Screening,Cloning And Heterologous Expression Of Halogenase-related Genes From Metagenomic Library

Over the decades,microorganisms have become an important biological resource for discovering new natural products.The natural products from microorganisms are widely used in agriculture,animal husbandry and medicine indusries.Studies have shown that humans could just study less than 1%of microorganisms by direct isolation.Nearly 99%of microorganisms could not be cultured,and their biological potential has not yet been recognized and excavated.Therefore,it is very important to establish a set of methods for the study of uncultured microorganisms.Through metagenomics the microbial genetic information in specific environoment could be obtained to the maximum extent.The problems of isolation and culture of microbe that have plagued humans for a long time might be overcome by the metagenomics.More functional enzymes genes and biosynthesis gene clusters were found by sequence-drived screening the metagenomic library,and new type of halogenases and natural halides were obtained by means of molecular techniques later.The tryptophan halogenase,which use tryptophan as its primary substrate,is widely present in the biosynthesis gene clusters of halides.And the characterized halides have good bioactivity.Humans need to find more new compounds because of the spread of antibiotic-resistant microbe.So the discover of tryptophan halogenase,which as a modifier in the halide biosynthetic gene cluster,could increase the possibility of discovering novel halogenated secondary metabolites.In this thesis,a pair of degenerate primers,which designed based on the conservative region of FADH2-dependent tryptophan halogenases was used to screen a total of 1.6 million clones from the metagenomic library of Mount Qomolangma soil.Totally 22 positive clones containing tryptophan halogenase genes were obtained.The phylogenetic tree display that the halogenase genes from clones 1765,1867,1875 and 1905 have close sequence similarity(70%)to the known tryptophan-halogenases,and the remaining eDNA derived halogenase genes,although far from known tryptophan halogenases,might belong to an unknown class of tryptophan halogenases.A total of 13 intact sequences of tryptophan halogenase gene were obtained by primer-walking sequencing on the positive clones,and eleven genes were expressed.By co-expression with pKJE7 companion plasmid,the proteins of tryptophan halogenase genes in 1717,1765,1867,1976 clone was successfully expressed.And by co-expression with pGro7 companion plasmid,protein of the tryptophan halogenase gene in the 1907 clone was successfully expressed and purified.Twenty clones were transferred to Streptomyces albus and S.coelicolor hosts,and clone specific peaks were found by HPLC analysis of S.albus harboring 1905 clone.In this study,we successfully identied the tryptophan halogenase positive clones from the Mount Qomolangma metagenomic library by sequence-drived screening,and established a new method for the discovery of novel tryptophan halogenase and halide by metagenomics.This method lays a foundation for the subsequent study of tryptophan halogenase,and also provides a new idea for discovering new functional enzymes and compounds from microbial resources.