A kind of genetically engineered acetic acid bacteria overexpressing ATPase and its construction method and application
An overexpression and genetic engineering technology, applied in the field of genetic engineering, can solve the problems of low ATPase concentration, restricting the efficiency of fermentation, etc., and achieve the effects of high fermentation rate, improved production efficiency, and improved stability.
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[0070] The preparation of primers involved in the present invention, PCR reaction, purification of nucleotide fragments, recovery, digestion, ligation, DNA introduction, artificial synthesis of nucleotide sequences and other operations are well known to those skilled in the art, and can be based on, for example, "Molecular Cloning" (Science Press, second edition, 2002) described in the method.
[0071] The present invention also provides the genetically engineered acetic acid bacteria overexpressing ATPase obtained by the above construction method.
[0072] The invention also provides the application of the genetically engineered acetic acid bacteria overexpressing ATPase in acetic acid fermentation, wherein the acetic acid fermentation uses ethanol or wine mash containing ethanol as raw material.
[0073] In the present invention, the application of the genetically engineered acetic acid bacteria overexpressing ATPase in acetic acid fermentation specifically includes the foll...
Embodiment 1
[0084] A kind of genetic engineering acetic acid bacterium of overexpressing ATPase, its construction method comprises the steps:
[0085] a. The acetaldehyde dehydrogenase promoter, the ATPase gene and the plasmid that can be stably replicated in acetic acid bacteria are sequentially connected to obtain a recombinant plasmid;
[0086] b. Transfer the recombinant plasmid obtained in step a into acetic acid bacteria to obtain.
[0087] Specifically, the step a specifically includes the following steps:
[0088] (1) Construction of recombinant plasmid pBBR-Paldh
[0089] Using the genome of Acetobacter pasteurianus CGMCC 3089 (purchased from the General Microorganism Center of China Microbiological Culture Collection Management Committee, Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip Code 100101.) genome as a template, using primers The PCR reaction of paldh-1 and paldh-2 was performed to ampli...
Embodiment 2
[0121] Fermentation of Acetic Acid by Acetobacter acetiCGMCC1.1809 Genetic Engineering Bacteria Containing Recombinant Plasmid pBBR-Paldh-ATPase
[0122] (1) Plasmid transformation
[0123] ① Preparation of Acetobacter acetiCGMCC1.1809 competent cells:
[0124] Pick Acetobacter acetiCGMCC1.1809 and inoculate it in YPG medium, pre-cultivate it at 30°C and 220 rpm for 12 hours, until the OD550 is about 0.6, take 1 mL of the pre-cultured bacterial liquid into a 250 mL Erlenmeyer flask containing 100 mL of YPG medium , 30°C, 220 rpm for 8 hours, until the OD600 is about 0.6; place the Erlenmeyer flask containing the bacterial solution on an ice bath for 20 minutes, centrifuge at 5000 rpm for 5 minutes at 4°C, discard the supernatant; add 80 mL Resuspend the cells in a 10% glycerol (mass ratio) solution pre-cooled to 0°C, make the cells fully diffuse, and centrifuge at 5000 rpm for 8 minutes at 4°C, discard the supernatant; add 3 mL of pre-cooled 10% glycerol solution Shake well ...
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