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Genetic engineering bacterium for producing L-leucine, and application of genetic engineering bacterium

A technology of genetically engineered bacteria and leucine, applied in the field of metabolic engineering, can solve the problems of unstable fermentation, slow growth of L-leucine-producing strains, nutrient deficiencies, etc., and achieves fast growth, short fermentation period and high transformation rate. Effect

Active Publication Date: 2019-12-10
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to overcome the shortcomings of the current wild-type isopropylmalate synthase being inhibited by L-leucine feedback and the existing L-leucine production strains such as slow growth, nutritional deficiencies, and unstable fermentation, the invention provides a method for releasing L-leucine. Leucine feedback-inhibited isopropylmalate synthase mutant and its coding gene, and using the gene to construct a genetically engineered bacterium producing L-leucine

Method used

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  • Genetic engineering bacterium for producing L-leucine, and application of genetic engineering bacterium
  • Genetic engineering bacterium for producing L-leucine, and application of genetic engineering bacterium
  • Genetic engineering bacterium for producing L-leucine, and application of genetic engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Isopropylmalate Synthase Encoding Gene leuA Relieving L-Leucine Feedback Inhibition M the acquisition

[0058] (1) Screening of mutants resistant to L-leucine structural analogues

[0059] ① Preparation of Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 bacterial suspension

[0060] Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 was inoculated into LB liquid medium, cultured at 32°C, 200rpm for 12h, collected by centrifugation, washed with sterile saline for 3 times and then resuspended to make the OD 600 =0.6-0.8, take 10 μL of the bacterial suspension and spread it on the slide.

[0061] ② Plasma mutagenesis at atmospheric pressure and room temperature

[0062] The mutagenesis parameters were as follows: the slide was placed at 2mm from the airflow port, the power was 120W, the airflow was 10SLM, and the action time was 20s.

[0063] ③ Screening of mutant strains resistant to L-leucine structural analog α-aminobutyri...

Embodiment 2

[0085] Example 2 Release the acetohydroxyacid synthase coding gene ilvBN of L-isoleucine feedback inhibition M the acquisition

[0086] (1) Screening of mutants resistant to L-isoleucine structural analogues

[0087] ① Preparation of Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 bacterial suspension

[0088] Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 was inoculated into LB liquid medium, cultured at 32°C, 200rpm for 12h, collected by centrifugation, washed with sterile saline for 3 times and then resuspended to make the OD 600 =0.6-0.8, take 10 μL of the bacterial suspension and spread it on the slide.

[0089] ② Plasma mutagenesis at atmospheric pressure and room temperature

[0090] The mutagenesis parameters were as follows: the slide was placed at 2mm from the airflow port, the power was 120W, the airflow was 10SLM, and the action time was 25s.

[0091] ③ Screening of mutant strains resistant to L-isoleucine structural analog ...

Embodiment 3

[0110] Embodiment 3: Construction of L-leucine producing bacteria TE03

[0111] (1) Recombinant fragment UHF-leuA M - Construction of DHF

[0112] synthetically containing leuA M The plasmid of the gene is used as a template, and LEUA-3 and LEUA-4 are used as primers for PCR amplification to obtain leuA M ;

[0113] Using the Escherichia coli W3110 genome as a template, using primers LEUA-1 and LEUA-2 and LEUA-5 and LEUA-6 to amplify the fragments UHF and DHF respectively, UHF and DHF are the upper and lower homology arms of the lacI gene respectively; UHF, DHF and leuA M As a template, use primers LEUA-1 and LEUA-6 for PCR amplification, and after recovery, it is the recombinant fragment UHF-leuA M -DHF.

[0114] (2) Recombinant fragment UHFA-ilvBN M - Construction of DHFB

[0115] Synthetic containing ilvBN M The plasmid of the gene is used as a template, and IlvB-3 and IlvB-4 are used as primers to perform PCR amplification to obtain ilvBN M ; Using the E. coli W3...

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Abstract

The invention relates to a genetic engineering bacterium for producing L-leucine, and application of the genetic engineering bacterium, and belongs to the field of metabolic engineering. The genetic engineering bacterium is obtained by an isopropylmalate synthetase encoding gene leuA[M] capable of relieving L-leucine feedback inhibition through overexpression in a host cell, an acetolactate synthase encoding gene ilvBN[M] capable of relieving L-leucine feedback inhibition, a 3-isopropylmalate dehydrogenase encoding gene leuB and a 3-isopropylmalate dehydratase encoding gene leuCD. Acetohydroxyacid synthase encoded by the leuA[M] relieves the feedback inhibition function of the L-leucine for the acetohydroxyacid synthase, in addition, the activity of the acetohydroxyacid synthase is not obviously lowered than wild leuA encoded isopropylmalate synthetase. The L-leucine genetic engineering bacterium does not have nutritional deficiencies, is quick in growth, and has a short fermentation period, a high yield and a high conversion rate. After fermentation is conducted for 40-44h, the concentration of the L-leucine in fermentation liquor achieves 60.5-69.6g / L.

Description

Technical field: [0001] The invention relates to a genetically engineered bacterium for producing L-leucine and an application thereof, belonging to the field of metabolic engineering. Background technique: [0002] L-leucine belongs to branched-chain amino acids and is one of the eight essential amino acids for the human body. L-leucine is a raw material for the synthesis of proteins and hormones, and plays a vital role in human life activities. Therefore, L-leucine has very broad market and application prospects in industries such as food and medicine. [0003] Industrial L-leucine synthesis methods include hair extraction and fermentation. However, the extraction method has the disadvantages of limited source of raw materials, high production cost, and environmental pollution. Therefore, the fermentation method is the mainstream method for producing L-leucine. At present, the industrial production of L-leucine is mainly obtained by mutagenesis, which has deficiencies s...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/10C12N15/70C12P13/06C12R1/19
CPCC12N9/0006C12N9/1022C12N9/1025C12N9/88C12N15/52C12N15/70C12P13/06C12Y101/01085C12Y202/01006C12Y203/03013C12Y402/01033
Inventor 张成林徐庆阳李燕军张宇李英滋朱福周卢楠韩世宝董解荣王子申徐昊李子翼
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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