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Method for producing l-lysine by fermentation and modified corynebacterium

A technology of Corynebacterium glutamicum and Corynebacterium, applied in the field of amino acid fermentation, can solve the problems of unclear utility, protein biological function has not been revealed biological function, etc., and achieve the effect of increasing yield

Active Publication Date: 2017-07-18
NINGXIA EPPEN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still approximately 960 genes encoding proteins whose biological functions have not been revealed, and their utility in the fermentative production of L-lysine is not clear

Method used

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  • Method for producing l-lysine by fermentation and modified corynebacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 NCgl1751 Gene expression downregulation experiments

[0041] According to the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, two pairs of amplification were synthesized NCgl1751 Primers for fragments at both ends of the gene coding region, as upstream and downstream homology arm fragments. Primers were designed as follows (synthesized by Shanghai Yingjun Company):

[0042] P1: 5' CCCAAGCTTCGACAGGGCTTGGATTG 3' (Hind3)

[0043] P2: 5' ATGGAGAAAT ACGTCAAGGT TTTTCCTGCT CTTTAACACC 3'

[0044] P3: 5' GGTGTTAAAG AGCAGGAAAA ACCTTGACGT ATTTCTCCAT 3'

[0045] P4: 5' CGGGATCCCGGTGGGTTTGTTGATGT 3' (BamH1)

[0046] Using Corynebacterium glutamicum ATCC13032 as a template, PCR amplification was carried out with primers P1 / P2 and P3 / P4 respectively to obtain a 660bp upstream homology arm fragment and a 780bp downstream homology arm fragment, and then OVER PCR with primers P1 / P4 to obtain The entire homology arm fragment is 1440bp, and the ...

Embodiment 2

[0051] Example 2 NCgl0097 Gene expression downregulation experiments

[0052] According to the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, two pairs of amplification were synthesized NCgl0097 Primers for fragments at both ends of the gene coding region, as upstream and downstream homology arm fragments. Primers were designed as follows (synthesized by Shanghai Yingjun Company):

[0053] P11: 5' CCCAAGCTTCGCAGCAGGTATGTAGTCAC 3' (Hind3)

[0054] P12: 5' CACTTCATAG GGTTGAATAC AGCACGCGCA CGGAAAGCCA 3'

[0055] P13: 5' TGGCTTTCCG TGCGCGTGCT GTATTCAACC CTATGAAGTG 3'

[0056] P14: 5' GCTCTAGAGCGGGCATCCACAATCAT 3' (Xba1)

[0057] Using Corynebacterium glutamicum ATCC13032 as a template, PCR amplification was carried out with primers P11 / P12 and P13 / P14, respectively, to obtain a 740bp upstream homology arm fragment and a 640bp downstream homology arm fragment, and then OVER PCR with primers P11 / P14 to obtain The entire homology arm fragment is 13...

Embodiment 3

[0062] Example 3 NCgl1751 and NCgl0097 Gene double expression down-regulation experiment

[0063] Based on the YPL-1-001 strain, the Ncgl0097 gene coding region on the genome was knocked out. The specific process was the same as the above-mentioned knockout of the Ncgl0097 gene coding region. PCR was performed on the strain with identification primers P5 / P6 and P11 / P12 For verification, fragments of 740bp and 645bp were obtained respectively (the PCR sizes of the original strain were 1000bp and 1195bp respectively), and the genetically engineered strains in which the NCgl1751 and NCgl0097 gene coding regions were knocked out were named YPL-1-003, which is the valley Corynebacterium glutamicum was preserved on August 16, 2016 in the General Microbiology Center of China Committee for the Collection of Microorganisms (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), and the preservation number is CGMCC No. 12856.

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Abstract

The invention provides a method for producing L-lysine by fermentation. The method comprises the following steps: modifying a gene for coding an NCBI reference sequence NP_601029.1 and / or NP_599350.1 on a corynebacterium lilium bacterial chromosome to enable the activity and / or expression quantity of NP_601029.1 and / or NP_599350.1 to be reduced; fermenting germs obtained by modified to produce the L-lysine. Furthermore, the invention further provides a method derived from the method and application, and a germ applied to the methods and the application.

Description

technical field [0001] The invention belongs to the field of amino acid fermentation, in particular, the invention relates to methods and applications for fermentative production of L-lysine, and bacteria that can be used in these methods and applications. Background technique [0002] Production of L-lysine by fermentation of L-lysine-producing bacteria (eg, Escherichia coli of the genus Escherichia and rod-shaped bacteria of the genus Corynebacterium) has been industrially applied. These bacteria can be bacteria isolated from nature, bacteria obtained through mutagenesis or genetic engineering, or both. [0003] L-lysine-producing bacteria include bacteria of the genus Corynebacterium. For example, Chinese patent CN1017906B discloses a method for producing L-lysine, including using Corynebacterium bacterium containing recombinant DNA for synthesizing dihydrodipyridinecarboxylic acid synthase and / or succinyltetrahydropyridinecarboxylic acid synthetase to ferment A step of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/77C12N1/21C12P13/08C12Q1/68C12Q1/02C12R1/15
CPCC07K14/34C12N15/77C12N2800/101C12P13/08C12Q1/689C12Q2600/158
Inventor 孟刚魏爱英马风勇贾慧萍马吉银
Owner NINGXIA EPPEN BIOTECH
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