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Method for producing L-arginine, L-ornithine or L-citrulline

A technology of amino acid and alanine, applied in botany equipment and methods, biochemical equipment and methods, chemical instruments and methods, etc.

Active Publication Date: 2007-08-29
KYOWA HAKKO BIO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, up to now, there has been no report about which mutations are introduced into the DNAs encoding ArgB and ArgR to increase the yield of L-arginine in strains in which mutations are introduced into the DNAs encoding ArgB and ArgR.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0147] When the recombinant DNA of the present invention used in the preparation of the microorganism of the present invention has an inducible promoter, an inducer compatible with the promoter can be added to the medium as necessary. For example, when using recombinant DNA with a lac promoter as recombinant DNA, isopropyl-β-D-thiogalactoside, etc. can be added to the medium; trp In the case of recombinant DNA of a promoter, indoleacrylic acid or the like can be added to the medium.

[0148] The culture time is usually 1-6 days, and L-arginine, L-ornithine or L-citrulline are produced and accumulated in the culture.

[0149] After the cultivation, the precipitates such as bacterial cells are removed from the culture, and the accumulated L-arginine, L-ornithine or L- citrulline.

Embodiment 1

[0152] Preparation of plasmid for ArgB amino acid substitution

[0153] (1) Preparation of vector for homologous recombination

[0154] Plasmid pHSG299 [Gene, 61, 63-74 (1987)] having a gene conferring resistance to kanamycin was treated with PstI, and Bacillus subtilis-derived fructan sucrose was linked at the cutting site A kilobase pair (hereinafter, abbreviated as kb) DNA fragment of the enzyme gene sacB [Mol. Microbiol., 6, 1195 (1992)] was used to obtain plasmid pESB30.

[0155] pESB30 was treated with BamHI (manufactured by Takara Shuzo Co., Ltd.), subjected to agarose gel electrophoresis, extracted and purified using a GENECLEAN kit (manufactured by BIO101 Co., Ltd.). Both ends of the obtained DNA fragment were blunted using a DNA Blunting Kit (manufactured by Takara Shuzo Co., Ltd.) according to the attached manual. The smoothed DNA fragments were treated with phenol and chloroform, provided for ethanol precipitation and concentrated, and reacted at 70°C for 2 hours...

Embodiment 2

[0175] Introduction of amino acid substitutions into chromosome ArgB

[0176] (1) Introduction of amino acid substitutions into ArgB on the chromosome of the wild strain

[0177] Using the plasmids pEargB26, pEargB31 and pEargB2631 prepared in Example 1, respectively, according to Rest et al. [Appl.Microbiol.Biotech., 52, 541 (1999)] by electroporation into Corynebacterium glutamicum ATCC13032 strains, and kanamycin-resistant strains were selected. As a result of preparing chromosomes from one of the kanamycin-resistant strains and studying them by Southern hybridization (Molecular Cloning Version 3), it was confirmed that pEargB26, pEargB31, and pEargB2631 were integrated into each strain by Cambell-type homologous recombination in the chromosome. In this strain, the DNA encoding ArgB originally present on the chromosome and the DNA of the present invention coexist adjacently on the chromosome, and the second homologous recombination easily occurs between them.

[0178] Si...

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PUM

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Abstract

It is intended to provide a polypeptide having (i) an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO:1 by substitution of one or more amino acids in the amino acid sequences of from the 20- to the 38-th positions from the N end, or (ii) an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO:1 by substitution of one or more amino acids in the amino acid sequences of from the 20- to the 38-th positions from the N end and deletion, substitution or addition of one or more amino acids in the amino acid sequence of from the first to the 19-th positions or from the 39-th to the 294-th positions, and having an N-acetylglutamate kinase activity.

Description

technical field [0001] The invention relates to a preparation method of L-arginine, L-ornithine or L-citrulline. Background technique [0002] In microorganisms, L-arginine is biosynthesized from L-glutamic acid through 8-stage reactions. L-ornithine and L-citrulline are intermediates on the biosynthetic pathway of L-arginine. The biosynthesis of L-arginine, L-ornithine and L-citrulline is regulated like other amino acids. [0003] For example, in coryneform bacteria, an arginine repressor (hereinafter referred to as ArgR) suppresses an operon (hereinafter referred to simply as an arginine operon) composed of a gene encoding an enzyme related to the biosynthesis of L-arginine transcription (see Patent Document 1). In addition, it is also known that in coryneform bacteria, the second enzyme N-acetylglutamate kinase (EC: 2.7.2.8, hereinafter referred to as ArgB) is subject to feedback inhibition by L-arginine (see Non-Patent Document 2). [0004] Like other amino acids, L...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/00C12N9/12C12N15/09C12P13/10C12N1/21C07K14/34
CPCC12P13/10C12N9/1217C12Y207/02008
Inventor 池田正人中野哲朗三桥敏林干郎田中建儿
Owner KYOWA HAKKO BIO CO LTD
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