Genetically engineered bacterium for producing L-valine with high yield and establishment method and application of genetically engineered bacterium

A technology of genetically engineered bacteria and valine, applied in the field of microorganisms and molecular biology, can solve the problems of low L-valine yield, insufficient supply of pyruvate, weak metabolic flow, etc.

Active Publication Date: 2019-11-19
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is that in the prior art, the synthetic metabolic flow of L-valine is relatively weak, and the supply of key precursor pyruvate is insufficient, resulting in low yield of L-valine

Method used

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  • Genetically engineered bacterium for producing L-valine with high yield and establishment method and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing L-valine with high yield and establishment method and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing L-valine with high yield and establishment method and application of genetically engineered bacterium

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Embodiment 1

[0026] The efficient production of L-valine strains VHY01, VHY02, and VHY03 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–Cas9 mediated genome editing. Metabolic engineering, 2015,31:13-21.), the specific method is as follows:

[0027] 1 The acetolactate synthase coding gene alsS was integrated into the pseudogene site ydeU to construct strain VHY01

[0028] 1.1 Preparation of recombinant DNA fragments

[0029] 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 (SEQ ID NO: 1) was amplified by PCR; using the genome of E. 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 upstream primer of the alsS gene. The integrated fragment P was obtained by overlapping PCR with the upper and lower homology...

Embodiment 2

[0071] The method for producing L-valine by fermentation of genetically engineered bacteria VHY01, VHY02, and VHY03 is as follows:

[0072] (1) Shake flask fermentation of strains VHY01, VHY02, and VHY03:

[0073] Slant culture: Streak inoculation of -80°C preserved strains on the activated slant, culture at 37°C for 12 hours, and subculture twice;

[0074] 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;

[0075] Shake flask fermentation culture: Inoculate 10-15% inoculum into a 500mL Erlenmeyer flask containing fermentation medium (final volume is 30mL), 37°C, 200r / min shaking culture, during the fermentation process, maintain the pH at 6.7-7.0; add 60% (m / v) glucose solution to maintain fermentation;

[0076] The composition of the slant medium is: glucose 5g / L, peptone 10g / L, beef extract 10g / L, yeast powder 5g / L, NaC...

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Abstract

The invention provides a genetically engineered bacterium for producing L-valine with high yield, which is established by changing the metabolic pathway of escherichia coli. Starting from escherichiacoli W3110, an encoding gene alsS of acetolactate synthase of bacillus subtilis is integrated on a genome of the escherichia coli W3110 and is intensively expressed; in addition, an encoding gene spoTof an escherichia coli ppGpp 3'-pyrophosphoric acid hydrolase mutant [R290E, K292D] is integrated on the genome of the escherichia coli W3110 and is intensively expressed; and furthermore, an encoding gene thiE of thiamine phosphate synthase is knocked off, and thus the genetically engineered bacterium is established. By adopting the genetically engineered bacterium, the problem that L-valine islow in yield as L-valine is weak in synthesis metabolic flux and a key precursor pyruvic acid is insufficient in supply can be solved.

Description

technical field [0001] The invention belongs to the field of microbiology and molecular biology, and relates to a genetically engineered strain of high-yield L-valine and its construction method and application. Background technique [0002] L-valine belongs to branched-chain amino acid and is an essential amino acid for human body. L-valine is widely used in the fields of medicine, food and feed, and has high commercial value. L-valine can be used as an antitumor drug in medicine and a food additive in food. In addition, L-valine has become an important feed amino acid. [0003] At present, the production method of L-valine mainly adopts the microbial fermentation method with glucose as raw material. The L-valine synthesis branch pathway starts from pyruvate and undergoes the following steps to form L-valine. Pyruvate is catalyzed by acetolactate synthase to form α-acetolactate, and α-acetolactate is catalyzed by dicarboxylic acid reductoisomerase to form α, β-dihydroxy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/90C12P13/08C12R1/19
CPCC12N9/1022C12N9/1085C12N9/14C12N15/902C12P13/08C12Y202/01006C12Y205/01003C12Y306/01001C12N2310/20
Inventor 谢希贤郝亚男刘晓倩蒋帅门佳轩刘益宁文晨辉李旋
Owner TIANJIN UNIV OF SCI & TECH
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