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Method for improving production capacity of L-arginine strain

A production capacity, arginine technology, applied in the field of genetic engineering, can solve the problems of low fermentation level, weakened branched metabolic pathways, etc., and achieve the effect of improving production capacity

Inactive Publication Date: 2019-12-13
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Abstract
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  • Application Information

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Problems solved by technology

[0005] In order to overcome the defect of low fermentation level of existing L-arginine production strains, the present invention uses genetic engineering technology to transform Corynebacterium glutamicum, by enhancing genes related to L-arginine production and weakening branched metabolic pathways, it can Significantly increase the L-arginine production capacity of the strain

Method used

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  • Method for improving production capacity of L-arginine strain
  • Method for improving production capacity of L-arginine strain
  • Method for improving production capacity of L-arginine strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1: Preparation of strain ATCC13032 (ΔargR) with argR gene knockout

[0082] 1.1 ATCC13032 genome extraction: connect a small amount of ATCC13032 glycerol bacteria (purchased from Shanghai Institute of Industrial Microbiology) to the BHIS test tube, culture on a constant temperature shaker at 30°C, 220rpm for 18 hours, collect the bacteria by centrifugation at 12000rpm, and use Axygen bacterial genome small-scale extraction reagent Cassette extraction of the ATCC13032 genome.

[0083] 1.2 Construction of knockout plasmid pK18mobsacB-argR

[0084] 1.2.1 Using the ATCC13032 genome extracted in step 1.1 as a template, use primers argR-aL-F and argR-aL-R to perform PCR amplification of the argR-aL fragment. The PCR system is as follows (the following PCR reagents were purchased from Toyobo TOYOBO KOD series): KOD Buffer 5μl, dNTP 5μl, MgSO 4 4 μl, primer argR-aL-F 0.5 μl, primer argR-aL-R 0.5 μl, KOD Plus Neo 1 μl, template 0.4 μl, ddH 2 O to make up 50 μl. The ...

Embodiment 2

[0095] Example 2: Preparation of strain ATCC13032 (ΔargRargBmut (A26V M31V) with mutant argB gene

[0096] 2.1 Construction of mutant plasmid pK18mobsacB-argBmut:

[0097] 2.1.1 Using the ATCC13032 genome extracted in step 1.1 as a template, use the primers argB-aL-F and argB-aL-R to amplify the argB-aL fragment according to the same PCR conditions as step 1.2.1, about 1kb, for the purpose of gel recovery fragment.

[0098] 2.1.2 Using the ATCC13032 genome extracted in step 1.1 as a template, use the primers argB-aR-F and argB-aR-R to amplify the argB-aR fragment according to the same PCR conditions as step 1.2.1, about 1kb, for the purpose of gel recovery fragment.

[0099] 2.1.3 Use Axygen's plasmid extraction kit to extract plasmid pK18mobsacB (gifted by Liu Shuangjiang, Institute of Microbiology, Chinese Academy of Sciences, its structure is as follows figure 1 shown), the plasmid was digested with HindIII and EcoRI, and the gel was recovered to obtain a 5.7kb vector fr...

Embodiment 3

[0109] Embodiment 3: the preparation of the bacterial strain CIBTS1512 (NCgl2620mut6) of the nucleotide sequence of the upstream regulatory region of the start codon of the mutation polyphosphate kinase gene (NCgl2620)

[0110] 3.1: Construction of recombinant plasmid pK18mobsacB-NCgl2620mut6

[0111] 3.1.1 Using the ATCC13032 genome extracted in step 1.1 as a template, use primers NCgl2620mut6-aL-F and NCgl2620mut6-aL-R to amplify the NCgl2620mut6-aL fragment, about 0.5kb, under the same PCR conditions as step 1.2.1, and recover from the gel target fragment.

[0112] 3.1.2 Using the ATCC13032 genome extracted in step 1.1 as a template, use primers NCgl2620mut6-aR-F and NCgl2620mut6-aR-R to amplify the NCgl2620mut6-aR fragment, about 0.5kb, according to the same PCR conditions as step 1.2.1, and recover from the gel target fragment.

[0113] 3.1.3 Use Axygen's plasmid extraction kit to extract plasmid pK18mobsacB (gifted by Liu Shuangjiang, Institute of Microbiology, Chinese...

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Abstract

The invention discloses a method for improving production capacity of an L-arginine strain through genetic engineering, which comprises the following steps: knocking out an argR gene in a corynebacterium glutamicum ATCC13032 genome to obtain a gene knockout strain ATCC13032 (delta argR); carrying out A26V and M31V mutation on the argB gene in the genome of the gene knockout strain to obtain a genemutation strain ATCC13032 (delta argR, argBmut (A26V M31V)); and mutating a base sequence of an upstream regulation region of an initiation codon of a polyphosphate kinase gene (NCgl2620) in a genomeof a gene mutation strain to obtain a gene engineering strain ATCC13032 (delta argR, argBmut (A26V M31V) and NCgl2620mut6). According to the method, the L-arginine production capacity of the strain can be improved by at least 15 times.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for improving the production capacity of L-arginine strains, in particular to a method for improving the production capacity of L-arginine-producing bacteria through genetic engineering. Background technique [0002] L-arginine (L-Argnine, referred to as L-Arg) is one of the semi-essential basic amino acids required in the human body. As a basic amino acid containing guanidinium, it is an important intermediate metabolism of the urea cycle in organisms It has a variety of unique physiological and pharmacological effects, and has good curative effects on treating physiological functions, cardiovascular diseases, stimulating the immune system, maintaining the nutritional balance of infants, and promoting detoxification of the human body. It is an important carrier for storing amino acids and is extremely important in intramuscular metabolism. It is an essent...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P13/10C12R1/15
CPCC07K14/34C12N9/1217C12P13/10C12Y207/02008
Inventor 杨晟蒋宇杨俊杰董枫刘映淼
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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