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A kind of recombinant bacteria with high lipopeptide production and its application

A technology of recombinant bacteria and lipopeptide, which is applied in the field of genetic engineering, can solve the problem of not increasing the yield of surfactin, and achieve good industrial application prospects and the effect of increasing yield.

Active Publication Date: 2021-01-05
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, (Qun Wu, Yan Zhi, Yan Xu, Systematically engineering the biosynthesis of a green biosurfactantsurfactin by Bacillus subtilis 168[J]. Metabolic Engineering, 2019, 52:87-97) overexpressed in surfactin-producing host bacteria Acetolactate synthase AlsS, ketol acid reductoisomerase IlvC, dihydroxyacid dehydratase IlvD, 2-isopropylmalate synthase LeuA, 3-isopropylmalate dehydrogenase in the leucine synthesis pathway Enzymes LeuB and 3-isopropylmalate dehydratase LeuCD, Surfactin production did not increase
There is no report on the research on enhancing the synthesis of leucine in cells to effectively increase the production of surfactin

Method used

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  • A kind of recombinant bacteria with high lipopeptide production and its application
  • A kind of recombinant bacteria with high lipopeptide production and its application
  • A kind of recombinant bacteria with high lipopeptide production and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Construction of plasmids carrying genes leuABCD and ilvK in the leucine synthesis pathway

[0053] Pick a single colony of Bacillus subtilis B.subtilis THY-7, inoculate it in LB liquid medium, place it in a shaker at 37°C and 200rpm for overnight culture, and collect the bacteria under the condition of centrifugation at 12000rpm and 5min. Bacterial Genome Extraction Kit to extract THY-7 genome. Using the obtained genome as a template, the upstream primer leuABCD-F (sequence shown in SEQ ID NO.8) and the downstream primer leuABCD-R (sequence shown in SEQ ID NO.9) were used for PCR amplification to obtain a leuABCD fragment. Use upstream primer ilvK-F (sequence shown in SEQ ID NO.10) and downstream primer ilvK-R (sequence shown in SEQ ID NO.11) to carry out PCR amplification to obtain ilvK fragment primers provided by Boshang Biotechnology (Shanghai) Co., Ltd., dissolved in sterile water and diluted to 10 μM for use. The polymerase, buffer and restriction endo...

Embodiment 2

[0058] Example 2 Construction of the genetically engineered bacteria B.subtilis THY-7 / Pg3-srfA (leuABCD-ilvK) overexpressing the leucine synthesis pathway

[0059] The Bacillus subtilis constructed in Example 1 carrying 2-isopropylmalate synthase LeuA, 3-isopropylmalate dehydrogenase LeuB, 3-isopropylmalate dehydratase LeuCD and branched-chain amino acid transaminase IlvK The Bacillus-Escherichia coli shuttle plasmid pJMP-leuABCD-ilvK is used to transform the competent cells of Bacillus subtilis THY-7 / Pg3-srfA by electroporation, and the genetically engineered bacteria THY-7 / Pg3-srfA (leuABCD-ilvK). Wherein, the preparation and electrotransformation of Bacillus subtilis THY-7 / Pg3-srfA competent cells adopt the method in Chinese patent document CN105400784A.

[0060] After resuscitation, take 100uL of bacterial liquid and spread it on LB solid medium containing 10-30μg / mL kanamycin, place it upside down in a 37°C incubator for overnight culture, pick a single colony, and use ...

Embodiment 3

[0061]Embodiment 3 uses genetically engineered bacteria THY-7 / Pg3-srfA (leuABCD-ilvK) to produce lipopeptide surfactant-surfactin

[0062] The genes obtained in Example 2 for overexpressing 2-isopropylmalate synthase LeuA, 3-isopropylmalate dehydrogenase LeuB, 3-isopropylmalate dehydratase LeuCD and branched-chain amino acid transaminase IlvK The engineering bacteria THY-7 / Pg3-srfA (leuABCD-ilvK) was inoculated in LB liquid medium (containing chloramphenicol and kanamycin), cultured at 37°C and 200rpm for 16h, and inserted into the container at a ratio of 5%. In a shake flask with 100 mL of fermentation medium, add IPTG when culturing at 37°C and 200 rpm for 2-6 hours, and continue culturing for 2-3 days to obtain a lipopeptide-containing fermentation broth.

[0063] The detection of surfactin in the fermentation broth adopts the method of Yu Huimin et al. (Chinese patent CN 105400784A). The statistical results of surfactin concentration in genetically engineered bacteria THY...

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Abstract

The invention discloses a recombinant strain with a high lipopeptide yield and application thereof, belonging to the technical field of genetic engineering. The recombinant strain is constructed by transforming a 2-isopropylmolic acid synthase gene in a leucine synthesis pathway into an original strain. Compared with the original strain, the recombinant strain which over-expresses a part of the leucine synthesis pathway (2-isopropylmolic acid synthase, 3-isopropylmolic acid dehydrogenase, 3-isopropylmolic acid dehydratase and branched chain amino acid transaminase) has a surfactin yield increased by 55.6%, can be used for production of lipopeptide type biosurfactants, and has a good industrial application prospect. The average lipopeptide yield in fermentation liquor obtained by shake flask fermentation is 13-16 g / L.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a high-production lipopeptide recombinant bacterium and its application. Background technique [0002] Lipopeptide biosurfactants are a kind of amphoteric substances connected by hydrophilic cyclic oligopeptides and hydrophobic fatty acid chains with lactone bonds, and are mainly synthesized by microorganisms such as Bacillus and Streptomyces. Due to the difference in amino acid composition and ring formation in the peptide rings that make up lipopeptides, lipopeptides from Bacillus can be classified into surfactin, phenmusin, iturin, etc. Among them, the unique "saddle" conformation is formed at the air / liquid interface. Surfactin has good surface activity, biodegradability and antibacterial activity, and has broad application prospects in the fields of oil exploration, biological control, medicine and daily chemicals. However, the low yield of surfactin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P21/00C12R1/125C12R1/085C12R1/38
CPCC12N9/0006C12N9/1025C12N9/1096C12N9/88C12P21/00C12Y101/01085C12Y203/03013C12Y206/01042C12Y402/01033
Inventor 于慧敏王苗苗许春梦
Owner TSINGHUA UNIV
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