# US 8,771,997 B2 · Method for producing monatin using an L-amino acid aminotransferase

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- Number: US 8,771,997 B2
- Document ID: US8771997B2
- Status: Granted
- Kind: B2
- Application number: 13/271,280
- Filed: 2011-10-12
- Published: 2012-10-25
- Granted: 2014-07-08
- Earliest claimed: 2011-04-20
- Assignees: Ajinomoto Co., Inc.
- Applicants: Yasuaki Takakura; Hiroomi Ogino; Masakazu Sugiyama; Kenichi Mori; Eri Tabuchi; Koki Ishikawa; Uno Tagami; Hidemi Fujii
- Inventors: Yasuaki Takakura; Hiroomi Ogino; Masakazu Sugiyama; Kenichi Mori; Eri Tabuchi; Koki Ishikawa; Uno Tagami; Hidemi Fujii
- Web page: https://patentgrep.com/patent/US8771997B2

## CPC

- C12P 13/04: Alpha- or beta- amino acids {(other amino acids C12P13/005)}
- C12N 9/1096: {transferring nitrogenous groups (2.6)}
- C12P 17/10: Nitrogen as only ring hetero atom
- 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

The present invention provides a methodology for improving a yield of 2R,4R-Monatin. Specifically, the present invention provides a method for producing 2S,4R-Monatin or a salt thereof, comprising contacting 4R-IHOG with an L-amino acid aminotransferase in the presence of an L-amino acid to form the 2S,4R-Monatin; a method for producing 2R,4R-Monatin or a salt thereof, comprising isomerizing the 2S,4R-Monatin to form the 2R,4R-Monatin; and the like. These production methods may further comprise condensing indole-3-pyruvate and pyruvate to form the 4R-IHOG, and deaminating a tryptophan to form the indole-3-pyruvate.

## Claims

### Claim 1

A method for producing 2S,4R-Monatin or a salt thereof, comprising contacting 4R form of 4-(indole-3-yl-methyl)-4-hydroxy-2-oxoglutaric acid (4R-IHOG) with an L-amino acid aminotransferase in the presence of an L-amino acid to form the 2S,4R-Monatin,
wherein the L-amino acid aminotransferase consists of an amino acid sequence having 95% or more identity to the amino acid sequence of SEQ ID NO: 2.

### Claim 2 (depends on 1)

The production method of claim 1, wherein a keto acid formed from the L-amino acid due to the action of the L-amino acid aminotransferase is contacted with a decarboxylase to degrade said keto acid.

### Claim 3 (depends on 1)

The production method of claim 1, wherein the L-amino acid is L-aspartate.

### Claim 4 (depends on 3)

The production method of claim 3, wherein oxaloacetate formed from the L-aspartate due to the action of the L-amino acid aminotransferase is contacted with an oxaloacetate decarboxylase to degrade said oxaloacetate to irreversibly form pyruvate.

### Claim 5 (depends on 1)

The production method of claim 1, wherein the L-amino acid aminotransferase is derived from a microorganism belonging to the genus Arthrobacter, Bacillus, Candida, Corynebacterium, Lodderomyces, Micrococcus, Microbacterium, Nocardia, Pseudomonas, Rhizobium, Stenotrophomonas, Dietzia, Ochrobactrum, Brevundimonas, Burkholderia, Carnimonas, Yarrowia, Clostridium, Deinococcus, Eubacterium, Lactobacillus, Methanothermobacter, Phormidium, Pyrococcus, Rhodococcus, Saccharomyces, Saccharophagus, Sinorhizobium, Thermoanaerobacter, Thermotoga or Thermus.

### Claim 6 (depends on 5)

The production method of claim 5, wherein the L-amino acid aminotransferase is derived from Arthrobacter sp., Bacillus altitudinis, Bacillus cellulosilyticus, Bacillus pumilus, Bacillus sp., Candida norvegensis, Candida inconspicua, Corynebacterium ammoniagenes, Corynebacterium glutamicum, Lodderomyces elongisporus, Micrococcus luteus, Microbacterium sp., Nocardia globerula, Pseudomonas chlororaphis, Pseudomonas citronocllolis, Pseudomonas fragi, Pseudomonas putida, Pseudomonas synxantha, Pseudomonas taetrolens, Pseudomonas sp., Rhizobium radiobacter, Rhizobium sp., Stenotrophomonas sp., Dietzia marls, Ochrobactrum pseudogrignonense, Brevundimonas diminuta, Burkholderia sp., Carnimonas sp., Yarrowia lypolytica, Clostridium cellulolyticum, Deinococcus geothermalis, Eubacterium rectale, Lactobacillus acidophilus, Methanothermobacter thermautotrophicus, Phormidium lapideum, Pyrococcus horikoshii, Rhodococcus erythropolis, Saccharomyces cerevisiae, Saccharophagus degradans, Sinorhizobium meliloti, Thermoanaerobacter tengcongensis, Thermotoga maritima, or Thermus thermophilus.

### Claim 7 (depends on 1)

The production method of claim 1, wherein the L-amino acid aminotransferase comprises one or more mutations of amino acid residues selected from the group consisting of the amino acid residues at position 39, position 109, position 128, position 150, position 258, position 287, position 288, position 289, position 303, position 358 and position 431 in the amino acid sequence of SEQ ID NO: 2.

### Claim 8 (depends on 7)

The production method of claim 7, wherein the one or more mutations of amino acid residues are selected from the group consisting of:
i) substitution of the lysine at position 39 with an arginine;
ii) substitution of the serine at position 258 with a glycine;
iii) substitution of the glutamine at position 287 with a glutamic acid;
iv) substitution of the threonine at position 288 with a glycine;
v) substitution of the isoleucine at position 289 with an alanine;
vi) substitution of the aspartic acid at position 109 with a glycine;
vii) substitution of the histidine at position 150 with a tyrosine;
viii) substitution of the phenylalanine at position 303 with a leucine;
ix) substitution of the aspartic acid at position 358 with a tyrosine;
x) substitution of the serine at position 431 with a threonine; and
xi) substitution of the glutamic acid at position 128 with a glycine.

### Claim 9 (depends on 1)

The production method of claim 1, wherein the 4R-IHOG is contacted with the L-amino acid aminotransferase using a transformant that expresses the L-amino acid aminotransferase.

### Claim 10 (depends on 1)

The production method of claim 1, further comprising condensing indole-3-pyruvate and pyruvate to form the 4R-IHOG.

### Claim 11 (depends on 10)

The production method of claim 10, wherein the indole-3-pyruvate and the pyruvate are condensed by contacting the indole-3-pyruvate and the pyruvate with an aldolase.

### Claim 12 (depends on 10)

The production method of claim 10, wherein at least part of the pyruvate used in the formation of the 4R-IHOG is from pyruvate formed from the oxaloacetate due to the action of an oxaloacetate decarboxylase.

### Claim 13 (depends on 10)

The production method of claim 10, further comprising deaminating a tryptophan to form the indole-3-pyruvate.

### Claim 14 (depends on 13)

The production method of claim 13, wherein the tryptophan is deaminated by contacting the tryptophan with a deamination enzyme.

### Claim 15 (depends on 10)

The production method of claim 10, wherein the production of the 2S,4R-Monatin or the salt thereof is carried out in one reactor.

### Claim 16 (depends on 13)

The production method of claim 13, wherein the production of the 2S,4R-Monatin or the salt thereof is carried out in one reactor.