Genetically engineered bacterium for high-yielding L-valine and method for producing L-valine by fermentation

A technology of genetically engineered bacteria and valine, applied in the field of microorganisms, can solve the problems of unbalanced coenzyme supply and demand, low sugar-acid conversion rate, etc.

Active Publication Date: 2019-12-24
TIANJIN UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] One of the purposes of the present invention is to provide a new idea for the construction of L-valine engineering bacteria, which solves the problems of unbalanced supply and demand of coenzyme and low conversion rate of sugar and acid in the synthesis and metabolism of L-valine, and obtains through directional transformation A Genetically Engineered Strain of Escherichia coli with High Production of L-Valine

Method used

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  • Genetically engineered bacterium for high-yielding L-valine and method for producing L-valine by fermentation
  • Genetically engineered bacterium for high-yielding L-valine and method for producing L-valine by fermentation
  • Genetically engineered bacterium for high-yielding L-valine and method for producing L-valine by fermentation

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Experimental program
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Effect test

Embodiment 1

[0038] The efficient L-valine production strains VXR01, VXR02, and VXR03 were constructed, and the gene editing method used was referred to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR–Cas9meditated genome editing. Metabolic engineering, 2015 ,31:13-21.), the specific method is as follows:

[0039] 1 The acetolactate synthase coding gene alsS was integrated into the pseudogene site ydeU to construct strain VXR01

[0040] 1.1 Preparation of recombinant DNA fragments

[0041] The alsS gene was integrated into the ydeU pseudogene locus. Using the genome of Bacillus subtilis B.subtilis 168 as a template, the gene alsS (NCBI-Protein ID: NP_391482.2) was amplified by PCR; using the genome of Escherichia coli W3110 as a template, the upstream and downstream homology arm fragments of ydeU (primer UP -ydeU-S, UP-ydeU-A; DN-ydeU-S, DN-ydeU-A); promoter P trc Designed in the downstream primer of the upstream homology arm and the u...

Embodiment 2

[0103] Shake flask fermentation of strains VXR02 and VXR05:

[0104] Slant culture: Streak inoculation of -80°C preserved strains on the activated slant, culture at 37°C for 12 hours, and subculture twice;

[0105] Shake flask seed culture: use the inoculation loop to scrape two rings of slant seeds and inoculate them into a 500mL Erlenmeyer flask containing 30mL of seed medium, culture at 37°C and 200rpm for 8-10h;

[0106] Shake flask aerobic fermentation: Inoculate 10-15% of the inoculum into a 500mL Erlenmeyer flask with fermentation medium (final volume is 30mL), culture at 37°C with shaking at 200r / min, maintain pH by adding ammonia water during fermentation At 6.7-7.0, add 60% (m / v) glucose solution to maintain the fermentation, and the fermentation period is 24h;

[0107] Aerobic-anaerobic two-stage fermentation: Inoculate 10-15% of the inoculum into a 500mL Erlenmeyer flask with fermentation medium (final volume is 30mL), 37°C, 200r / min shaking culture; after 12-16h,...

Embodiment 3

[0115] 5L fermenter fermentation of strains VXR02 and VXR05:

[0116] Aerobic fermentation: activate the strains on the slope for two generations, insert 15-20% of the inoculum into the fermentation medium, and start fermentation. During the fermentation process, the pH is controlled to be stable at about 6.7, the temperature is maintained at 35°C, and the dissolved oxygen is controlled at 25-30%; when the glucose in the medium is consumed, add 80% (m / v) glucose solution to maintain the glucose concentration in the fermentation medium at 0.1-5g / L; the fermentation period is 40h;

[0117] Aerobic-anaerobic two-stage fermentation: Activate the strains for two generations on a slant, insert 15-20% of the inoculum into the fermentation medium, and start fermentation. During the fermentation process, the pH is controlled to be stable at around 6.7, and the temperature is maintained at 35°C , control dissolved oxygen at 25-30%; when the glucose in the medium is consumed, add 80% (m / ...

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Abstract

The invention provides a genetically engineered bacterium for high-yielding L-valine. A construction method of the genetically engineered bacterium comprises the steps that starting from an escherichia coli W3110, an acetolactate synthase gene alsS of a bacillus subtilis is integrated on a genome of the escherichia coli W3110 and subjected to high expression; an escherichia coli ppGpp 3'-pyrophosphoric acid hydrolytic enzyme mutant R290E/K292D gene spoT is integrated on the genome of the escherichia coli W3110 and subjected to high expression; genes of frdA, frdB, frdC and frdD of four subunits of a lactic dehydrogenase gene ldhA, a pyruvate formate lyase I gene pflB and fumaric reductase on the genome of the escherichia coli W3110 are knocked out; a branched chain amino acid transaminasegene ilvE of the escherichia coli is replaced with leucine dehydrogenase gene bcd of the bacillus subtilis; and an acetyl-hydroxyl acid isomerized reductase gene ilvC of the escherichia coli is replaced with an encoding gene of a mutant L67E/R68F/K75E. According to the genetically engineered bacterium for the high-yielding L-valine, an L-valine fermentation method is further modified. Double-phasedissolved oxygen control is adopted, and the L-valine yield and the saccharic acid conversion rate are improved.

Description

technical field [0001] The invention relates to the field of microorganisms, genetic engineering and fermentation engineering, in particular to a genetically engineered bacterium with high yield of L-valine and its application. Background technique [0002] L-valine (L-valine) is a branched chain amino acid (Branched chain amino acids, BCAA), widely used in food, medicine, cosmetics and feed and other fields. As an essential amino acid for the human body, it can not only promote muscle formation, strengthen liver function, and reduce muscle fatigue, but also serve as an intermediate in the synthesis of immune antibiotics and drugs, and it can also promote skin glial protein synthesis. In recent years, as an important dietary amino acid variety, the demand for L-valine continues to increase. [0003] The synthetic branch of L-valine uses pyruvate as the immediate precursor. Pyruvate undergoes a four-step reaction catalyzed by acetohydroxyacid synthase AHAS, acetohydroxyacid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P13/08C12R1/19
CPCC12P13/08C12N9/1022C12Y202/01006C12N9/14C12N9/0016C12N9/0004C12Y306/01005C12Y104/01009C12R2001/19C12N15/52C12R2001/125C12N15/70
Inventor 谢希贤刘晓倩郝亚男门佳轩刘益宁李旋田道光文晨辉熊博
Owner TIANJIN UNIV OF SCI & TECH
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