Method for producing gamma-aminobutyric acid by whole cell transformation method

A production method and amino acid technology, applied in the biological field, can solve the problems of inability to meet the needs of industrial production, low yield of γ-aminobutyric acid, etc., and achieve the effect of reducing the cost of separation and extraction, single component, and high-efficiency expression

Inactive Publication Date: 2019-05-07
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, currently reported food-safe strains such as lactic acid bacteria or Corynebacterium glutamicum produce low yields of GABA, which cannot meet the needs of industrial production

Method used

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  • Method for producing gamma-aminobutyric acid by whole cell transformation method
  • Method for producing gamma-aminobutyric acid by whole cell transformation method
  • Method for producing gamma-aminobutyric acid by whole cell transformation method

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Experimental program
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Embodiment 1

[0058] The vector construction used in the present invention preferably following method:

[0059] Guided by primers gad-F (SEQ ID No.3) / gad-R (SEQ ID No.4), preferably using the codon-optimized BmGAD gene (SEQ ID No.2) as a template for PCR amplification reaction, amplified The augmented fragment was digested with HindIII and BamHI and then ligated into the plasmid pXMJ19 digested with HindIII and BamHI to construct the plasmid pXMJ19-BmGAD.

[0060] The vector pXMJ19-BmGAD is transformed into a Corynebacterium glutamicum strain to obtain a Corynebacterium glutamicum strain CG-BmGAD capable of catalytically producing γ-aminobutyric acid in whole cells.

[0061] Preferably, in the present invention, the Corynebacterium glutamicum strain is preferably CG-BmGAD.

[0062] Those skilled in the art should understand that Corynebacterium glutamicum and Bacillus megaterium exhibit different degrees of codon preference when expressing proteins. The codon optimization of the glutamat...

Embodiment 2

[0067] Seed medium: glucose 10-15g / L, corn steep liquor 1-3g / L, ammonium sulfate 10-20g / L, magnesium sulfate 0.5-1g / L, potassium dihydrogen phosphate 0.5-1g / L, sodium citrate 0.5- 2g / L.

[0068] Fermentation medium: glucose 40-60g / L, corn steep liquor 5-8g / L, ammonium sulfate 10-30g / L, magnesium sulfate 0.5-5g / L, potassium dihydrogen phosphate 0.5-5g / L, sodium citrate 0.5- 2g / L.

Embodiment 3

[0070] This example is used to illustrate the high-density fermentation culture of recombinant Corynebacterium glutamicum CG-BmGAD.

[0071] In the present invention, the method for fermenting culture may comprise the following steps:

[0072] (1) Inoculate the recombinant Corynebacterium glutamicum expression strain CG-BmGAD constructed as described above into the seed medium containing chloramphenicol as a selection marker, and cultivate it for 16 hours at 30 degrees Celsius to obtain recombinant glutamic acid Corynebacterium acid CG-BmGAD seed solution;

[0073] (2) Inoculate the recombinant Corynebacterium glutamicum CG-BmGAD seed liquid obtained in step (1) into the fermentation medium containing the chloramphenicol as the screening marker, and cultivate it at 30 degrees Celsius, when the OD 600Add isopropyl-β-D-thiogalactopyranoside (IPTG) when the temperature reaches 5-10, and induce culture at 16-20 degrees Celsius for 3-6 hours.

[0074] Those skilled in the art sho...

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Abstract

The invention discloses a method for producing gamma-aminobutyric acid by a whole cell transformation method. Corynebacterium glutamicum is used as a production bacterial strain, glutamate decarboxylase of bacillus megaterium is in heterologous expression, and through high-density fermentation culture, protein over-expressed corynebacterium glutamicum thalli cells are obtained. L-glutamic acid orL-glutamate is used as a substrate, and the gamma-aminobutyric acid is produced through corynebacterium glutamicum whole cell catalyzing. According to the method disclosed by the invention, glutamatedecarboxylase high in catalytic activity and a production bacterial strain namely the corynebacterium glutamicum in food safety grade are used for producing the gamma-aminobutyric acid, and the methodhas the advantages that the yield of the gamma-aminobutyric acid is high, transformation liquid is single in ingredients, the production cycle is short, the operation is simple, the production cost is low, and potential safety hazard does not exist.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to high-efficiency production of gamma-aminobutyric acid by transforming L-glutamic acid or L-glutamate through whole cells using Corynebacterium glutamicum as a production strain. Background technique [0002] γ-Aminobutyric acid (GABA) is a naturally occurring functional non-protein amino acid, which has physiological functions such as improving brain activity, lowering blood pressure, anti-anxiety, regulating hormone secretion, treating epilepsy and protecting liver and kidney. Health care, beverage processing [1] , cosmetics and other fields have broad application prospects and market demand [2] . The preparation methods of γ-aminobutyric acid mainly include chemical synthesis, plant enrichment, microbial fermentation and biotransformation. The chemical synthesis method has harsh conditions, high energy consumption, high cost, low yield and poor safety, so it cannot be used in food...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/77C12P13/00C12R1/15
Inventor 刘君周威徐宁
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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