Study On The Lantibiotics And Leinamycin Analogue Biosynthetic Gene Clusters In Streptomycrs Sp.SH-62

This article mainly includes the heterologous expression and disruption of the lantibiotic biosynthetic gene cluster and the key gene knockout of the leinamycin analogue biosynthetic gene cluster in Streptomyces sp.SH-62.1.Lantibiotics produced by Gram-positive bacteria have good biological activity against Gram-positive bacteria,and some of them also exhibit potential anti-tumor and anti-virus effects.A putative lantibiotic biosynthetic gene cluster(named lah)was found in the genome of Streptomyces sp.SH-62.To investigate function of the lah cluster,4 BAC plasmids,15H2,21C4,21D6 and 21D7,were introduced into S.lividans ZX1 and S.lividans ZAY3 for heterologous expression through conjugation in this study.The heterologous expression strains in S.lividans ZX1 displayed a weak inhibition against Gram positive bacteria,while no inhibition effect was detected in the heterologous expression strains in S.lividans ZAY3 in which that three endogenous antibiotic biosynthetic gene clusters of actinorhodine,undecylprodigiosin and and calcium dependent antibiotic were abrogated.It was speculated that other genes on the BAC plasmid stimulated the expression of originally silent antibiotic synthetic gene clusters in S.lividans ZX1.In order to a improve the expression of the lah cluster,all the essential biosynthetic genes of the cluster were cloned into a high copy number and self-replicative Streptomyces vector and on the control of strong promotors.Meanwhile,the His-tag was fused to the C terminu of LahA protein for purification of the target lantibiotic peptide.On the other hand,the lantibiotic dehydratase gene lahB was disrupted by single crossover in the wild type strain Streptomyces sp.SH-62.And the plasmid for gene knockout of the precursor peptide synthetase gene lahA by double crossover was also constructed.2.Leinamycin(LNM)is a kind of macrolide antibiotic with anticancer effect.Bioinformatics analysis found that there is a gene cluster with high similarity to the LNM biosynthetic gene cluster(named lem)in the genome of Streptomyces sp.SH-62.The corresponding product of this cluster was supposed to be leinamycin analogue.Bioassay of the heterologous expressin strains of the lem cluster found a small amount of antibacterial product,but the absorption peak of this compound was not traced by HPLC.Presumably,the yield of the heterologous expression product is too low to be detected.At the same time,the key NRPS-PKS gene lemL for the product biosynthesis was successfully knocked out in SH-62 by double crossover.Metabolic defferences between the wild type strain and the gene knockout mutant strain SH-62?lemL need to be studied in details by TLC and HPLC in the future.