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A METHOD FOR PRODUCING AN L-AMINO ACID USING A BACTERIUM OF THE FAMILY ENTEROBACTERIACEAE HAVING OVEREXPRESSED THE yajl GENE

An amino acid, overexpression technology, applied in microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve problems such as yajL genes that have not been reported and proven

Active Publication Date: 2016-10-12
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] To date, no data have been reported demonstrating the effect of overexpression of the yajL gene on the production of L-amino acids by modified Enterobacteriaceae bacterial strains

Method used

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  • A METHOD FOR PRODUCING AN L-AMINO ACID USING A BACTERIUM OF THE FAMILY ENTEROBACTERIACEAE HAVING OVEREXPRESSED THE yajl GENE
  • A METHOD FOR PRODUCING AN L-AMINO ACID USING A BACTERIUM OF THE FAMILY ENTEROBACTERIACEAE HAVING OVEREXPRESSED THE yajl GENE
  • A METHOD FOR PRODUCING AN L-AMINO ACID USING A BACTERIUM OF THE FAMILY ENTEROBACTERIACEAE HAVING OVEREXPRESSED THE yajl GENE

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0141] Example 1. Construction of Escherichia coli producing L-valine strains modified in the manner of overexpressing the yajL gene

[0142] 1.1 Construction of the Escherichia coli MG1655 strain that has modified the yajL regulatory region

[0143] Using a method called "λRed / ET-mediated integration" developed by Datsenko K.A. and Wanner B.L. (Datsenko K.A. and Wanner B.L., Proc.Natl.Acad.Sci.USA, 2000,97(12):6640-6645 ) changed the expression of the yajL gene in Escherichia coli. According to this procedure, PCR primers P1 (SEQ ID NO:3) and P2 (SEQ ID NO:4) were constructed, which are adjacent to the yajL gene and confer chloramphenicol resistance (Cm R ) region of the gene and the region adjacent to the promoter in the template chromosome are both homologous. The chromosome of chloramphenicol-resistant Escherichia coli MG1655 strain, which contains a tac-like promoter with lacZ upstream -35 region as TGGCCA (from the 5'-end to the 3 '-end) structure (see Table 1, Katash...

Embodiment 2

[0149] Example 2. Passage of E. coli H81P tac7 - Production of L-valine by the yajL strain

[0150] The modified Escherichia coli H81P tac7 -yajL and control E. coli H81 strains were cultured in LB medium (also known as lysogenic broth or Luria-Bertani medium, as described in Sambrook, J. and Russell, D.W. "Molecular Cloning: A Laboratory Manual", 3 rd ed., Cold Spring Harbor Laboratory Press (2001)) at 32°C for 18 hours. Then, 0.2 mL of the obtained culture was inoculated into 2 mL of fermentation medium in a 20 × 200-mm test tube and cultured on a rotary shaker (250 rpm) at 32 °C for 66 h to OD 550 to about 29 until glucose is consumed.

[0151] The composition (g / L) of fermentation medium is as follows:

[0152]

[0153] Fermentation medium was sterilized at 116 °C for 30 min except for glucose and CaCO 3 Sterilize separately as follows: Glucose was sterilized at 110 °C for 30 min while CaCO 3 Sterilize at 116°C for 30 minutes. The pH was adjusted to 7.0 by addin...

Embodiment 3

[0158] Example 3. Construction of an L-phenylalanine-producing Escherichia coli strain modified in a manner of overexpressing the yajL gene

[0159] Will P tac7 The -yajL fragment was introduced into Escherichia coli DV269 (TyrA-LAA) strain, also known as Escherichia coli MG1655htrE:(P L -yddG)[MUDaroG4-pheA fbr -aroL], as described in Example 1, subsection 1.3. Escherichia coli MG1655P tac7 -yajL strain (see Example 1.1) was used as donor. The construction of Escherichia coli DV269 (TyrA-LAA) strain is described in Doroshenko V.G. et al., Construction of an L-phenylalanine-producing tyrosine-prototrophic Escherichia coli strain using tyrA ssrA-like tagged alleles, Biotechnol. Lett., 2010, 35:1117-1121. On the plate containing LB medium, agar (1.5%) and chloramphenicol (20mg / L), selection containing P tac7 - E. coli DV269 (TyrA-LAA) mutant of the yajL cassette. Escherichia coli H81DV269(TyrA-LAA)P thus obtained tac7 -yajL strain.

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Abstract

The present invention provides a method for producing L-amino acids or salts thereof by fermentation using a bacterium of the family Enterobacteriaceae, particularly a bacterium belonging to the genus Escherichia, which has been modified to overexpress the yajL gene.

Description

technical field [0001] The present invention relates to the microbiology industry, and in particular to a method for producing L-amino acids by fermentation of Enterobacteriaceae bacteria that have been modified in such a way as to overexpress the yajL gene. Background technique [0002] Conventionally, L-amino acids are industrially produced by fermentation methods using microbial strains obtained from natural sources or mutants thereof. Typically, the microorganism is modified to increase the production yield of the L-amino acid. [0003] Many techniques for increasing the yield of L-amino acid production have been reported, including microbial transformation using recombinant DNA (see, for example, U.S. No. 4,278,765A) and changes in regulatory regions, such as promoters, leader sequences, and / or attenuators ( attenuator), or other regulatory regions known to those skilled in the art (see eg US20060216796 A1 and WO9615246 A1). Other techniques for increasing production ...

Claims

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Application Information

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IPC IPC(8): C12P13/08C12P13/22C12P13/10C12P13/12C12P13/14C12P13/06C12P13/24C12P13/20C12P13/04C12N1/21C12R1/19
CPCC12N9/16C12N9/80C12P13/04C12P13/06C12P13/08C12P13/10C12P13/12C12P13/14C12P13/20C12P13/22C12P13/24C12Y301/02C12N15/70C12P13/222C12P13/227
Inventor V.V.萨姆索诺夫N.S.厄瑞米娜N.V.斯托诺瓦V.G.多罗申科
Owner AJINOMOTO CO INC
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