The Exploration Of Secondary Metabolite Biosynthetic Genes From Soil Microorganisms And Marine Streptomycetes

Microorganisms are an important source of new drugs.In the past few decades,new drugs including cephalosporins,lovastatin,and rapamycin have been discoveried from microorganisms.During the subsequent development of natural products,some known structures of compounds were found repeatedly,resulting in waste of material resources.Researches on unculturable microorganisms and microorganisms in unexplored environments have become new ways to obtain new active naturai products.According to studies,there are at least 107-109 species of bacteria per gram of soil on Earth,and only about 1%of microorganisms can be cultured under laboratory conditions,which limits the discovery of new genes from environmental microbial resources.In metagenome approach,the genome of environmental microorganisms was extracted directly,ligated with a vector and transferred into a suitable host to construct a metagenomic library.Then,the metagenomic library was screened for novel compounds.The screening methods include functional-based screening methods and sequence-based screening methods.Metagenomics breaks through the barriers of unculturable bacteria and can acquire more novel genes,providing resources for discovering novel active products.This thesis focuses on the study of biosynthetic genes of natural products from microorganisms in two directions.First,discovery of new aromatic polyketides by using metagenomics.Second,the biosynthetic genes of secondary metabolites of 7 marine Streptomycetes strains were analyzed to provide guidance for the subsequent acquisition of active products.Positive clones were identified from library by using degenerate primers designed according to the conserved KS? sequences.A total of 19 positive clones were obtained.Homologous analysis was performed base on the KS? sequences of positive clones.The analysis results showed that the KS? sequences on the positive clones,except 775,783,and 2077 are brand new.The KS? sequences on 775,783 and 2077 showed homolgy to the KS?responsible for chartreusin,hedamycin,and actinhordin,respectively.Fifteen positive clones were expressed and fermentation broth were analyzed by HPLC.Analysis results showed that the crude extract of the 886 fermentation broth contained 2 clone-special peaks.End-sequence analysis revealed that the synthetic gene cluster in the 886 clone was incomplete.From Everest library clones 198 and 1718,which share overlapping DNA frament with 886,were identified.End-sequence analysis of 1718 overlapping clones indicates that it may contain a complete gene cluster.Therefore,the 1718 clone was subjected to sequencing,analysis and functional expression.The sequence analysis of the 1718 clone revealed that the failed functional expression might be due to the lack of some biosynthetic genes in the 886 clone.In this study,the DNA fragments within conserved sequences of type I polyketide synthase,type ? polyketide synthase and NRPS synthase from seven marine Streptomycetes strains are amplified by PCR and sequenced.The sequencing results showed that some of secondary metabolite biosynthetic genes derived from H3 strain showed high homolog to these of the known compounds.At the same time,homology analysis of 16S rDNA sequences of 7 marine strains was performed and the evolutionary tree was constructed.Phylogenetic tree analysis showed that H2,H3,H4 and H10 strains were related to Streptomycetes sp.WMMB518,Streptomycetes sp.NEAE-126,Streptomycetes krainskii strain RSU51,Streptomycetes yangpuensis strain fd2-tb and that H5,H7 and H9 were related to Streptomycetes sp.HDI001.