Genetic recombinant method of pseudomonas aeruginosa for high-yield producing rhamnolipid

Abstract

A genetic recombinant method of pseudomonas aeruginosa for high-yield producing rhamnolipid includes following steps: (1) designing a primer for amplifying RhlAB gene according to the RhlAB gene sequence of rhamnolipid synthetase; (2) with chromosome of wild pseudomonas aeruginosa producing rhamnolipid as a template, performing PCR amplification to the RhlAB gene and performing rubber cutting purification to target genes through a purification kit; (3) performing enzyme digestion to the purified RhlAB gene to introduce the purified RhlAB gene into an escherichia coli-pseudomonas aeruginosa shuttle expression vector pAK1900 to obtain new plasmid pAK1900-AB; (4) introducing the constructed shuttle expression vector pAK1900-AB into a recipient bacterium pseudomonas aeruginosa competent cell through a heat-shock conversion method, coating a carboxyl-benzyl resistance plate with the competent cell and performing PCR verification to the constructed engineering bacteria; and (5) performing fermentation in a shake flask and measuring the yield of the rhamnolipid in a sulfuric acid-anthranone manner. The method can greatly reduce the production cost of the rhamnolipid, provides basis for industrial application of the rhamnolipid and can promote high-yield production of the rhamnolipid.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a gene recombination method for high-production rhamnolipids of Pseudomonas aeruginosa. Background technique [0002] Rhamnolipid is a glycolipid anionic surfactant, it has good biocompatibility and efficient emulsifying, solubilizing and reducing surface tension, etc. And food processing and other fields have broad application prospects. [0003] At present, the production strain of rhamnolipid is mainly Pseudomonas aeruginosa (Pseudomonas aeruginosa). It is very easy to reverse the mutation, resulting in the instability of the production performance of the strain. In 1994, the research found that the genes rhlA and rhlB involved in the synthesis of rhamnolipids were considered to be the glycosyltransferase genes necessary for the synthesis of rhamnolipids. Further research was carried out in 2003 Determined that RhlA is an acyltransferase and is required fo...

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Problems solved by technology

[0003] At present, the production strain of rhamnolipid is mainly Pseudomonas aeruginosa (Pseudomonas aeruginosa). It is very easy to reverse the mutation, resulting in the instability of the production performance of the strain. In 1994, the research found that the genes rhlA and rhlB involved in the synthesis of rhamnolipids were considered to be the glycosyltransferase genes necessary for the synthesis of rhamnolipids. Further research was carried out in 2003 Determined that RhlA is an acyltransferase and is required for the synthesis of HAA (alkyl acyl acid), while HAA (β-hydroxydecanoyl-β-hydroxydecanoic acid) and TDP-rhamnose are precursors for the synthesis of rhamnolipids body substance, and HAA is also an extracellular biological surface active substance. RhlB is the key gene that catalyzes the reaction of TDP-rhamnose and HAA to generate rhamnolipid containing a rhamnosyl group. In Pseudomonas aeruginosa, rhlAB It exists in the form of an operon. Under this operon, a total of three proteins can be encoded, RhlA, RhlB and RhlR. These three genes have their own stop codons, but due to the presence of ribose in front of their promoters The body binding site, so that the next gene is expressed, we can use this feature to directly amplify the RhlAB gene and express it in other bacterial species

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