Method for producing l-lysine

An amino acid and nucleotide technology, applied in biochemical equipment and methods, botanical equipment and methods, applications, etc., can solve problems such as improving growth, no L-lysine biosynthesis genes are reported, and achieve yield improvement. Effect

Inactive Publication Date: 2004-09-01
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is also no report of growth improvement by enhancing L-lysine biosynthesis genes

Method used

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  • Method for producing l-lysine
  • Method for producing l-lysine
  • Method for producing l-lysine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0122] Example 1: Preparation of wild-type lysC gene and mutant lysC gene from Brevibacterium lactofermentum

[0123] 1 Preparation of wild-type and mutant lysC and preparation of plasmids containing them

[0124] The Brevibacterium lactofermentum ATCC 13869 strain and the L-lysine-producing mutant strain AJ3445 (FERM P-1944) obtained from the ATCC 13869 strain by mutation treatment were used as chromosomal DNA donors. The AJ3445 strain has undergone mutations that alter lysC to substantially desensitize the synergistic inhibition by lysine and threonine (J. Biochem., 68, 701-710 (1970)).

[0125] A DNA fragment containing lysC was amplified from chromosomal DNA by the PCR method (polymerase chain reaction; see White, T.J. et al., Trends Genet., 5, 185 (1989)). A 1,643bp region encoding lysC was added based on the known sequence of Corynebacterium glutamicum (see Molecular Microbiology (1991), 5(5), 1197-1204; and Mel.Gen.Genet. (1990), 224, 317-324) to synthesize 23-mer and...

Embodiment 2

[0136] Example 2: Preparation of dapB from Brevibacterium

[0137] 1 Preparation of dapB and construction of a plasmid containing dapB

[0138] Brevibacterium lactofermentum wild-type strain ATCC 13869 was used as the chromosomal DNA donor. Chromosomal DNA was prepared from the ATCC 13869 strain according to an ordinary method. A DNA fragment containing dapB was amplified from chromosomal DNA according to PCR. As for the DNA primers used for amplification, in order to amplify the 2.0 kb region encoding DDPR, based on the known sequence of Brevibacterium lactofermentum (see Journal of Bacteriology, 175(9), 2743-2749(1993)), respectively 23-mer DNA (with the nucleotide sequences shown in SEQ ID NO: 8 and 9 in the Sequence Listing) was synthesized, and DNA synthesis and PGR were performed in the same manner as described in Example 1. pCR-Script (manufactured by Invitrogen) was used as a cloning vector for amplifying a gene fragment of 2,001 bp, which was ligated with the ampli...

Embodiment 3

[0144] Example 3: Preparation of dapA from Brevibacterium

[0145] 1 Preparation of dapA and construction of a plasmid containing dapA

[0146] Brevibacterium lactofermentum wild-type strain ATCC 13869 was used as the chromosomal DNA donor. Chromosomal DNA was prepared from the ATCC 13869 strain according to an ordinary method. A DNA fragment containing dapA was amplified from chromosomal DNA according to PCR. As for the DNA primers used for amplification, in order to amplify the region of 1.5kb encoding DDPS, based on the known sequence of Corynebacterium glutamicum (Nucleic Acid Research, 18 (21), 6421 (1990); EMBL deposit number X53993 ) to synthesize 23 strands of DNA respectively having the nucleotide sequences shown in SEQ ID NO: 12 and 13 in the sequence listing, and DNA synthesis and PCR were carried out in the same manner as described in Example 1. pCR-1000 (manufactured by Invitrogen, see Biotechnology, 9, 657-663 (1991)) was used as a cloning vector for amplifyin...

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Abstract

A coryneform bacterium harboring an aspartokinase in which feedback inhibition by L-lysine and L-threonine is substantially desensitized, and comprising an enhanced DNA sequence coding for a dihydrodipicolinate reductase, an enhanced DNA sequence coding for dihydropicolinate reductase, an enhance DNA sequence coding for dihydropicolinate synthase, an enhanced DNA sequence coding for diaminopimelate decarboxylase and an enhanced DNA sequence coding for aspartate aminotransferase; a method for producing L-lysine comprising the steps of cultivating the coryneform bacterium in an appropriate medium to allow L-lysine to be produced and accumulated in a culture of the bacterium, and collecting L-lysine from the culture; and a recombinant DNA usable for production of the coryneform bacterium.

Description

[0001] This application is a divisional application of the invention application whose filing date is December 5, 1997, application number is 97120820.4, and the title of the invention is "method for producing L-lysine". technical field [0002] The present invention relates to a method for producing L-lysine by culturing microorganisms obtained by modifying coryneform bacteria and the like for fermentative production of amino acids by means of techniques based on genetic engineering. Background technique [0003] Lysine as a feed additive is generally produced by fermentation using an L-lysine-producing mutant strain belonging to Corynebacterium. Various L-lysine-producing bacteria are now known as those produced by artificial mutation starting from wild-type strains belonging to coryneform bacteria. [0004] In the case of coryneform bacteria, vector plasmids are disclosed which are autonomously replicable in bacterial cells and have drug resistance marker genes (see US Pa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N9/02C12N9/10C12N9/12C12N9/88C12N15/53C12N15/54C12N15/60C12N15/77C12P13/08
CPCC12P13/08C12N9/001C12N9/88C12N9/1096C12N9/1217C12N15/77
Inventor 荒木政行杉本雅一吉原康彦中松亘
Owner AJINOMOTO CO INC
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