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Recombinant corynebacterium glutamicum for high production of gamma-aminobutyric acid as well as construction method and application thereof

A technology of Corynebacterium glutamicum and GABA, applied in the direction of microorganism-based methods, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of complex components of fermentation broth, high risk factor, and many side reactions, etc. Achieve the effect of good industrial application prospect, high substrate conversion rate and simple fermentation process

Active Publication Date: 2014-02-05
NANJING UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The composition of the fermentation broth obtained by the direct fermentation method to synthesize GABA is relatively complex, which will increase the cost of downstream separation and extraction, and is one of the bottleneck problems in the industrial production of GABA
[0004] Although the chemical method has the characteristics of rapid reaction, due to the many side reactions and residual chemical substances in this method, even if the pure product is obtained, it is not a natural product. At the same time, the reaction has strict requirements on environmental conditions and high energy consumption. high risk factor

Method used

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  • Recombinant corynebacterium glutamicum for high production of gamma-aminobutyric acid as well as construction method and application thereof
  • Recombinant corynebacterium glutamicum for high production of gamma-aminobutyric acid as well as construction method and application thereof
  • Recombinant corynebacterium glutamicum for high production of gamma-aminobutyric acid as well as construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1: Construction of recombinant Corynebacterium glutamicum C. glutamicum NJ-M6.

[0035] Strains: Corynebacterium glutamicum C. glutamicum CICC10240.

[0036] Exogenous gene: the glutamate decarboxylase gene gad derived from Arthrobacter (strain number; CGMCC No.3584).

[0037] Vector: E.coli / C. glutamicum shuttle vector pXJ19.

[0038] Using the Arthrobacter CGMCC3584 genome as a PCR template, the glutamic acid decarboxylase gene gad was amplified by PCR; figure 1 , the size of the gad gene is 1392bp. Connect the two ends of the gad fragment to EcoRI and HindIII sites respectively, and the primers are:

[0039] Sense strand: 5'-TCGCGGATCCGAATTCATGTCACACGGCGACGACGA-3';

[0040] Antisense strand: 3'-TGCGGCCGCAAAGCTTTCAGCTTCCGCGTACTGCGG-5'.

[0041] The obtained glutamic acid decarboxylase gene gad product was double-enzymatically digested and loaded into the shuttle vector pXJ19 to form a fusion fragment, which was then identified by enzyme digestion and sequ...

Embodiment 2

[0045] This example illustrates the process of producing γ-aminobutyric acid by fed-feed fermentation of Corynebacterium glutamicum C. glutamicum NJ-M6.

[0046] The medium formula described in this embodiment (% is mass percent):

[0047] Solid plate medium: 1L distilled water contains: 1L distilled water contains 10g peptone, 5g yeast powder, 10g sodium chloride, 15g agar powder, pH7.0.

[0048] Seed medium: 1L distilled water containing 30g glucose, 10g ammonium sulfate, 5g peptone, 3.5g corn steep liquor, 1.0g potassium dihydrogen phosphate, 1.0g sodium chloride, pH=7.0.

[0049] Fermentation medium: 1L distilled water containing 30g glucose, 10g sodium glutamate, 26g ammonium sulfate, 20g corn steep liquor, 4.5g potassium dihydrogen phosphate, 2.5g magnesium sulfate, pH=7.0.

[0050] Inoculate the Corynebacterium glutamicum NJ-M6 preserved in the glycerol tube onto the LB medium at a culture temperature of 30 degrees for 8-12 hours; culture the Corynebacterium glutamicum...

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Abstract

The invention discloses recombinant corynebacterium glutamicum for high production of gamma-aminobutyric acid. The recombinant corynebacterium glutamicum is classified and named as corynebacterium glutamicum NJ-M6 and is collected at the China Center for Type Culture Collection (CCTCC) with the collection number of CCTCC No. M2013486 on October 22, 2013. Corynebacterium glutamicum of a glutamate decarboxylas gene derived from arthrobacterium is guided into the recombinant corynebacterium glutamicum. The invention also discloses a construction method and an application of the recombinant corynebacterium glutamicum. By the method, the concentration of gamma-aminobutyric acid in feed-batch fermentation liquid can reach 36.1g / L, while C. glutamicum CICC10240 basically does not produce gamma-aminobutyric acid under the same culture condition.

Description

technical field [0001] The invention relates to a recombinant Corynebacterium glutamicum capable of high-yielding gamma-aminobutyric acid, its construction method and application in gamma-aminobutyric acid production, belonging to the technical field of biological fermentation. Background technique [0002] γ-aminobutyric acid (γ-aminobutyric acid, GABA), also known as aminobutyric acid, belongs to amino acid derivatives, and its molecular formula is C 4 h 9 NO 2 , the relative molecular mass is 103.12. γ-aminobutyric acid is an important inhibitory neurotransmitter, which has important physiological regulation effects on mammals, such as lowering blood pressure, improving sleep and anti-arrhythmia; the Ministry of Health of my country has approved γ-aminobutyric acid as a new resource food. In recent years, the market demand for γ-aminobutyric acid is increasing day by day. [0003] The synthesis methods of γ-aminobutyric acid mainly include chemical synthesis and biosy...

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 谢婧婧郭亭周韬玉陈可泉周治冯晓慈
Owner NANJING UNIV OF TECH
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