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Genetic engineering bacterium for producing L-isoleucine and application thereof

A technology of genetically engineered bacteria and isoleucine, applied in the direction of microorganism-based methods, bacteria, microorganisms, etc., can solve the problems of slow growth of L-isoleucine production strains, unstable fermentation, nutritional deficiencies, etc., and achieve fermentation The effect of short cycle, fast growth and high conversion rate

Active Publication Date: 2019-10-08
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 acetohydroxyacid synthase being inhibited by L-isoleucine feedback and the existing L-isoleucine production strains such as slow growth, nutritional deficiencies, and unstable fermentation, the invention provides a method for releasing L-isoleucine. Acetohydroxyacid synthase mutant and its coding gene for isoleucine feedback inhibition, and using the gene to construct genetically engineered bacteria producing L-isoleucine

Method used

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  • Genetic engineering bacterium for producing L-isoleucine and application thereof
  • Genetic engineering bacterium for producing L-isoleucine and application thereof
  • Genetic engineering bacterium for producing L-isoleucine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Gene ilvBN Encoding Acetohydroxyacid Synthase Relieving L-Isoleucine Feedback Inhibition M the acquisition

[0052] (1) Screening of mutants resistant to L-isoleucine structural analogues

[0053] ① Preparation of Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 bacterial suspension

[0054] 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.

[0055] ② Plasma mutagenesis at atmospheric pressure and room temperature

[0056] 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.

[0057] ③ Screening of mutant strains resistant to L-isoleucine structural analog α-...

Embodiment 2

[0076] Example 2: Construction of L-isoleucine-producing bacteria TUIE03

[0077] (1) Construction of recombinant fragment UHF-ilvA-DHF

[0078] Using the genome of Escherichia coli W3110 as a template, the primers Ilv-3, Ilv-4, Ilv-5 and Ilv-6 were amplified by overlapping PCR to obtain the threonine dehydratase coding gene ilvA that relieves L-isoleucine feedback inhibition (shown in SEQ ID NO.5). Using the Escherichia coli W3110 genome as a template, primers Ilv-1, Ilv-2, Ilv-3, Ilv-6, Ilv-7 and Ilv-8 were used for overlapping PCR amplification, and the recombinant fragment UHF-ilvA- DHF, UHF and DHF are the upper and lower homology arms of the lacY gene, respectively.

[0079] (2) Construction of recombinant fragment UHF-ilvBN-DHF

[0080] 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 W3110 genome as a template, using primers IlvB-...

Embodiment 3

[0085] Embodiment 3: Fermentation tank fermentation experiment of L-isoleucine producing bacteria TUIE03

[0086] (1) Seed cultivation

[0087] Use an inoculation loop to inoculate 3-5 TUIE03s activated on fresh slopes into a 5L fermenter with 1L of seed medium, add 25% (W / V) ammonia water to adjust the pH of the fermentation broth to 6.8-7.2, dissolve oxygen Maintain at 20-40%, ventilation volume 3-5m 3 / h, the stirring speed is 400rpm, and cultivated at 37°C for 6-8h.

[0088] (2) Fermentation tank fermentation

[0089] Connect the seed culture of step (1) with 5% inoculum to a 5L fermenter with 3L fermentation medium for fermenting. The fermentation temperature is 35°C and the ventilation rate is 3-5m 3 / h, the stirring speed is 500rpm, the dissolved oxygen is maintained at 20-40%, and the glucose solution with a concentration of 70% (W / V) is added to maintain the concentration of residual sugar at 0.1-0.5% (W / V), and 25% is added (W / V) ammonia water adjusts the pH of t...

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Abstract

The invention relates to a genetic engineering bacterium for producing L-isoleucine and an application thereof, which belong to the field of metabolic engineering. The genetic engineering bacterium isobtained by overexpressing an acetohydroxyacidsynthase encoding gene ilvBN<M>, a threonine dehydratase encoding gene ilvA, and a threonine operon thrABC releasing feedback inhibition of L-isoleucine,the acetohydroxyacidsynthase encoded by ilvBN<M> releases the feedback inhibition of L-isoleucine, and its activity is not significantly lower than that of the wild-type ilvBN-encoded acetohydroxyacidsynthase; The L-isoleucine genetic engineering bacterium of the invention has no nutritional deficiency, rapid growth, short fermentation cycle, high yield and high conversion rate, and after 26-36hfermentation, the concentration of L-isoleucine in a fermentation broth reaches 42.5-51.6g / L.

Description

Technical field: [0001] The invention relates to a genetically engineered bacterium for producing L-isoleucine and an application thereof, belonging to the field of metabolic engineering. Background technique: [0002] L-isoleucine is a branched-chain amino acid and is one of the eight essential amino acids for the human body. L-Isoleucine is a raw material for the synthesis of human hormones and enzymes. It can promote protein synthesis and inhibit its decomposition, and plays a vital role in human life activities. Therefore, L-isoleucine has very broad market and application prospects in industries such as food and medicine. [0003] The synthesis methods of L-isoleucine include extraction method, chemical synthesis method and fermentation method. Industrial production mainly uses hair extraction method and fermentation method to synthesize L-isoleucine. Because the extraction method has the disadvantages of limited source of raw materials, high production cost, and env...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P13/06C12R1/19
CPCC12N9/1022C12N9/88C12N15/52C12N15/70C12P13/06C12Y202/01006C12Y403/01019
Inventor 张成林李英滋韩世宝徐庆阳李燕军张宇芦楠朱福周董解荣陈亭利
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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