Screening And Heterologous Expression Of Halogenase Harboured Gene Clustersfrom Metagenomic Library

Soil is a large and complex ecosystem,in 1g of which containing 105 different species of microorganisms.Different microbial species contain different natural product synthetic gene clusters,regarding to lots of microbial gene resources.Microorganisms are one of the main sources of natural bioactive substances(anticanceragents and antineoplastic agents,antibiotics,biological enzymes).But according to the existing laboratory conditions as well as traditional microbial isolation and cultivation methods,only about less than 1%of microbes can be cultivated.These circumstances largely limited the excavation and utilization of environmental microbial resources.In order to discover more new gene information and natural active products from environmental microbes,the whole microorganisms genome can be transfered into cultured microbial bymetagenomic method.This method can largely increase the utilization of microbial resources and the potential of discovering new nature bioactive products.Up to now,there are more than 4700 kinds of halogen containing natural products was found,and a variety of active substances such as antibiotics,antitumor drugs,immune modulators and enzyme inhibitors.Halogen substituent profoundly affected the bioactivities of natural product.The halogenation process of natural products is usually catalyzed by halogenated enzymes,so the screening of halogenated enzyme gene is extremely important for the discovering of new halogenases.In this study we constructed a metagenomic library of Yunnan soil.We screenning halogenase harboured gene clusters by PCR amplification and halogenase primers.According to this technical route.In the end 31 positive clones were obtained.Aligned clones sequence with the known halozyme sequences on NCBI.All of clones,only No.1076 fragment size is about 800 bp.Designed the specific primers and screen 1076 mixed bacteria again,the sequence result with degenerate primer's similarity is 95%,andthought the two ought to be the same sequence.All of the clones sequence blast on the NCBI,and the result show the screened clones contain FAHD2-halohenase gene or tryptophan halogenated gene.All of screened clones,2032-6,2165-1,2264-7,2270,2280-9,2295-5,2401,2435-1,2439-7,2472-4 have higher similarity with know Compounds,it's above 85%.The Neighbor Joining phylogenetic tree was established on MEGA according to the obtained clones sequence.Based on the branch analysis of phylogenetic tree,almost clones on the branch analysis of phylogenetic tree far way with know compounds.But No.2024-7 belong to a branch with clorobiocin.According to the end sequencing results,No.2024-7 is similar with 5-oxoprolinase and they substrate has the same structure.No.2032-6 branch contains the four known compounds balhimycin,ansamitocin,vancomycin,teicoplaincin,glycopeptide antibiotic.In the NCBI sequence alignment results,the similarity of No.2032-6 to the known compound was 95%.Conversion screened clones,and heterologous expression in Streptomyces albus.The crude extract of the obtained fermentation broth was detected by high performance chromatography,no specific peaks were found.Analysis of this result,we got a conclsion which clones do not contain a complete halogenase gene cluster or the transferred host is not suitable.