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  • C12N9/10—Transferases (2.)
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/88—Lyases (4.)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
  • C12P13/12—Methionine; Cysteine; Cystine

Definitions

• 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.
• fermentation methods for methionine synthesis comprising the use of reduced sulfur compounds instead of sulfate as the fermentation sulfur source and/ ⁇ r comprising re-designing and thereby simplifying the biochemical pathway.
• 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.
• the reduced sulfur source is hydrogen sulfide, methyl mercaptan or salts thereof.
• Figure la is the ccranon biosynthetic pathway to Lysine, Methionine and
• 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.
• the present invention relates to methods for the fermentation synthesis of methionine and homocysteine. To understand why a cost-effective
• methionine that serves as the sulfur donor in the biosynthesis of methionine (Fig. 1).
• 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.
• CH3-THF 5-methyl-tetrahydrofolate
• Neidhardt Chapter 27 in Escherichia coli and Salmonella typhimurium.
• homoserine is first activated either by succinyl-CoA (EU. coli and S. typhimurium) or acetyl-CoA (fungi, yeast, and bacteria such as Brevibacterium and
• succinyltransferase (EC 2.3.1.46) and homoserine acetyltransferase (EC 2.3.1.31), respectively.
• O-phosphohomoserine is the branchpoint between the methionine and threonine pathways, whereas in microbes the brandhpoint is homoserine.
• cystathionine ⁇ -lyase (thiol)-lyase (EC 4.2.99.9) and cystathionine ⁇ -lyase (EC 4.4.1.8), accepts reduced sulfur frcm cysteine to give homocysteine.
• O- Succinylhomoserine (thiol)-lyase is also known as cystathionine ⁇ - synthase.
• 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.
• methionine is produced directly from acylhomoserine and methyl mercaptan by O- succinylhomoserine (thiol)-lyase or O-acetylhomoserine (thiol)-lyase.
• cystathionine ⁇ -sy ⁇ thase catalyzed by cystathionine ⁇ -sy ⁇ thase.
• the plant enzyme cystathionine ⁇ -synthase is distinct from EC 4.2.99.9 and is unique in vising O phosphchomoserine as a substrate.
• 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 pyrid ⁇ xal 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.
• sulfide or methyl mercaptan instead of sulfate reduces the metabolic cost of methionine synthesis to the levels of lysine and threonine.
• two ATP and three NADPH are required since the active transport of sulfate, reduction of sulfate, arri synthesis of cysteine are all eliminated.
• 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 ⁇ -lyase activity.
• This cculd be accomplished by structurally .modifying microbial O-acylhomoserine (thiol)-lyase or by expressing plant cystathionine ⁇ -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 inc ⁇ rp ⁇ rati ⁇ n. Or the O-acetylhomoserine (thiol)-lyase from S. pombe could be used without
• oxidized forms such as sulfate, sulfite, and thiosulfate may be provided as sulfur sources and biochemically reduced to sulfide.
• 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.
• metabolism for example, any effect on microbial self-regulation by feed-back inhibition or repression.
• 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.
• 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-a ⁇ etylhomoserine (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
• Table I indicates the relative amount of methionine that is produced by each strain.
• microbes are then transformed with plasmid(s) encoding homoserine
• Table II indicates the relative amcu ⁇ t of homocysteine that is produced by each strain.
• methylmercaptan is supplied as the supplemental sulfur source.
• Table III indicates the relative amount of methionine that is produced by each strain.
• the parent strains of Exanple 1 are transformed with plasmid(s) encoding homoserine kinase, plant cystathionine 7-synthase and homocysteine methylase.
• Table IV indicates the relative amount of methionine that is produced by each strain.
• Exanple 2 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.
• microbes are then transformed with
• Table VI indicates the relative amount of methionine that is produced by each strain.
• Example 6 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.

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• 1993
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