiology, Washington D.C., 19 B? , M. Dixton and E. C. Webb, "Enzymes", 3rd edition, Akademitsu. Kupres, New York, 1979. S, Yamagata, Paiokimi71, (19 89) 1125-1143) 1) In the methionine biosynthesis pathway of all microorganisms. and succinyl-CoA (E. coli and murine Salmonella) or acetyl-CoA. Ru-CoA (bacteria such as Brevibacterium and Bacillus) homoserine is first activated by either Ru. These reactions each involve homoserine succinyltransferase (EC 2,3,1,46) and homoserine acetyltransferase (EC2,3 , 1, 31).

2) In the plant methionine biosynthesis pathway, homoserine kinase (EC2,7 , 139) homoserine is activated by ATP.

The homoserine kinase reaction also occurs in microorganisms, but the O-phosphorus produced Homoserine is an intermediate in threonine synthesis rather than methionine synthesis.

Therefore, in plants, 0-phosphohomoserine is linked to the methionine pathway and It is a branching point between the nin pathway, and in all living organisms, the branching point is homoserine.

3) In the microbial transsulfurylation pathway to methionine, O- Succinylhomoserine (thiol)-lyase (EC4,2,99,9) and The reaction triggered by cismethionine β-lyase (EC4,4,1,8) The acyl homoserine that produces homoserine accepts reduced sulfur from cysteine and generates homoserine. do. (0-succinylhomoserine (thiol)-lyase is cystathionine γ -Also known as synthase. )4) Microbial sulfhydration Homocystine is 0-succinylhomoserine (thiol) -ase or O-acetylhomoserine (thiol)-lyase (EC4,2,99 , 10) directly from acylhomoserine and sulfide. 0-acety Lehomoserine (thiol)-lyase is homocysteine synthase and methyl Also known as onine synthase 5) Microbial methyl sulfhydrylase In the cation pathway, methionine is converted into 0-succinylhomoserine (thiol)- lyase or O-acetylhomoserine (thiol)-lyase. Produced directly from moserin and methyl mercaptan.

6) Trans-sulfhytylation and sulfhytylation in plants tion is triggered by cystathionine γ-synthase. plant enzyme cystachio Nin γ-synthase is completely different from EC4,2,99,9, and has a substrate It is unique in the use of 0-phosphohomoserine as

7) Except for enzymes from fission yeast pump (poa+be), homocerate is an essential component of O-acetylhomoserine (thiol)-lyase (S, Yama). Gata, supra).

The methionine biosynthetic enzymes mentioned above belong to the group of pyridoxal phosphate-containing enzymes.

These enzymes are soft enzymes known to perform a variety of removal and substitution reactions. It is a flexible catalyst (C. Walsh, "Enzyme Reaction Mechanism", Chapter 24, W. .

H., Freeman & Company, San Francisco (1979)').

Another enzyme in the group mentioned above, tryptophan synthase, is found in very high proportions in serine. Converts carbon and sulfide to cysteine (K, Ishiwata, T, Nakamura, M, Shimada) , and N. Makiguchi, “The tryptophan synthase system of Escherichia coli” "Enzyme production of phosphorus", J1 Fermentation and Biotechnology 67.169-], 72.1989 ). This reaction is similar to that of homoserine and sulfide.

In designing a viable fermentation method, the link between sulfur uptake and methionine biosynthesis should be considered. Various reactions are still being considered. Sulfide or Methyl Mel instead of Sulfate By using captan, the metabolic cost of methionine synthesis can be reduced to lysine and threo. reduce to the level of nin. In the present invention, active transport of sulfate, reduction of sulfate, Since all original and cysteine synthesis is removed, two ATP and three Does not require NADPHL.

By using sulfide or methyl mercaptan, cysteine biosynthesis and In the case of methyl mercaptan, CH3-THF biosynthesis is deleted. , the metabolic complexity of methionine biosynthesis is reduced. Further simplification is possible. , by adapting plant biosynthetic pathways to microorganisms by methods known to those skilled in the art. Naturalization would be desirable. Homoserine kinase functions in the microbial threonine pathway This modification involves the use of plant cystathionine γ- Only lyase activity is required. This suggests that the microbial O-acylhomose is structurally by modifying phospho(thiol)-lyases or when producing microorganisms. This is done by expressing plant cystathionine γ-lyase. or , to effectively use homoserine directly as a substrate for sulfur uptake. These enzymes or other candidates such as tryptophan synthase Structural changes are made in the monkey phosphate enzyme. Or fission yeast pump Using O-acetylhomoserine (thiol)-lyase from You can also do it.

Although the reduced form of sulfur is preferred to minimize biochemical energy requirements, sulfur Other more oxidized forms of are also useful. As mentioned above, than cysteine Rather, when sulfide is incorporated directly into homoserine or activated derivatives, Improvements will also be made by simplifying the metabolism. Therefore, sulfates, sulfites, More oxidized forms such as sulfur and thiosulfates may be provided as sulfur sources May be chemically reduced to sulfide. Sulfite and thiosulfate are less reduced than sulfate. They also reduce the need for biochemical energy relative to sulfate. less, but its energy requirements are lower than that of sulfides or methyl mercaptans. It's also big.

Reduces methionine overproduction by reducing the complexity of the methionine biosynthetic pathway The involvement of microbial metabolism in this process is reduced. Because of this and to de-regulate methionine biosynthesis. must be introduced into the producing microorganism by systematic or genetic engineering methods. Reduce the number of genetic changes and generally limit disruption of microbial metabolism. used here "Deregulation" refers to any influence on the autoregulation of microbial metabolism (e.g. (any effect on microbial self-regulation by back-inhibition or suppression) . Such deregulation can be achieved by, for example, traditional mutagenesis and selection or genetic engineering. This can be done by methods known to those skilled in the art, such as those skilled in the art.

The end result is that the methionine biosynthetic pathway is compared to lysine and lysine in terms of metabolic cost and complexity. The goal is to switch to a biosynthetic pathway that is favorable and comparable to that of threonine and threonine. . In this way, a viable fermentation method for methionine production can be realized.

X-ray The following disclosure is intended to represent embodiments and is intended to limit the scope of this application. It should not be thought of as something to do.

Example 1 Production of methionine by acyl homoserine (sulfhydration pathway) Overproduction of homoserine has been largely investigated by traditional or genetic engineering methods. It deregulates C. enterica, C. glutamicularia, and B. flavus. child These microorganisms were treated with homoserine acetyltransferase, 0-acetyl homoserine Encodes serine (thiol)-lyase and homocysteine methylase sulfhydrylase to methionine by transforming with a plasmid that tion pathway into these microorganisms. Parent organism and transformed microorganisms , in a fermentation medium containing glucose, soybean hydrolysate, and inorganic nutrients. Cultivate this. Sulfate or sulfide as a source of sulfur for methionine production Supplement the medium with either Table ■ shows the relative amount of methionine produced by each strain. It shows the amount.

Escherichia coli mother body sulfate − Escherichia coli mother body sulfide − Escherichia coli transformant sulfate 10 E. coli transformant sulfide ++ C, glutaa+icum parent sulfate -C, glutaa+icum parent Sulfide -C, glutaaiicum transformant Sulfate -C, glut amicum transformant sulfide ++ B, flavui + parent sulfate − B, flavum matrix sulfide - B, flavui transformant sulfate B, flavum transformant sulfide ++＊ Less (-), Medium (+), More (++) Example 2 Production of homocysteine by acylhomoserine (sulfhydration pathway) The parent strain of Example 1 lacks homocysteine methylase activity. Next II X, homoserine acetyltransferase and O-acetylhomoserine Transforming the above microorganism with a plasmid encoding (thiol)-lyase. . The homocysteine methylase-negative mother and the transformed microorganism were treated as described in Example 1. Cultivate like this. Table II shows the relative amount of homocysteine produced by each strain. Indicates that E. coli mother body sulfate − Escherichia coli mother body sulfide − Escherichia coli transformant sulfate 10 E. coli transformant sulfide ++ C, glutamic parent sulfate - C, glutamicua + parent sulfate -c, glutamicum transformant sulfate 10C, glutam icum transformant sulfide ++ B, flavua + parent sulfate - B, flavum matrix sulfide- B, flavum transformant sulfate 10B, flavum transformant sulfate All strains lack homocysteine methylase activity.

** Low (-), Medium (+), High (++) Example 3 Production of methionine by acylhomoserine (methyl sulfhydration pathway) ) strain of Example 2 except supplemented with methyl mercaptan as a supplemental sulfur source (1 and culture as in Example 1. Table III shows the methionine produced by each strain. It shows the relative amount of nin. The production of methionine is a function of 0 methyl suluryl tion route.

E. coli mother body − E. coli transformant ++ C. glutamicum mother body - C. glutacicum transformant ++B. flavum mother body - B. flavuI11 transformant +1* All strains contain homocysteine Chylase activity (lack).

** Few (-), many (++) Production of methionine by phosphohomoserine (sulfhydration pathway) The parent strain of Example 1 was combined with homoserine kinase, plant cismethionine γ-synthesis Transformation with plasmids encoding homocysteine methylase and homocysteine methylase Ru. The parent microorganism and the transformed microorganism are cultured as in Example 1. Table IV is The relative amount of methionine produced by each strain is shown.

Escherichia coli mother body sulfate − Escherichia coli mother body sulfide − Escherichia coli transformant sulfate 10 E. coli transformant sulfide ++ C, glutamicum parent sulfate-'C, glutamicum parent Sulfide -C, glutasjcum transformant Sulfate -C, glutam icum transformant sulfide ++B, flavuI parent sulfate - B, flavus matrix sulfide - B, flavui transformant sulfate B, flavui transformant sulfide 10+* Less (-), Medium (+), More (++) Example 5 Production of methionine by phosphohomoserine (methyl sulfhydration pathway) ) The deletion parent strain of Example 2 was transformed into homoserine kinase and plant cystathionine γ. - Transform with a plasmid encoding the synthase. Parent microorganisms and transformation The transformed microorganisms are cultured as in Example 3. Table ■ is produced by each strain. The relative amount of methionine is shown.

Methionine 0a produced by microorganisms E. coli mother body − E. coli transformant +10 C0glutamicum mother body - C9 glutamiqua + transformant + 10B, flavum mother - B, flavum transformant + ten *All strains lack homocysteine methylase activity, It is supplemented with captan.

** Few (=), many (++) The parent strain of Example 1 lacks homoserine acyltransferase activity. . Microorganisms were treated with 0-acetylhomoserine (thiol)-lyase from fission yeast. and a plasmid encoding homocysteine methylase.

Parent microorganisms and transformed microorganisms are cultured as in Example 1. Table VI shows each bacteria. The relative amount of methionine produced by the strains is shown.

Escherichia coli mother body sulfate − Escherichia coli mother body sulfide − Escherichia coli transformant sulfate 10 E. coli transformant sulfide ++ C, glutamicum parent sulfate -C, glutamicum parent sulfate -C, glutamicum transformant sulfate Toc, glutamicum icum transformant sulfide ++B, flavul mother sulfate- B, flavum matrix sulfide- B, flavu armpit transformant sulfate 10B, flavu I11 transformant Sulfide ++ * All strains lack homocysteine methylase activity (all Ru.

** Low (-), medium (+), high (++) Example 7 Production of methionine by homoserine (Methylsulfhydration pathway) The deletion parent strain of Example 6 was obtained from fission yeast. A plasmid encoding 0-acetylhomoserine (thiol)-lyase of Transform. Parent microorganisms and transformed microorganisms are cultured as in Example 3. . Table VII shows the relative amount of methionine produced by each strain.

E. coli mother body − E. coli transformant ++ C, glutamicuI11 mother - Coglutamicum transformant ++B, flavum mother body - B, flavum transformant ++ *All strains lack homocysteine methylase activity.

** Few (-), many (10+) Mouth G, 1a Mouth G, Ib Ki S Ao 1 Mouth G, 1d Enter ・-111・-1 needle 6+1 cotton 10. PCT/lJs93101351

Claims (17)

[Claims] 1. By changing the methionine biosynthetic pathway of microbial cells, A method for increasing methionine production in a fermentation process, the method comprising: i) homoserine activation; Expression of homoserine activating enzyme gene fragment and sulfur uptake enzyme capable of expressing the enzyme Transforming or transducing the sulfur uptake enzyme gene fragment into the microbial cell. death, ii) Conditions that allow transformation or transduction of both enzyme gene fragments described above growing the microbial cells under; iii) harvesting the transformed or transduced microbial cells; iv ) External factors other than cysteine and methionine as sulfur sources to produce methionine a sulfur compound into the transformed or transduced microbial cell. add, A method characterized by comprising each step. 2. The exogenous sulfur compound may be hydrogen sulfide, methyl mercaptan or the like. The method according to claim 1, wherein the reduced sulfur compound is a salt. . 3. Oxidation in which the exogenous sulfur compound consists of sulfate, sulfide or thiosulfate The method according to claim 1, characterized in that it is a sulfur compound. 4. The homoserine activating enzyme is homoserine kinase, homoserine acetylate, A group consisting of transferases and homoserine succinyltransferases. 4. A method according to claim 2 or 3, characterized in that the method is selected from: 5. The sulfur uptake enzyme is 0-succinylhomoserine (thiol)-lyase. , 0-acetylhomoserine (thiol)-lyase and plant cystathioninga Claims 2 or 3, characterized in that the enzyme is selected from the group consisting of or the method described in Section 3. 6. The sulfur uptake enzyme homoserine and hydrogen sulfide or their salts 3. A method according to claim 2, characterized in that the stain is directly converted. 7. The sulfur uptake enzyme is homoserine and methyl mercaptan or their 3. Process according to claim 2, characterized in that the salt is directly converted to methionine. 8. The sulfur uptake enzyme converts homoserine directly to homocysteine. 3. The method according to claim 3. 9. By changing the methionine biosynthetic pathway of microbial cells, A method for increasing the production of homocysteine in a fermentation process, the method comprising: i) homocysteine; A homoserine activating enzyme gene capable of expressing a homoserine activating enzyme other than methylase. The gene fragment and the sulfur uptake enzyme gene fragment capable of expressing the sulfur uptake enzyme are transformed or transduced into biological cells, ii) both enzyme gene fragments as described above; growing the microbial cell under conditions that allow for the transformation or transduction of iii) harvesting the transformed or transduced microbial cells; iv ) Other than cysteine and methionine as a sulfur source to produce homocysteine Exogenous sulfur compounds are added to the transformed microbial cells or transduced microbial cells. add to the cell, A method characterized by comprising each step. 10. Reductive sulfurization in which the exogenous sulfur compound is hydrogen sulfide or a salt thereof 10. The method according to claim 9, which is a compound. 11. The exogenous sulfur compound is an acid consisting of sulfate, sulfide or thiosulfate. 10. The method according to claim 9, which is a sulfur compound. 12. The homoserine activating enzyme is homoserine kinase, homoserine acetyl A group consisting of transferases and homoserine succinyltransferases The method according to claim 10 or 11, characterized in that the method is selected from . 13. The sulfur uptake enzyme is 0-succinylhomoserine (thiol)-rea lyase, 0-acetylhomoserine (thiol)-lyase and plant cystathionine Claim 10, characterized in that it is selected from the group consisting of gamma synthase. or the method described in paragraph 11. 14. The sulfur uptake enzyme homoserine and hydrogen sulfide or its salt. 11. Process according to claim 10, characterized in that it is directly converted into a stain. 15. The sulfur uptake enzyme directly converts homoserine to homocysteine. 12. The method according to claim 11, characterized in that: 16. The transformed microbial cell or transduced microbial cell is transformed Amino acids greater than those in microbial cells that do not transduce or transduce microbial cells 10. The method according to claim 1 or 9, characterized in that an acid is produced. 17. The transformed microbial cell or transduced microbial cell is selected from the group consisting of Brevibacteria, Brevibacteria or Escherichia coli. 10. A method according to claim 1 or claim 9, characterized in that: