Cloning And Heterologous Expression Of The Bioactive Natural Product Biosynthetic Gene Cluster From Streptomyces SH-62 And Preliminary Study On Regulation Of The Azinomycin B Biosynthesis

Two different research works are described in this study:1.Cloning and heterologous expression of the bioactive natural product biosynthetic gene cluster from Streptomyces SH-62.Streptomyces SH-62 isolated from the soil shows very high active biological activities and genome sequence analyses showed that there are many PKS and NRPS gene clusters in its genome.In order to study on these natural product biosynthetic gene clusters,we established a genetic manipulation system for SH-62.By using E.coil DCDH/pUZ8002 as the donor host strain,the temperature sensitive plasmid pKC1139 could be transferred into SH-62 through conjugation with the highest conjugation efficiency of 4.1×10-8 conjugants/recipet,which is still very low.Meanwhile,132 positive clones containing polyketide synthase(PKS)and/or nonribosomal peptide synthetase(NRPS)genes were fished out from the genomic BAC library of SH-62 by PCR screening.These 132 BAC plasmids were introduced into S.lividans ZX1 through high-throughput conjugation for heterologous expression and bioassay.31 of them could inhibit the growth of Bacillus subtilis.According to BAC terminal sequencing and restriction map analysis,we speculated that these 31 BAC spaned on seven NRPS/PKS natural product biosynthetic gene clusters.A hybird NRPS/PKS biosynthetic gene cluster(named lem)shows high homology with the leinamycin(Inm)biosynthetic gene cluster.By bioinformatics analyses,we found three related contigs,filled the gaps and assembled them together to obtain the complete sequence of the lem cluster.Compared the lem cluster with the Inm cluster,we found that their organization of the active domains in the hybrid NRPS/PKS and post-modification genes somehow were different.the lem cluster could be responsible for a new macrolactam antibiotic biosynthesis.Heterologous expression of 15E11 and 18D5 properly containing the entire lem cluster was failed in S.lividans and S.albus,but successful in ?aziD and?aziU3.The chemical structure of the heterologous expression product will be elucidated by further research in the future.2.Preliminary study on regulation of the azinomycin B biosynthesisAzinomycin B is produced by Streptomyces sahachiroi ATCC3318.Its unique structure,remarkable antitumor activity and novel mode of action have attracted increased research interests.Bioinformatics analyses show that three regulatory genes are located in azinomycin B biosynthetic gene cluster.orf7 is a TetR-family transcriptional regulator gene,orf9 is a HxlR-family transcriptional regulator gene and orf1213 is a two-component regulator gene.In the study,we made a preliminary investigation on regulation of the azinomycin B biosynthesis.By gene knockout and overexpression of orf7,orf9 and orfl2,it demonstrated that these three regulator genes were not involved in the azinomycin B biosynthetsis.RT-PCR assays also confirmed the transcriptional levels of aziA4?aziE?aziC7?aziB?aziU1?orf7?orf9 and orf12 from the azinomycin B biosynthetic gene cluster were no obviously different between the wild type strain and the gene knockout mutant strains.Using a promoter-probe vector,14 putative promoter regions were cloned and detected by expression of the reporter gene,the enhanced green fluorescent protein gene(egfp).It demonstrated that 12 of them exhibited promoter activities.Four promotors,Pazi/A2,PaziG,PaziC4,and Porf3 were introduced into the orginal host Streptmyces sahachiroi and the heterologous host Streptomyces lividans ZX1,respectively,for dynamic changes analysis of promoter activities through fluorescent intensity assay of mycelia.The change trends of their activities are similar with the strong constitutive promoter PermE*·We speculated that genes involved in the azinomycin biosynthesis could be constitutive expression,not controlled by pathway-specific regulators.