Process for producing L-amino acids

Details Process for producing L-amino acids Process for producing L-amino acids

1998-04-17

2001-11-20

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing alpha or beta amino acid or substituted amino acid...

C435S116000

Reexamination Certificate

active

06319696

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a method for producing L-amino acid. In particular, the present invention relates to a method for efficiently producing amino acid for which the demand rapidly increases, such as those used for a raw material for a sweetener aspartame (L-phenylalanine), feed additives (L-tryptophane, L-threonine), and raw materials for pharmaceuticals such as infusion solution (L-tryptophane, L-phenylalanine, L-tyrosine, L-threonine, and L-isoleucine).
BACKGROUND ART
There are a large number of methods for producing amino acid based on the use of the microorganism.
For example, methods for producing L-phenylalanine are known, including those based on the use of recombinants of
Escherichia coli
(
E. coli
) as disclosed in Japanese Patent Publication No. 2-4276, Japanese Laid-Open Patent Publication (PCT) No. 4-501813, Japanese Laid-Open Patent Publication No. 5-244956, and International Publication WO87/00202.
Methods for producing L-phenylalanine or L-tyrosine are also known, including those based on the use of a mutant strain belonging to the genus Corynebacterium as disclosed in Japanese Laid-Open Patent Publication No. 61-128897, and those based on the use of a recombinant of Corynebacterium strain as disclosed in Japanese Laid-Open Patent Publication Nos. 60-34197, 60-24192, 61-260892, and 61-124375.
Methods for producing L-tryptophane have been reported, including those based on the use of a recombinant of
Escherichia coli
as disclosed in Japanese Laid-Open Patent Publication No. 57-71397 and U.S. Pat. No. 4,371,614, those based on the use of a mutant strain of
Bacillus subtilis
as disclosed in Japanese Patent Publication Nos. 53-39517 and 62-34399, those based on the use of a recombinant of
Bacillus subtilis
as disclosed in Japanese Laid-Open Patent Publication Nos. 61-104790 and 1-67179, those based on the use of a mutant strain belonging to the genus Brevibacterium as disclosed in Japanese Laid-Open Patent Publication No. 57-174096, and those based on the use of a recombinant belonging to the genus Brevibacterium as disclosed in Japanese Laid-Open Patent Publication No. 62-51980.
Methods for producing L-threonine have been reported, including those based on the use of a mutant strain belonging to the genus Escherichia as disclosed in Japanese Laid-Open Patent Publication No. 5-304969, those based on the use of a recombinant of
Escherichia coli
as disclosed in Japanese Patent Publication No. 1-29559, Japanese Laid-Open Patent Publication Nos. 2-109985 and 56-15696, and Japanese Laid-Open Patent Publication (PCT) No. 3-501682. Further, there have been reported those based on the use of a mutant strain of a bacterium belonging to the genus Corynebacterium as disclosed in Japanese Laid-Open Patent Publication No. 62-239996, and those based on the use of a recombinant bacterium belonging to the genus Corynebacterium as disclosed in Japanese Laid-Open Patent Publication No. 61-195695.
Methods for producing L-isoleucine have been reported, including those based on the use of
Escherichia coli
as disclosed in Japanese Laid-Open Patent Publication No. 5-130882, and those based on the use of a recombinant of
Escherichia coli
as disclosed in Japanese Laid-Open Patent Publication No. 2-458. Further, there have been reported those based on the use of a mutant strain of a bacterium belonging to the genus Corynebacterium as disclosed in Japanese Patent Publication No. 3-62395, and those based on the use of a recombinant belonging to the genus Corynebacterium as disclosed in Japanese Patent Publication No. 5-47196.
The microorganisms, which have been used in the methods for producing amino acid as described above, have been bred principally on the basis of the enhancement of enzymes for catalyzing reactions in common pathways for various amino acids and in inherent pathways subsequent thereto for individual amino acids, or on the basis of the avoidance of control effected by final products or the like (feedback inhibition and suppression). Specifically, those which have been employed for the breeding include, for example, addition of auxotrophy to the microorganism, addition of drug resistance, and amplification of enzyme genes concerning the biosynthesis system and introduction of mutation aimed at desensitization of control based on the recombinant DNA technique.
The enzyme, which is in charge of the first reaction in the common pathway of aromatic amino acid biosynthesis, is 3-deoxy-D-arabino-hepturonate-7-phosphate (DAHP) synthase (DS). DS of
Escherichia coli
includes three types of isozymes which are encoded by genes called aroF, aroG, and aroH respectively and which undergo feedback inhibition by L-tyrosine, L-phenylalanine, and L-tryptophane respectively. Concerning these genes, a technique for improving the productivity of aromatic amino acid is known, which is based on the introduction, into
Escherichia coli
, of a combination of a gene coding for a desensitized type enzyme (enzyme substantially not subjected to feedback inhibition) originating from aroF or aroG involving high enzyme activity and a tryptophane operon containing a gene coding for a desensitized type anthranilate synthase (AS) of the inherent system of L-tryptophane biosynthesis.
Phosphoenclpyruvic acid (hereinafter referred to as “PEP” and D-erythrose 4-phosphate (E4 P) are used as substrates in the synthetic reaction for DAHP. PEP also serves as precursors for biosynthesis of L-aspartic acid, L-threonine, L-isoleucine and the like. The substrates as described above are supplied from a carbon source such as glucose via the glycolytic pathway and the pentoses phosphate pathway. However, no case has been hitherto known in the breeding of amino acid-producing strains, in which the ability to supply such substrates is enhanced to improve the productivity of amino acid.
Phosphoenolpyruvate synthase (hereinafter abbreviated as “PPS” is an enzyme which is broadly found in the microorganism. This enzyme plays an important role to supply PEP from pyruvic acid in glyconeogenesis.
Escherichia coli
, which is a bacterium belonging to the genus Escherichia, has been used to perform cloning of a gene (pps) coding for PPS, determination of the nucleotide sequence of the gene, and functional analysis for the enzyme. Besides, it has been reported that DAHP is produced at a value approximate to the theoretical yield by means of simultaneous amplification of the pps gene and the transketorase gene in a dehydroquinate synthase-deficient strain (Patnaik, R. et al.,
Appl. Environ. Mircobiol
., Vol. 60, No. 11, 3903-3908 (1994)). However, there has been known no case in which amplification of the pps gene enhances the productivity of aromatic amino acids and other amino acids.
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide a method for producing various amino acids represented by aromatic amino acids inexpensively and at a high yield.
The present inventors have found out that the productivity of L-amino acids can be improved by enhancing the phosphoenolpyruvate-producing ability in cells of a microorganism which has the L-amino acid-producing ability. Thus, the present invention has been completed.
That is, the present invention lies in a method for producing L-amino acid comprising the steps of cultivating, in a medium, a microorganism having an L-amino acid-producing ability, producing and accumulating the L-amino acid in the medium, and collecting the L-amino acid, wherein:
a phosphoenolpyruvate-producing ability of the microorganism is enhanced.
The L-amino acid, which is preferably produced by the production method described above, includes L-tryptophane, L-phenylalanine, L-tyrosine, L-threonine, and L-isoleucine.
The microorganism having the L-amino acid-producing ability is exemplified by bacteria belonging to the genes Escherichia and coryneform bacteria. The coryneform bacteria referred to herein include bacteria which have been hitherto classified into those belonging to the genus Brevibacterium but which are unified into bacteria belonging to

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