# US 7,611,873 B1 · Method for producing L-methionine by fermentation

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- Number: US 7,611,873 B1
- Document ID: US7611873B1
- Status: Granted
- Kind: B1
- Application number: 09/441,055
- Filed: 1999-11-16
- Granted: 2009-11-03
- Earliest claimed: 1998-11-17
- Assignees: Ajinomoto Co., Inc.
- Inventors: Yoshihiro Usuda; Osamu Kurahashi
- Web page: https://patentgrep.com/patent/US7611873B1

## CPC

- C12P 13/12: Methionine; Cysteine; Cystine
- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12N 9/1018: {Carboxy- and carbamoyl transferases (2.1.3)}
- C12N 9/1085: {transferring alkyl or aryl groups other than methyl groups (2.5)}
- C12N 9/1205: {Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases}
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

L-Methionine is produced by culturing a microorganism which is deficient in repressor of L-methionine biosynthesis system and/or enhanced intracellular homoserine transsuccinylase activity is cultured in a medium so that L-methionine should be produced and accumulated in the medium, and collecting the L-methionine from the medium. The microorganism preferably further exhibits reduced intracellular S-adenosylmethionine synthetase activity, L-threonine auxotrophy, enhanced intracellular cystathionine γ-synthase activity and enhanced intracellular aspartokinase-homoserine dehydrogenase II activity. The present invention enables breeding of L-methionine-producing bacteria, and L-methionine production by fermentation.

## Claims

### Claim 1

A method for producing L-methionine which comprises culturing a recombinant Escherichia bacterium in a medium to produce and accumulate L-methionine in the medium in an amount in excess of the corresponding unmodified Escherichia bacterium, and collecting the L-methionine from the medium, wherein
the bacterium is deficient in repressor of L-methionine biosynthesis system encoded by the endogenous metJ gene and has L-methionine productivity,
activity of intracellular homoserine transsuccinylase encoded by the metA gene of a Escherichia bacterium is increased compared to an unmodified Escherichia bacterium by increasing copy number of the metA gene including its own promoter, or replacing the native promoter with a stronger promoter, and
the bacterium comprises at least one characteristic selected from the group consisting of:
(a) exhibits reduced activity of intracellular S-adenosylmethionine synthetase encoded by the endogenous metK gene as compared to an unmodified Escherichia bacterium;
(b) exhibits L-threonine auxotrophy;
(c) exhibits enhanced activity of intracellular cystathionine γ-synthase encoded by the metB gene of a Escherichia bacterium and enhanced activity of intracellular aspartokinase-homoserine dehydrogenase II encoded by the metL gene of a Escherichia bacterium as compared to an unmodified Escherichia bacterium by increasing copy number of each of the genes including their own promoters, or replacing the native promoter with a stronger promoter; and
(d) has a homoserine transsucinylase for which concerted inhibition by L-methionine and S-adenosylmethionine is desensitized, wherein the homoserine transsuccinylase comprising the amino acid sequence of SEQ ID NO: 26 contains at least one amino acid replacement wherein said at least one amino acid replacement is independently selected from the group consisting of replacement of the amino acid residue Arg-27 with cysteine, replacement of the amino acid residue Ile-296 with serine, and replacement of the amino acid residue Pro-298 with leucine.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the bacterium is Escherichia coli.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the bacterium comprises at least the characteristic (a).

### Claim 4 (depends on 1)

The method according to claim 1, wherein the bacterium comprises at least the characteristic (b).

### Claim 5 (depends on 1)

The method according to claim 1, wherein the bacterium comprises at least the characteristic (c).

### Claim 6 (depends on 1)

The method according to claim 1, wherein the bacterium comprises at least the characteristic (d).

### Claim 7 (depends on 1)

The method according to claim 1, wherein the bacterium comprises the characteristics (a) and (b).

### Claim 8 (depends on 1)

The method according to claim 1, wherein the bacterium comprises the characteristics (a) and (c).

### Claim 9 (depends on 1)

The method according to claim 1, wherein the bacterium comprises the characteristics (a) and (d).

### Claim 10 (depends on 1)

The method according to claim 1, wherein the bacterium comprises the characteristics (b) and (c).

### Claim 11 (depends on 1)

The method according to claim 1, wherein the bacterium comprises the characteristics (b) and (d).

### Claim 12 (depends on 1)

The method according to claim 1, wherein the bacterium comprises the characteristics (c) and (d).

### Claim 13 (depends on 1)

The method according to claim 1, wherein the bacterium comprises the characteristics (a), (b), and (c).

### Claim 14 (depends on 1)

The method according to claim 1, wherein the bacterium comprises the characteristics (a), (b), and (d).

### Claim 15 (depends on 1)

The method according to claim 1, wherein the bacterium comprises the characteristics (a), (c), and (d).

### Claim 16 (depends on 1)

The method according to claim 1, wherein the bacterium comprises the characteristics (b), (c), and (d).

### Claim 17 (depends on 1)

The method according to claim 1, wherein the bacterium comprises the characteristic (a), (b), (c), and (d).

### Claim 18 (depends on 1)

The method according to claim 1, wherein the activity of intracellular S-adenosylmethionine synthetase is reduced due to that the bacterium has S-adenosylmethionine synthetase which contains amino acid substitution which is selected from the group consisting of replacement of the amino acid residue Ile-303 with leucine, replacement of the amino acid residue Val-185 with glutamic acid, and replacement of amino acid residues 378-384 with the amino acid sequence of SEQ ID NO: 29, respectively in the amino acid sequence of SEQ ID NO: 18.

### Claim 19 (depends on 3)

The method according to claim 3, wherein the activity of intracellular S-adenosylmethionine synthetase is reduced due to that the bacterium has S-adenosylmethionine synthetase which contains amino acid substitution which is selected from the group consisting of replacement of the amino acid residue Ile-303 with leucine, replacement of the amino acid residue Val-185 with glutamic acid, and replacement of amino acid residues 378-384 with the amino acid sequence of SEQ ID NO: 29, respectively in the amino acid sequence of SEQ ID NO: 18.

### Claim 20 (depends on 1)

The method according to claim 1, wherein the L-threonine auxotrophy is due to deletion of the thrBC genes.

### Claim 21 (depends on 4)

The method according to claim 4, wherein the L-threonine auxotrophy is due to deletion of the thrBC genes.