The Mutant Library Construction Of Burkholderia Sp.WGB30Biontransformating Trans-anethole And Analysis

Trans-anethole, a simple phenylpropanoid compound, is abundant in aniseand easy to obtain from the plants. China is the main origin of the anise, and90%of the anise is produced in GuangXi. Trans-anethole was used in flavor and foodproducts, and as a natural product, was loved by cosumers, biotransformation ofthe trans-anethole becomes a new hot direction in recent years.So far, there are some strains that can biotransform trans-anethole wereisolated. And the biotransformating conditions of these strains were improved andefficiency has been promoted. But the studies of the gene that involved incapabilibty of biotransformating trans-anethole were rare.This article based on the isolation of a strain named Burkholderia sp.WGB30can bioconverse trans-anethole, a mutation library of Burkholderia sp.WGB30were construted by the mini transposon through triparental cross usingmutation vector pTnMod-KmO, help plasmid pRK2073, a mutant librarycontaining11,000mutants was obtained using antibiotics and PCR screening.When use trans-anethole as the sole carbon source,34mutants that can not usetrans-anethole were found from the mutant library. The screened mutant strainswere culture in M9liquid medium, and the trans-anethole were added into themedium when the mutant strains growed to a certion concentration and culturedfor24hours at28℃right along, the liquid of transformation was examined bythin layer chromatography. Mutant1th-8th,14th-16thhave a small amount ofanisic acid produced; while30th-32thhave a small amount of anisaldehydeproduced and there are some anisic acid in the mutant30, thus the insertion sitesof transposon may be on the genes related anisic acid transforming in thesemutants. There was no anisic acid and no anisaldehyde produced in mutant strains9th-13th,17th-28th,33th-34th, the insertion sites of transposon may be on the genesrelated trans-anethole transforming in these mutants supposedly. There was a kindof intermediate in the way of trans-anethole to anisaldehyde, the insertion sites oftransposon may be on the genes related this intermediate transforming supposedly. There was anisaldehyde but no anisic acid in mutant32th, suggesting that in thismutant strain the way transforming anisaldehyde to anisic acid may be disrupted.The results lay a foundation for analyzing the further identificating genesassociated with transforming anethole and the metabolic pathways.