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Novel aspartokinase mutant and method for production of l-amino acid using same

A technology of aspartate kinase, aspartate, applied in the field of aspartate kinase variants

Active Publication Date: 2019-08-23
CJ CHEILJEDANG CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this regard, although applications related to feedback inhibition have been submitted (US Patent No. US 8062869B and Japanese Patent No. JP3473042B), continued research is needed to improve the productivity of aspartic acid-derived products

Method used

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  • Novel aspartokinase mutant and method for production of l-amino acid using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1: Preparation of bacterial strains introducing lysC variants

[0070] A variant whose activity can be enhanced compared with the wild type was selected by structural modeling of aspartokinase, and a strain into which the variant was introduced was prepared as described below.

[0071] In the gene lysC (SEQ ID NO: 2) encoding the aspartokinase (SEQ ID NO: 1) derived from Corynebacterium glutamicum ATCC13032 (hereinafter referred to as WT), the amino acid at position 377 was selected as the mutation site , and selected the basic amino acid L-lysine and the nonpolar amino acid L-methionine as representative examples of other amino acids for substitution.

[0072] In order to prepare a vector for introducing variation, a pair of primers (SEQ ID NO: 7 and 8 or SEQ ID NO: 7 and 10) and a pair of primers for amplifying the 5' upstream region were designed with the variation site as the center. A pair of primers (SEQ ID NO: 9 and 12 or SEQ ID NO: 11 and 12) for the ...

Embodiment 2

[0080] Example 2: Confirmation of the ability of strains introducing lysC variants for the production of aspartic acid-derived amino acids

[0081] In order to compare the abilities of the strains CA01-2307 and CA01-2308 obtained from Example 1 and the strain WT for producing amino acids mainly derived from aspartic acid, the strains were cultured using the following method, and the components in the medium were analyzed.

[0082] Each strain was inoculated into a baffled flask (250 mL) containing a seed medium (25 mL), and cultured at 37° C. for 20 hours while shaking at 200 rpm. A seed culture solution (1 mL) was inoculated into a corner-baffled flask (250 mL) containing a production medium (24 mL), and then cultured at 37° C. for 24 hours while shaking at 200 rpm. The concentrations of L-lysine and L-threonine, which are representative amino acids derived from L-aspartic acid and aspartic acid, were analyzed by HPLC, and the analysis The concentrations are shown in Table...

Embodiment 3

[0093] Example 3: Preparation of L-threonine enhanced strain and confirmation of L-threonine productivity

[0094] In order to clearly confirm the change in L-threonine productivity by introducing the lysC(L377K) variant, which has the higher lysine productivity identified in Example 2, a variant was introduced into the homoserine encoding the production of homoserine Among the genes of dehydrogenases, this gene is a common intermediate in the biosynthetic pathway of L-threonine, L-isoleucine, L-methionine and homoserine derivatives. Specifically, the strain was prepared by introducing the hom(G378E) mutation known in the art (Appl. Microbiol. Biotechnol. 45, 612-620 (1996)) into the strain CA01-2307 prepared in Example 1. In addition, a strain in which the hom(G378E) mutation was introduced into WT was also prepared as a control group. To prepare a hom(G378E)-introducing vector, PCR was performed using SEQ ID NOs: 13 and 14 and SEQ ID NOs: 15 and 16 using WT genomic DNA as...

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Abstract

The present application relates to an aspartokinase mutant, a microorganism including the mutant, and a method for production of an aspartate-derived L-amino acid or homoserine derivative, using the microorganicm.

Description

technical field [0001] The present disclosure relates to aspartokinase variants, microorganisms comprising the variants, and methods of producing aspartic acid-derived L-amino acids or amino acid derivatives thereof by using the microorganisms. Background technique [0002] Microorganisms of the genus Corynebacterium, particularly Corynebacterium glutamicum, are Gram-positive microorganisms widely used for the production of L-amino acids and other useful substances. In order to produce L-amino acids and other useful substances, various studies have been conducted to develop efficient production microorganisms and techniques for fermentation processes. For example, a method of specifically approaching a target substance by increasing the expression of a gene encoding an enzyme involved in L-lysine biosynthesis or by knocking out a gene unnecessary for biosynthesis is mainly used (Korean Patent No. 10-0838038). [0003] Meanwhile, among L-amino acids, L-lysine, L-threonine, L...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/77C12P13/02
CPCC12N15/77C12N9/1217C12Y207/02004C12P13/08C12P13/12C12P13/06C12P13/001C12P13/02C12N9/12C12Y207/02003
Inventor 金亨俊金孝珍裵贤原金贤雅徐昌一李知宣张真淑
Owner CJ CHEILJEDANG CORP
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