Engineering bacterium for highly expressing lipopeptide biosurfactant and application thereof

A technology of biological surface and engineering bacteria, applied in the field of biotechnology and biochemical industry, to achieve good industrial application prospects, increase production, and increase output

Active Publication Date: 2014-07-02
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Engineering bacterium for highly expressing lipopeptide biosurfactant and application thereof
  • Engineering bacterium for highly expressing lipopeptide biosurfactant and application thereof
  • Engineering bacterium for highly expressing lipopeptide biosurfactant and application thereof

Examples

Experimental program
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Embodiment 1

[0057] Construction of Bacillus subtilis-Escherichia coli shuttle plasmid carrying lipopeptide carrier protein gene ycxA

[0058] Pick a single colony of Bacillus subtilis 1012wt (purchased from MoBiTec), inoculate it in LB liquid medium, cultivate it for 16 hours at a temperature of 37°C and a shaker speed of 200 rpm, and centrifuge at 12,000 rpm for 5 minutes to collect the bacterial precipitate. Bacillus subtilis 1012wt genomic DNA was extracted with the Bacterial Genome Extraction Kit from Omega. Using the above obtained Bacillus subtilis 1012wt genomic DNA as a template, the upstream primer ycxAFB (sequence shown in SEQ ID No: 2) and the downstream primer ycxARS (sequence shown in SEQ ID No: 3) were used for PCR gene amplification. Primers were synthesized by Platinum Biotechnology (Shanghai) Co., Ltd., dissolved in sterile water, and diluted to 10 μM for use. The polymerase, buffer and dNTP used in PCR amplification were purchased from TaKaRa Company. The PCR amplifica...

Embodiment 2

[0062] Construction of Genetic Engineering Bacteria Overexpressing Lipopeptide Carrier Protein YcxA

[0063] The Bacillus subtilis-Escherichia coli shuttle plasmid pHT-ycxA carrying the lipopeptide carrier protein gene ycxA constructed in Example 1 was transformed into competent cells of Bacillus subtilis THY-7 by electroporation to obtain an overexpressed lipopeptide carrier protein Genetic engineering strain TS589 of YcxA. Wherein, the preparation of Bacillus subtilis THY-7 competent cells adopts the method of Xue et al. (Journal of Microbiological Methods, 1999, 34:183-191). Add 2.5 μL of recombinant plasmid pHT-ycxA and 50 μL of Bacillus subtilis THY-7 competent cells to a 1.5 mL centrifuge tube, mix well, add a 0.1 cm electroporation cup, and ice-bath for 30 min; adjust the voltage of the electroporator to 1.25 kV, and turn the Put the cup into the electroporation instrument and press the electric shock button; after the electroporation, add 1mL recovery medium (see Xue ...

Embodiment 3

[0065] Production of lipopeptide surfactant—surfactin by using genetically engineered bacteria TS589

[0066] The overexpressed lipopeptide carrier protein ycxA genetically engineered bacterium TS589 obtained in Example 2 was inoculated in LB liquid medium (containing 5 μg / mL chloramphenicol), cultured at 37°C and 200 rpm for 16 hours, and inserted into the fermentation at a ratio of 5%. In the culture medium, cultivate at 37°C and 200 rpm for 4 hours, add IPTG (final concentration 1mM), and continue to cultivate for 24 hours to obtain lipopeptide-containing fermentation broth. Take 100 μL of the fermentation broth obtained in step 1, add 900 μL of deionized water, mix well, filter through a 0.22 μm filter membrane, and load the sample for HPLC analysis. The Shimadzu LC20A chromatographic system was used, the chromatographic column was ODS-SP250×4.6mm (GL Sciences), the mobile phase was acetonitrile:water:trifluoroacetic acid=93:7:0.1, the flow rate was 0.8mL / min, and the load...

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Abstract

The invention discloses an engineering bacterium for highly expressing a lipopeptide biosurfactant and application thereof, belonging to the fields of biotechnology and biochemical engineering. The engineering bacterium over-expressing transmembrane protein YcxA is constructed by adopting a gene engineering technology, and transmembrane transport of lipopeptide from the inner part of a cell to the outside of the cell is enhanced, so that the yield of the lipopeptide is significantly improved. Compared with an original strain, the obtained gene engineering bacterium over-expressing lipopeptide carrier protein YcxA has the advantages that the yield of surfactin is improved by 97%, the engineering bacterium can be used for producing the lipopeptide biosurfactant and has a good industrial application prospect, and the yield of lipopeptide in a fermentation broth obtained by fermentation is averagely 1-5 g/L.

Description

technical field [0001] The invention belongs to the fields of biotechnology and biochemical industry, and in particular relates to an engineering bacterium highly expressing a lipopeptide biosurfactant and its application. Background technique [0002] Lipopeptide (lipopeptide) biosurfactant is an amphoteric substance mainly synthesized by microorganisms such as Bacillus and Streptomyces. It consists of a hydrophilic cyclic oligopeptide and a hydrophobic fatty acid carbon chain. Different Bacillus strains synthesize different types of lipopeptides. According to the molecular structure of lipopeptides, they can be divided into surfactin, fengycin, plipastatin, iturin, bacillomycin, mycosubtilin, etc. . The difference between different types of lipopeptides mainly lies in the composition of amino acids and the way of ring formation. For example, the 7-peptide composed of nonpolar valine and leucine, and polar glutamic acid and aspartic acid constitutes the cyclic oligopeptid...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P21/02C12R1/125C12R1/085C12R1/38
Inventor 于慧敏李煦杨欢李雪沈忠耀
Owner TSINGHUA UNIV
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