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Method for production of L-lysine

A lysine, amino acid technology, applied in biochemical equipment and methods, enzymes, isomerases, etc., can solve problems such as reduction

Active Publication Date: 2010-06-30
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it was not expected that reducing the activity of enzymes belonging to the meso-DAP synthesis pathway is effective for L-lysine production when DDH is expressed in bacteria of the genus Escherichia

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0139] [Example 1] Construction of L-lysine-producing bacteria in which dapD is disrupted

[0140] Construction of dapD gene disrupted strain

[0141] First, the dapD-disrupted strain was constructed using the Escherichia coli wild-type strain MG1655.

[0142] Take pMW118(λattL-Km r -λattR) (refer to International Publication No. WO2006093322) plasmid as a template, using SEQ ID NO: 19 and 20, the sequences attL and attR corresponding to the sequence attL and attR at the 3' end of the primer 1. A synthetic oligonucleotide having a sequence corresponding to a part of the dapD gene as the target gene at the 5' end of the primer was used as a primer to carry out PCR, and the λ- MG1655ΔdapD::att-Km strain was constructed by red method. In the λ-red method, Km-resistant recombinants are obtained by plate culture on L-agar medium containing Km (kanamycin) (50mg / L) at 37°C and selecting Km-resistant recombinants. ongoing.

[0143] Transform L-lysine producing strain WC196LC / pC...

Embodiment 2

[0147] [Example 2] Evaluation of L-lysine production capacity of L-lysine-producing bacteria with dapD destroyed

[0148] Melt the glycerol preservation solution of the bacterial strain obtained in Example 1, take 100 μL and evenly spread it on an L plate containing 20 mg / L streptomycin, and incubate at 37° C. for 24 hours. About 1 / 8 of the bacterial cells on the obtained plate were suspended in 0.5 mL of physiological saline, and the turbidity at a wavelength of 600 nm was measured with a spectrophotometer U-2000 (Hitachi). Inoculate 20 mL of the fermentation medium (MS medium, composed as follows) containing 20 mg / L streptomycin in a 500 mL Sakaguchi flask with the resulting suspension containing the bacterium, wherein the amount of liquid inoculated is such that the turbidity at a wavelength of 600 nm was 0.15, and cultured for about 24 hours with a reciprocating shaking culture device at a stirring speed of 114 rpm and a temperature of 37°C. After the cultivation, the amo...

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PUM

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Abstract

L-lysine can be produced by culturing an Escherichia coli cell in a culture medium, and collecting L-lysine from the culture medium, wherein the Escherichia coli cell is capable of producing L-lysine, is so modified that the activities of one or more enzymes involved in the synthetic pathway of meso-a,e-diaminopimelic acid (e.g., 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase, succinyldiaminopimelate transaminase, succinyldiaminopimelate desuccinylase, diaminopimelate epimerase) are decreased, and has a gene encoding diaminopimelate dehydrogenase introduced therein.

Description

technical field [0001] The present invention relates to a method for producing L-lysine using Escherichia coli. L-Lysine is an essential amino acid and is used as a component of nutritional mixtures such as pharmaceuticals or feed supplements for animals. Background technique [0002] L-amino acids such as L-lysine are industrially produced by fermentation using amino acid producing bacteria such as coryneform bacteria or Escherichia bacteria capable of producing these L-amino acids. As these amino acid producing bacteria, strains isolated from nature, artificial mutants of the strains, recombinants in which the activity of L-amino acid biosynthetic enzymes have been enhanced by genetic recombination, etc. have been used in order to increase productivity. The production method of L-lysine includes methods described in Patent Documents 1 to 4, for example. [0003] As a method of improving the productivity of amino acids such as L-lysine, in addition to the method of increa...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N1/21C12P13/08
CPCC12P13/08C12N9/90C12N9/1096C12N9/1029
Inventor 土井秀高植田拓嗣
Owner AJINOMOTO CO INC
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