Recombinant bacteria producing γ-aminobutyric acid and its construction method and application

A technology of aminobutyric acid and recombinant bacteria, applied in the direction of microorganism-based methods, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problem of low yield of bacterial cells and active proteins, difficult production process control, strong cytotoxicity And other issues

Active Publication Date: 2017-09-22
农博士(福建)生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The lactose operon of the pET-28a(+) expression vector, the protein expression is not rigorous enough and the cells need to be cultured to a certain concentration before adding inducers, the production process is difficult to control; the high-copy replication initiation site and strong T7 promoter are used The progeny makes bacteria express many inclusion body proteins and the inducer IPTG has strong cytotoxicity, and the yield of bacterial cells and active proteins is low

Method used

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  • Recombinant bacteria producing γ-aminobutyric acid and its construction method and application
  • Recombinant bacteria producing γ-aminobutyric acid and its construction method and application
  • Recombinant bacteria producing γ-aminobutyric acid and its construction method and application

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Embodiment 1

[0091] Embodiment 1, the construction of producing γ-aminobutyric acid genetically engineered bacteria

[0092] One, construct the recombinant plasmid expressing the coding gene of glutamic acid decarboxylase B

[0093] Replace the DNA sequence between the NcoI and EcoRI recognition sites of the pBAD / HisB vector with the DNA sequence used to encode glutamic acid decarboxylase B shown in SEQ ID No.4, keep other DNA sequences unchanged, and obtain the recombinant vector pEcgadB ( figure 1 ). Restriction identification proved that the gene encoding glutamic acid decarboxylase B was successfully inserted between the NcoI and EcoRI recognition sites of the pBAD / HisB vector. The recombinant vector pEcgadB can express glutamic acid decarboxylase B shown in SEQ ID No.1, and the nucleotide sequence shown in SEQ ID No.4 is the coding sequence of glutamic acid decarboxylase B.

[0094] 2. Construction of Escherichia coli mutant strain K12ΔgadAB with knockout γ-aminobutyric acid catabo...

Embodiment 2

[0114] Embodiment 2, producing gamma-aminobutyric acid genetically engineered bacteria transforming L-glutamic acid to produce gamma-aminobutyric acid

[0115] 1. Induction culture of genetically engineered bacteria producing γ-aminobutyric acid

[0116] The genetically engineered bacteria KG01, KG02, KG03, KG04, KG05, Escherichia coli K12ΔgabT (referred to as K12ΔgabT strain) and Escherichia coli K12 (referred to as K12 strain) were streaked to agar containing 1.5% mass concentration respectively. and ampicillin with a mass concentration of 100 μg / mL on LB plates, and cultured at 37°C for 12 hours. Pick a single colony on the plate, inoculate it into the liquid LB medium containing ampicillin with a mass concentration of 100 μg / mL, culture it overnight at 37° C. with shaking at 220 rpm; The inoculation amount was inoculated into the self-inducing medium ZYM, and the shaking culture was carried out at 200 rpm and 30°C for 16 hours to obtain the induced KG01 strain, KG02 strai...

Embodiment 3

[0135] Example 3 Preparation of γ-aminobutyric acid by genetically engineered bacteria KG01

[0136] Pick a single bacterium colony of the genetically engineered bacterium KG01 and inoculate it into the liquid LB medium containing ampicillin with a mass concentration of 100 μg / mL, cultivate overnight at 37° C. with shaking at 220 rpm; inoculate the overnight culture with 1% (volume percentage) Inoculate a large amount into a 100L fermenter equipped with 70L self-inducing medium ZYM, with an aeration ratio of 0.6-0.8vvm, and ferment for 16 hours at 30°C and a rotation speed of 300rpm to obtain a fermentation broth. Then use a tube centrifuge to centrifuge the fermentation broth, collect KG01 cells into a 150L transformation tank, add about 60L of pure water to resuspend the strains, and obtain the resuspended strains, so that the content of the cells in the resuspended strains is calculated by the wet weight of the cells It was calculated as 20g / L, and glutamic acid was added t...

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Abstract

The invention discloses a recombinant bacterium producing γ-aminobutyric acid, its construction method and application. Adopt the method of the present invention to import glutamic acid decarboxylase B gene (gadB gene) into Escherichia coli mutant strain K12ΔgadABC through the expression vector pBAD / HisB. Glutamic acid: γ-aminobutyric acid antiporter is sensitive to pH, which effectively reduces the degradation of γ-aminobutyric acid and improves the yield and conversion efficiency of GABA. The transformation rate of KG01 strain was not affected by pH change, and the production of GABA was the highest, which was significantly higher than that of K12ΔgabT strain and K12 strain. Using glutamic acid with a concentration of 2M as the substrate, the KG01 strain was continuously transformed three times, and the transformation rate was above 99% each time, the yield was greater than 204g / L, and the substrate residue was low, which was convenient for downstream crystallization and purification. The gamma-aminobutyric acid produced by the genetically engineered bacteria constructed by the method of the invention has the advantages of low raw material, simple process, high production efficiency, etc., and has good industrial application prospect.

Description

technical field [0001] The invention relates to a recombinant bacterium producing gamma-aminobutyric acid in the field of biotechnology, a construction method and application thereof. Background technique [0002] γ-Aminobutyric acid (γ-aminobutyric acid, GABA) is a non-protein natural amino acid, which widely exists in microorganisms, plants and animal cells. GABA is an inhibitory neurotransmitter in the central nervous system of mammals. It has physiological functions such as reducing blood ammonia, anti-anxiety, lowering blood pressure, treating diabetes, promoting alcohol metabolism, improving kidney and liver functions, deodorization, and weight loss. It is widely used in In food health care, medicine and feed industry. In addition, GABA can also be polymerized to form nylon 4, which also has good application prospects in the chemical industry. [0003] At present, the ideal way for large-scale preparation of GABA is to convert L-glutamic acid or its sodium salt by mi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N1/21C12P13/00C12R1/19
Inventor 柯崇榕黄建忠陶勇杨欣伟胡美荣饶焕新曾文超
Owner 农博士(福建)生物技术有限公司
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