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Acid-resistant E.coli engineering bacteria

A technology of Escherichia coli and engineering bacteria, applied in the field of genetic engineering, can solve the problems of lack of gene manipulation tools, inability to realize the gene manipulation of Propionibacterium acidobacterium, and inability to verify the function of key enzymes, etc.

Active Publication Date: 2015-01-28
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Due to the lack of effective genetic manipulation tools, the genetic manipulation of P. acidigens is not yet possible, and it is impossible to verify the effect of key enzymes on the acid resistance of P. acidigens.

Method used

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Effect test

Embodiment 1

[0017] Determination of embodiment 1secretion neighborhood ATPase

[0018] Collect the bacterial cells of the original strain of Propionibacterium acidogenicum and the acid-resistant mutant strain in the mid-log phase, and extract the intracellular proteins respectively. Through the comparison of proteomics technology, it is found that the content of a protein spot in the acid-resistant strain is the same as that of the original strain. Compared with the up-regulation of more than 3 times, this point was dug out and identified by MALDI-TOF / MS mass spectrometry, and the protein was determined to be the secretion neighborhood ATPase. Secretion neighborhood ATPase may be related to the acid resistance of bacteria.

Embodiment 2

[0019] The construction of embodiment 2 acid-resistant escherichia coli engineering bacteria

[0020] Primers were designed based on the amino acid sequence of the secretion neighborhood ATPase on NCBI, and the whole genome sequence of P. acidipropionici was used as a template to amplify the sec target fragment by PCR. The sequence is shown in SEQ ID NO.1. The target gene was connected to the expression vector pACYCDuet-1 through restriction sites Nco I and Hind III to obtain the recombinant expression plasmid pACYCDuet-sec. The recombinant plasmid was transformed into Escherichia coli E.coli BL21(DE3), and spread to LB solid medium containing chloramphenicol resistance. The transformants were randomly selected for colony PCR verification, and positive transformants were obtained, and the recombinant E. coli E. coli BL21(DE3)-sec was confirmed by further isolation of the plasmid for double enzyme digestion verification.

Embodiment 3

[0021] Embodiment 3 Recombinant bacteria and control bacteria acid resistance experiment

[0022] E.coli BL21(DE3)-1 (control strain, containing plasmid pACYCDuet-1) and E.coli BL21(DE3)-sec were inoculated into LB medium respectively, and the bacterial concentration was measured after overnight culture as seed solution. Take the same OD 600 The above seed solution was inoculated into LB medium, and cultivated to OD at 37°C 600 When = 6.0, IPTG with a final concentration of 1 mg / mL was added for induction, and after 2 hours of induction culture at 30°C, 1 g / L propionic acid, 2 g / L propionic acid, 2 g / L acetic acid, 4 g / L propionic acid, and 4 g / L L acetic acid and no organic acid added. After adding 1g / L propionic acid, 2g / L propionic acid, 2g / L acetic acid, and 4g / L acetic acid, the pH of the medium was 4.75, 4.35, 4.15, and 3.85, respectively. Continue to cultivate at 30°C until the stationary phase, and take the fermentation broth for gradient After dilution, take the di...

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Abstract

The invention discloses acid-resistant E.coli engineering bacteria, belonging to the field of genetic engineering. According to proteomics, secretion neighborhood ATPase is likely to be related to acid resistance of P.acidipropionici. With a molecular measure, a gene for coding secretion neighborhood ATPase in P.acidipropionici is over-expressed in E.coli BL21 (DE3) to obtain recombinant bacteria with high expression of secretion neighborhood ATPase. Compared with a contrast bacterial strain, the acid-resistant E.coli engineering bacteria have the advantage that resistance to propionic acid and acetic acid is improved to varying degrees. A research idea disclosed by the invention provides a basis and a new idea for revealing acid resisting mechanisms of propionibacterium and E.coli and improving bacterial fermentation production capability.

Description

technical field [0001] The invention relates to an acid-resistant Escherichia coli engineering bacterium, which belongs to the field of genetic engineering. Background technique [0002] As an important bulk chemical product, propionic acid is mainly used as an esterification agent, a solvent for nitrocellulose, a plasticizer, a chemical reagent and a food raw material. Propionate is an important derivative of propionic acid, and is internationally recognized as a safe and reliable fungicide for grain, feed and food. With the development of feed industry and food industry, the consumption of propionic acid is increasing year by year, and its output is far from meeting the industrial demand. [0003] The production methods of propionic acid include chemical synthesis method and fermentation method. The chemical synthesis method uses petrochemical products as raw materials and is synthesized by heating and pressurizing catalysts. The microbial fermentation method is that prop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12R1/19
CPCC12N9/14
Inventor 刘龙陈坚堵国成李江华管宁子
Owner JIANGNAN UNIV
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