One-step method for synthesizing gamma-aminobutyric acid by using recombinant corynebacterium crenatum and with glucose as substrate

A technology of Corynebacterium bluff and aminobutyric acid, which is applied in the directions of microorganism-based methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as lack of γ-aminobutyric acid synthesis pathway, and reduce production. Cost, improved safety, effect of simplified preparation steps

Active Publication Date: 2013-07-24
南宁汉和生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The engineered bacterium lacks the γ-aminobutyric acid synthesis pathway, but can efficiently convert glucose into glutamic acid

Method used

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  • One-step method for synthesizing gamma-aminobutyric acid by using recombinant corynebacterium crenatum and with glucose as substrate
  • One-step method for synthesizing gamma-aminobutyric acid by using recombinant corynebacterium crenatum and with glucose as substrate
  • One-step method for synthesizing gamma-aminobutyric acid by using recombinant corynebacterium crenatum and with glucose as substrate

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Knockout Verification of the Corynebacterium blunt-toothed argB Gene

[0048] The identification of wild type / gene deletion type was carried out on the strains with the second homologous recombination. The chromosomes of transformants were extracted, and the upstream and downstream primers of the target gene argB were used for PCR identification. PCR identification results such as figure 1 shown.

Embodiment 2

[0049] Example 2: Expression of glutamic acid decarboxylase in E.coli JM109

[0050] The recombinant plasmid pGAD was transferred into E.coli JM109, and the positive transformant was picked on the kanamycin-resistant LB plate to liquid LB. After IPTG induction, the bacteria were collected and crushed by ultrasonic waves. The supernatant was detected by SDS-PAGE. A specific band with a molecular weight of about 53kDa ( figure 2 ), consistent with the reported size of the target protein, but neither the control strain E.coli JM109 / pJC-tac nor the uninduced E.coli JM109 / pGAD had this band. It shows that the recombinant plasmid pGAD is correctly expressed in Escherichia coli, so the constructed recombinant plasmid pGAD can be transformed into Corynebacterium bacillus to detect whether glutamic acid decarboxylase can be effectively expressed.

Embodiment 3

[0051] Embodiment 3: Identification of recombinant bacteria C.crenatum E01 / pGAD

[0052] The recombinant plasmid pGAD was electroporated to transform C.crenatum E01, and after the transformant grew out, it was transferred to LBG liquid medium containing kanamycin, and the plasmid was extracted from the bacterial solution for PCR verification and enzyme digestion verification. The PCR products all showed a size of 1410bp The target band, after verification by Sal I / BamH I enzyme digestion, the result is as follows image 3 , indicating that the recombinant strain C.crenatum E01 / pGAD was constructed successfully.

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Abstract

The invention belongs to the fields of genetic engineering and enzyme engineering, and relates to a one-step method for synthesizing gamma-aminobutyric acid by using recombinant corynebacterium crenatum (C.crenatum) and with glucose as a substrate. According to the method, a genetic engineering method is adopted, C.crenatum SYPA5-5 gene argB is knocked out, such that a safe strain C.crenatum E01 which can accumulate glutamic acid is obtained; a glutamic acid decarboxylase gene is cloned from L.Plantarum GB01-21 and is expressed in the C.crenatum E01, such that recombinant C.crenatum E01 / pGAD used for directly synthesizing gamma-aminobutyric acid with glucose as a substrate is obtained. A recombinant strain fermentation broth gamma-aminobutyric acid accumulation reaches 13.98g / L. According to the invention, glutamic acid and gamma-aminobutyric acid metabolic pathways are successfully integrated, such that one-step production from glucose to gamma-aminobutyric acid is realized. Therefore, preparation method is simplified, cost is reduced, safety is improved, and novel idea is provided for high-efficiency and low-cost amino acid preparation.

Description

technical field [0001] Using genetic engineering technology to construct safe and efficient Corynebacterium blunt-toothed engineering bacteria. The method for synthesizing gamma-aminobutyric acid by using glucose as a substrate in one step belongs to the field of genetic engineering and enzyme engineering, and specifically relates to a method for producing gamma-aminobutyric acid by genetic engineering recombinant Corynebacterium blunt tooth. technical background [0002] γ-aminobutyric acid is a non-protein amino acid naturally present in some organisms. It is an important inhibitory neurotransmitter in the central nervous system of mammals. Reproductive, diuretic, analgesic and other important physiological functions. It has a wide range of applications in food, feed, medicine and other fields. In 2009, the Ministry of Health approved γ-aminobutyric acid as a new resource food, which means that γ-aminobutyric acid has entered a new era in the domestic market. [0003] A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N1/21C12N15/60C12N15/63C12P13/00C12P13/14C12R1/15
Inventor 饶志明孙红梅徐美娟李秀鹏张显
Owner 南宁汉和生物科技股份有限公司
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