# US 8,785,161 B2 · Method for producing L-amino acids using bacteria of the enterobacteriaceae family

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- Number: US 8,785,161 B2
- Document ID: US8785161B2
- Status: Granted
- Kind: B2
- Application number: 13/466,266
- Filed: 2012-05-08
- Published: 2012-08-30
- Granted: 2014-07-22
- Earliest claimed: 2004-10-22
- Assignees: Ajinomoto Co., Inc.
- Applicants: Konstantin Vyacheslavovich Rybak; Ekaterina Aleksandrovna Slivinskaya; Ekaterina Alekseevna Savrasova; Valeriy Zavenovich Akhverdian; Elena Vitalievna Klyachko; Sergei Vladimirovich Mashko; Vera Georgievna Doroshenko; Larisa Gotlibovna Airikh; Tatyana Viktorovna Leonova; Mikhail Markovich Gusyatiner; Elvira Borisovna Voroshilova; Yury Ivanovich Kozlov; Yoshihiko Hara; Takuji Ueda
- Inventors: Konstantin Vyacheslavovich Rybak; Ekaterina Aleksandrovna Slivinskaya; Ekaterina Alekseevna Savrasova; Valeriy Zavenovich Akhverdian; Elena Vitalievna Klyachko; Sergei Vladimirovich Mashko; Vera Georgievna Doroshenko; Larisa Gotlibovna Airikh; Tatyana Viktorovna Leonova; Mikhail Markovich Gusyatiner; Elvira Borisovna Voroshilova; Yury Ivanovich Kozlov; Yoshihiko Hara; Takuji Ueda
- Web page: https://patentgrep.com/patent/US8785161B2

## CPC

- C12P 13/04: Alpha- or beta- amino acids {(other amino acids C12P13/005)}
- C12P 13/08: Lysine; Diaminopimelic acid; Threonine; Valine
- C12P 13/10: Citrulline; Arginine; Ornithine
- C12P 13/24: Proline; Hydroxyproline; Histidine
- 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)}

## Abstract

There is disclosed a method for producing L-amino acid, for example L-threonine, L-lysine, L-histidine, L-phenylalanine, L-arginine or L-glutamic acid, using a bacterium of the Enterobacteriaceae family, wherein the bacterium has been modified to enhance an activity of D-xylose permease.

## Claims

### Claim 1

A method for producing L-amino acid, which comprises cultivating a bacterium of Enterobacteriaceae family in a culture medium to cause accumulation of the L-amino acid in the culture medium, and isolating the L-amino acid from the culture medium, wherein said bacterium has been modified to enhance an activity of D-xylose permease and activities of xylose utilization proteins encoded by xylABFGHR locus by a method selected from the group consisting of:
a) increasing the copy number of a gene encoding D-xylose permease and the xylABFGHR locus,
b) placing the gene encoding D-xylose permease and the xylABFGHR locus under the control of a potent promoter, and
c) combinations thereof,
wherein the carbon source in said culture medium consists of glucose; and
wherein said D-xylose permease and said xylose utilization proteins encoded by xylABFGHR locus are both derived from a bacteria of the Enterobacteriaceae family.

### Claim 2 (depends on 1)

The method according to claim 1, wherein said gene encoding D-xylose permease is selected from the group consisting of:
(A) a protein comprising the amino acid sequence of SEQ ID NO:2; and
(B) a protein comprising an amino acid sequence which is at least 95% homologous to the sequence of SEQ ID NO:2 and has an activity of D-xylose permease.

### Claim 3 (depends on 1)

The method according to claim 1, wherein said gene encoding D-xylose permease is selected from the group consisting of:
a) a DNA comprising nucleotides 1 to 1476 in SEQ ID NO:1; and
b) a DNA which is hybridizable with nucleotides 1 to 1476 in SEQ ID NO:1 under stringent conditions comprising washing at 60° C. at a salt concentration of 1×SSC and 0.1% SDS for 15 minutes, and said DNA encodes a protein having an activity of D-xylose permease.

### Claim 4 (depends on 1)

The method according to claim 1, wherein said bacterium is an L-threonine producing bacterium.

### Claim 5 (depends on 4)

The method according to claim 4, wherein said bacterium has been additionally modified to enhance expression of a gene selected from the group consisting of
the mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I and is resistant to feedback inhibition by threonine,
the thrB gene which codes for homoserine kinase,
the thrC gene which codes for threonine synthase,
the rhtA gene which codes for a putative transmembrane protein, and
combinations thereof.

### Claim 6 (depends on 5)

The method according to claim 5, wherein said bacterium has been modified to increase expression of said mutant thrA gene, said thrB gene, said thrC gene, and said rhtA gene.

### Claim 7 (depends on 1)

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

### Claim 8 (depends on 1)

The method according to claim 1, wherein said bacterium is Pantoea ananatis.