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Method for producing gamma-aminobutyric acid through catalysis of saccharomyces cerevisiae cells and gamma-aminobutyric acid

A technology of Saccharomyces cerevisiae cells and aminobutyric acid, which is applied in the field of γ-aminobutyric acid, can solve the problems of incomparable GABA yield, low GABA synthesis efficiency, complex components, etc., and achieves a perfect exogenous gene expression system, easy control, and benefits. high rate effect

Inactive Publication Date: 2019-08-09
杭州唯铂莱生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The production conditions of glutamic acid are generally controlled in a neutral environment, while the pH range of GAD catalysis is between 4 and 6, which will lead to low GABA synthesis efficiency due to the different environments required by the two pathways.
Although GABA can also be synthesized by two-stage fermentation of glucose, the yield of GABA synthesized by microbial fermentation cannot be compared with that of enzyme-catalyzed method.
In addition, due to the complexity of the components in the fermentation broth, the fermentation method poses a considerable burden on downstream separation and extraction.

Method used

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  • Method for producing gamma-aminobutyric acid through catalysis of saccharomyces cerevisiae cells and gamma-aminobutyric acid
  • Method for producing gamma-aminobutyric acid through catalysis of saccharomyces cerevisiae cells and gamma-aminobutyric acid
  • Method for producing gamma-aminobutyric acid through catalysis of saccharomyces cerevisiae cells and gamma-aminobutyric acid

Examples

Experimental program
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Effect test

experiment example 1

[0051] Experimental Example 1: Expression and Characterization of SsGAD and ScGAD

[0052] The SsGAD and ScGAD genes from MJ654-NF4 and the two genes of Streptomyces rufosa ATCC 49982 were amplified from the Streptomyces genomic DNA sample, and connected into the yeast expression vector YEpADH2p-TRP to produce pHY24 (YEpADH2p-TRP-SsGAD) respectively and pHY25(YEpADH2p-TRP-ScGAD), the GAD gene was expressed in Saccharomyces cerevisiae BJ5464 by an ADH promoter and an ADH terminator. SDS-PAGE analysis showed that SsGAD and ScGAD were successfully expressed in yeast, such as Figure 1a , the relative molecular weights of SsGAD and ScGAD were calculated to be 54.7kDa and 56.3kDa, respectively. Therefore, although these two GADs are originally from Streptomyces, the findings suggest that they can be heterologously expressed in S. cerevisiae BJ5464.

experiment example 2

[0053] Experimental Example 2: Biotransformation of L-GLU into GABA by Engineering Saccharomyces cerevisiae BJ5464

[0054] In order to prove whether the expressed SsGAD and ScGAD work in yeast, the following method was used to prove: the corresponding Saccharomyces cerevisiae engineering strain BJ5464 reacted with L-GLU, and the wild type was used as a control. The reaction mixture contained 0.1M L-GLU, 0.2mM PLP and yeast cells (OD 600 10), at 0.2M Na 2 HPO 4 In citrate buffer, the pH is 5.4. The catalytic process was carried out at 50°C for 12 hours. GABA production as Figure 1b shown. S. cerevisiae BJ5464 / SsGAD and S. cerevisiae BJ5464 / ScGAD produced 74.8-fold and 19.5-fold higher GABA production than wild type, respectively, confirming the functional expression of these two enzymes in S. cerevisiae. The catalytic efficiency of Saccharomyces cerevisiae BJ5464 / SsGAD was significantly higher than that of BJ5464 / ScGAD, which was consistent with the results observed in ...

experiment example 3

[0055] Experimental Example 3: Comparison of GABA conversion efficiency between Saccharomyces cerevisiae BJ 5464 / SsGAD cells and SsGAD cells in different culture periods

[0056]The effect on decarboxylation efficiency of Saccharomyces cerevisiae BJ5464 / SsGAD was evaluated by incubation time. Using 0.1M L-GLU as substrate, in 0.2mM Na 2 HPO 4 0.2 mM PLP was added to citrate buffer (pH 5.4). Cell density is set to OD 600 10. The reaction was carried out at 50 °C for 12 h. Such as Figure 2a As shown, the production of GABA increased from 46.3mM to 89.9mM when the culture time of Saccharomyces cerevisiae BJ5464 / SsGAD cells was extended from 24 hours to 48 hours. However, further prolonging the culture time significantly reduced the transformation ability of S. cerevisiae BJ5464 / SsGAD. When cultured for 96h compared with cultured for 48h, the production of GABA decreased by 28.6%. The GABA metabolic pathway consists of three yeasts (including GAD, GABA aminotransferase (C...

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Abstract

The invention relates to the technical field of biology, and aims at providing a method for producing gamma-aminobutyric acid through catalysis of saccharomyces cerevisiae cells. The method comprisesthe steps of 1, extracting a target gene from glutamic acid decarboxylase, conducting PCR amplification on the target gene, and connecting the amplified target genes to plasmids for recombination to construct a cloning vector; 2, conducting enzyme digestion treatment on the cloning vector through a restriction enzyme to obtain target gene fragments, and connecting the target gene fragments into acerevisiae vector to generate recombined plasmids; 3, guiding the recombined plasmids into the saccharomyces cerevisiae cells through a lithium acetate quick-conversion method to obtain recombined saccharomyces cerevisiae cells; 4, culturing the recombined saccharomyces cerevisiae cells in a Na2HPO4 citrate buffer solution, and mixing L-glutamic acid and 5'-pyridoxal phosphate for a reaction to obtain the gamma-aminobutyric acid. The invention further provides the gamma-aminobutyric acid. The production speed of the saccharomyces cerevisiae cells is high, culture is simple, an exogenous gene expression system is complete, and the method is high in catalysis efficiency, low in cost and easy to control.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for catalyzing the production of gamma-aminobutyric acid by brewer's yeast cells and gamma-aminobutyric acid. Background technique [0002] γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid, white powder, widely present in nature, easily soluble in water, slightly soluble in hot ethanol, insoluble in cold ethanol, ether and benzene. GABA can reduce blood ammonia, promote brain metabolism, and regulate metabolic balance in the body; it can be used as an inhibitor of neurotransmitter signals, can regulate sleep, treat human headaches, hepatic coma, hypertension and other physiological diseases, and can also synthesize 2-pyrrolidone and 4-polyamide nylon, used in food and industrial production. Glutamate decarboxylase (GAD) is a 5'-pyridoxal phosphate (PLP)-dependent decarboxylase that exists in bacteria, yeast and filamentous fungi. [0003] At present, the biolog...

Claims

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Application Information

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IPC IPC(8): C12P13/00C12N15/60C12N15/81C12N1/19C12R1/865
CPCC12N9/88C12N15/81C12P13/005C12Y401/01015
Inventor 占纪勋
Owner 杭州唯铂莱生物科技有限公司
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