# US 7,608,437 B2 · Method of constructing amino acid producing bacterial strains, and method of preparing amino acids by fermentation with the constructed amino acid producing bacterial strains

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- Number: US 7,608,437 B2
- Document ID: US7608437B2
- Status: Granted
- Kind: B2
- Application number: 11/218,433
- Filed: 2005-09-06
- Published: 2006-01-05
- Granted: 2009-10-27
- Earliest claimed: 1998-09-25
- Assignees: Ajinomoto Co., Inc.
- Inventors: Yoko Asakura; Jun Nakamura; Sohei Kanno; Mikiko Suga; Eiichiro Kimura; Hisao Ito; Kazuhiko Matsui; Tsuyoshi Ohsumi; Tsuyoshi Nakamatsu; Osamu Kurahashi
- Web page: https://patentgrep.com/patent/US7608437B2

## CPC

- C12P 13/14: Glutamic acid; Glutamine
- C12N 15/67: General methods for enhancing the expression
- 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

A method of producing coryneform bacteria having an improved amino acid or nucleic acid-productivity comprises the steps of introducing a mutation in a promoter sequence of amino acid- or nucleic acid-biosynthesizing genes on a chromosome of a coryneform bacterium so that it is close to a consensus sequence or introducing a change in the promoter sequence of amino acid- or nucleic acid-biosynthesizing genes on the chromosome of a coryneform bacterium by gene recombination so that it is close to a consensus sequence, obtaining mutants of the coryneform amino acid- or nucleic acid-producing microorganism, culturing the mutants and select a mutant capable of producing the intended amino acid or nucleic acid in a large amount. This method allows one of skill in the art to construct a mutant capable of enriching or controlling the expression of an intended gene without using a plasmid and to promote production of amino acids in a high yield, by the recombination or mutation.

## Claims

### Claim 1

A method of producing L-glutamic acid, comprising:
culturing a coryneform bacterium expressing enzymes encoded by coryneform bacteria glutamic acid biosyntesizing genes, in a medium for a time and under conditions suitable to produce and accumulate said L-glutamic acid in the medium, and
collecting said L-glutamic acid from the medium;
wherein the glutamic acid biosynthesizing genes are located on the chromosome of the coryneform bacterium and the enzymes are glutamate dehydrogenase and citrate synthase,
wherein said glutamic acid biosynthesizing genes comprise a DNA sequence situated at about position −10 from the transcription start site of the glutamic acid biosynthesizing genes,
wherein said DNA sequence is TATAAT,
wherein said gene which encodes glutamate dehydrogenase further comprises TTGTCA situated at about position −35 from the transcription start site of the gene,
wherein said L-glutamic acid is produced at a level greater than the L-glutamic acid produced by the corresponding wild-type coryneform bacterium.

### Claim 2

A method of producing L-glutamic acid, comprising:
culturing a coryneform bacterium expressing enzymes encoded by coryneform bacteria glutamic acid biosynthesizing genes, in a medium for a time a time and under conditions suitable to produce and accumulate said L-glutamic acid in the medium, and
collecting said L-glutamic acid from the medium;
wherein the glutamic acid biosynthesizing genes are located on the chromosome of the coryneform bacterium and the enzymes are glutamate deydrogenase and citrate synthase,
wherein said glutamic acid biosynthesizing genes comprise a DNA sequence situated at about position −10 from the transcription start site of the glutamic acid biosythesizing genes,
wherein said DNA sequence is TATAAT,
wherein said gene which encodes said citrate sythase further comprises TTGACA situated at about −35 from the transcription start site of the gene,
wherein said gene which encodes said glutamate dehydrogenase further comprises TTGTCA situated at about position −35 from the transcription start site of the gene,
wherein said L-glutamic acid is produced at a level greater than the L-glutamic acid produced by the corresponding wild-type coryneform bacterium.