An l-cysteine-producing bacterium and a method for producing l-cysteine

A technology of cysteine ​​and cystine, which is applied in the field of L-cysteine ​​producing bacteria and L-cysteine ​​production, can solve the problems of undetermined genes and proteins, and achieve improved production capacity and high efficiency. The effect of production

Inactive Publication Date: 2011-06-01
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, it is speculated that there are two kinetically different cysteine ​​uptake systems in Legionella pneumophila (Non-Patent Document 9), but the genes and proteins have not yet been identified

Method used

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  • An l-cysteine-producing bacterium and a method for producing l-cysteine
  • An l-cysteine-producing bacterium and a method for producing l-cysteine
  • An l-cysteine-producing bacterium and a method for producing l-cysteine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] (Example 1) Identification of proteins with cysteine ​​or cystine uptake activity

[0155] (1) Obtain a mutant strain that cannot use S-thiocysteine ​​as a single cysteine ​​source (1-1) Obtain a cysE gene-deficient strain from Escherichia coli MG1655 strain (ATCC No. 47076). The cysE gene defect was passed The method called "Red-driven integration" developed by Datsenko and Wanner et al. (Proc. Natl. Acad. Sci. USA, 2000, vol. 97, No. 12, p6640-6645) and a lambda phage-derived excision system (J. Bacteriol. 2000 184.5200-5203 (2002)). Red drive integration and use synthetic oligonucleotides as primers to obtain PCR products. The PCR products can be used to construct gene-disrupted strains in one step. The synthetic oligonucleotides are designed with a part of the target gene on the 5'side, 3' The side design has a part of the anti-biotin resistance gene. In addition, by combining a cut-out system derived from lambda phage, the avidin resistance gene introduced in the ge...

Embodiment 2

[0187] (Example 2) Cysteine ​​production by Escherichia coli deficient in ydjN and / or fliY

[0188] (1) Construction of Escherichia coli cysteine ​​production strain

[0189] In order to give ydjN and / or fliY deficient E. coli cysteine ​​production capacity, a mutant cysE containing a mutant serine acetyltransferase encoding a reduced feedback inhibition of L-cysteine ​​was constructed (US20050112731(A1)) The plasmid. Specifically, first, in the pACYC-DES construction method described in JP 2005-137369 (US20050124049 (A1), EP1528108 (A1)), a mutation encoding a phosphoglycerate dehydrogenase that is not subject to serine feedback inhibition will be carried. The steps of type serA5 gene (described in US Patent No. 6,180,373) were omitted, and plasmid pACYC-DE1 was constructed. Compared with the pACYC-DES carrying the above-mentioned mutant serA5, the cysEX gene encoding the feedback-inhibited mutant SAT gene, and the ydeD gene encoding the cysteine ​​and acetylserine secretion fac...

Embodiment 3

[0218] (Example 3) Cysteine ​​production of P.ananatis deficient in ydjN and / or fliY

[0219] (1) Production of P.ananatis cysteine ​​producing strain EYPS1976(s)

[0220] The cysteine ​​producing strain of P.ananatis was constructed according to the following method: cysE5 (U.S. Patent Application Publication No. 20050112731) encoding mutant serine acetyltransferase and encoding mutant 3-phosphoglycerate dehydrogenase were introduced Chem., 1996, 271(38):23235-8), and enhanced yeaS (JP2000189180(A), which encodes secretion factors of various amino acids, and cysPTWA, which encodes sulfur uptake factors. Below, the construction method is described in detail.

[0221] (1-1) Introduce CysE5 and YeaS into P.ananatis SC17 strain

[0222] First, the plasmid used to construct the above strain was constructed. The method is as follows.

[0223] Using the chromosomal DNA of Escherichia coli MG1655 (ATCC No. 47076) as a template, using P1 (agctgagtcg acccccagga aaaattggtt aataac: SEQ ID NO: 3...

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Abstract

The invention provides a novel method for producing L-cysteine. The method adopts the gene having the ingesting activity of L-cysteine in the bacterium belonging to the family Enterobacteriaceae. The present invention provides a bacterium belonging to the family Enterobacteriaceae, which is able to produce L-cysteine, and has been modified to decrease activity of the YdjN protein, or the activities of the YdjN and the FliY protein. This bacterium is cultured in a medium, and L-cysteine, L-cystine, a derivative or precursor thereof, or a mixture of these can be collected from the medium.

Description

Technical field [0001] The present invention relates to a method for producing L-cysteine ​​or its related substances, in particular to bacteria suitable for the production of L-cysteine ​​or its related substances, and L-cysteine ​​or its related substances using the bacteria The production method of related substances. L-cysteine ​​and its related substances can be used in the fields of pharmaceuticals, cosmetics, and food. Background technique [0002] L-cysteine ​​can be obtained by extraction from keratin-containing materials such as hair, horns, and feathers, or by bacterial enzyme conversion using DL-2-aminothiazolin-4-carboxylic acid as a precursor. In addition, it is also planned to use new enzymes to produce L-cysteine ​​in large quantities through an immobilized enzyme method. [0003] An attempt has also been made to produce L-cysteine ​​by a fermentation method using bacteria. For example, the present inventors disclosed a method for producing L-cysteine ​​using the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P13/12C12R1/01C12R1/19
CPCC12P13/12
Inventor 野中源
Owner AJINOMOTO CO INC
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