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Genetically engineered bacterium for expressing human derived acetaldehyde dehydrogenase gene and application thereof

A technology of acetaldehyde dehydrogenase and engineering bacteria, which is applied in the direction of genetic engineering, application, plant gene improvement, etc., can solve the problems of product lack of food safety, loss of function of target protein, etc., to maintain enzyme activity and improve stability , Good acid resistance and ethanol resistance

Active Publication Date: 2018-03-20
JIANGNAN UNIV RUGAO FOOD BIOTECH RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problem in the prior art that the target protein loses some functions because it cannot be folded correctly, and the expression in eukaryotic cells often requires an inducer, resulting in the lack of food safety of the product, the present invention provides a human acetaldehyde dehydrogenase Gene and Saccharomyces cerevisiae engineering bacteria producing the acetaldehyde dehydrogenase

Method used

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  • Genetically engineered bacterium for expressing human derived acetaldehyde dehydrogenase gene and application thereof
  • Genetically engineered bacterium for expressing human derived acetaldehyde dehydrogenase gene and application thereof
  • Genetically engineered bacterium for expressing human derived acetaldehyde dehydrogenase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Synthesis of human ALDH2 gene and construction of recombinant plasmid pUC57-ALDH2

[0051] The human ALDH2 gene (shown in SEQ ID NO.1) and the recombinant plasmid pUC57-ALDH2 were synthesized and constructed by Sangon Bioengineering (Shanghai) Co., Ltd.

Embodiment 2

[0052] Example 2 Construction of expression plasmid pYX212-ALDH2

[0053] The recombinant plasmid pUC57-ALDH2 was double-digested with EcoR I and BamH I, and then ligated with the pYX212 vector that had been double-digested with the same enzymes. The enzyme digestion system is shown in Table 2:

[0054] Table 2 enzyme digestion system

[0055]

[0056] Place the enzyme digestion system in a water bath at 37°C for 20-30 minutes, and after agarose gel electrophoresis to detect the enzyme digestion effect, recover the ALDH2 target gene fragment and the carrier pYX212 fragment from the gel, and connect them according to the connection system in Table 3:

[0057] Table 3 Ligation system of T4 ligase

[0058]

[0059] Incubate overnight at 16°C, transform into E.coli JM109, select positive transformants, and obtain recombinant plasmid pYX212-ALDH2 through PCR amplification and sample sequencing verification.

[0060] PCR amplification program:

[0061] F5 / R5: Pre-denaturat...

Embodiment 3

[0062] Example 3 Construction of ALDH2 Gene Expression Module Trp1L-URA3-TPIp-ALDH2-TPIt-Trp1R

[0063] Extract the genome of Saccharomyces cerevisiae W303-1A (see the instruction manual of the MiniBEST Universal Genomic DNA Extraction Kit kit for specific operation methods) as a template for PCR, and use Prime STARDNA Polymerase to amplify the upstream Homology arm Trp1L (sequence shown in SEQ ID NO.2) and downstream homology arm Trp1R (sequence shown in SEQ ID NO.5); extract the genome of Saccharomyces cerevisiae N85 (see the MiniBEST Universal Genomic DNA Extraction Kit reagent for specific operation methods Instructions for use of the box) as a template for PCR, use the primer pair F2 / R2 in Table 3 to amplify the complete screening marker gene URA3 (sequence such as SEQID NO.3); extract the recombinant plasmid pYX212-ALDH2 in E.coli JM109 (specifically For the operation method, refer to the instruction manual of the SanPrep column type plasmid DNA mini-extraction kit) as a...

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Abstract

The invention discloses a genetically engineered bacterium for expressing a human derived acetaldehyde dehydrogenase gene and application thereof, and belongs to the technical field of gene engineering and enzyme. The ALDH2 gene efficient expression component Trp1L-URA3-TPIp-ALDH2-TPIt-Trp1R is integrated into the genome of Saccharomyces cerevisiae W303-1A, the stability of gene expression is improved compared with free plasmid expression, a constitutive promoter is used, no induction agent is added during fermentation, and the product is relatively safe. When fermentation is performed for 96hours, the specific activity of producing acetaldehyde dehydrogenase reaches 0.97 U / mg. The specific activity of the producing acetaldehyde dehydrogenase reaches 6.256 U / mg in ethanol with the concentration of 250 mg / 100 mL, and the specific activity of the producing aldehyde dehydrogenase in a buffer with pH of 3 reaches 0.301 U / mg. The Saccharomyces cerevisiae is selected as a host strain, on the one hand, the activity of the enzyme is kept, on the other hand, product safety is improved, and a new approach and a theoretical basis are provided for the research and development of antialcoholicdrugs.

Description

technical field [0001] The invention relates to a genetic engineering bacterium expressing human acetaldehyde dehydrogenase gene and application thereof, which belongs to the field of genetic engineering and enzyme technology. Background technique [0002] Alcohol culture has a long history in our country. Quite a few Chinese people have a hobby of drinking. However, long-term drinking and health problems caused by excessive drinking have attracted more and more attention. Alcohol dehydrogenase (Alcoholdehydrogenase) and acetaldehyde dehydrogenase (EC 1.2.1.10, Aldehyde dehydrogenase) in the human body play an important role in the metabolism of ethanol. Ethanol is oxidized into acetaldehyde under the action of alcohol dehydrogenase, and then oxidized into acetic acid under the action of acetaldehyde dehydrogenase. When the two enzymes in the human body can be fully expressed, ethanol will be fully metabolized in a short time, reducing alcohol consumption. Effects on the ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N1/19C12N9/02C12G3/02C12R1/865
CPCC12G3/02C12N9/0008C12Y102/0101
Inventor 陆健豆欣喜吴殿辉李晓敏孙军勇蔡国林
Owner JIANGNAN UNIV RUGAO FOOD BIOTECH RES INST
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