Preliminary Study On The Key Genes In The Hygrocins Biosynthetic Gene Cluster

Most of ansamycin antibiotics exhibit antibacterial and antitumor activities.Many of them have been applied for pharmaceutical products and some of them are possible to be developed as new medicines due to their special targets of action.Hygrocins newly isolated from the fermentation broth of Streptomyces hygroscopicus ATCC29253,were identified as new ansamycin macrolideswith the shortest aliphatic chains.Biosynthesis of hygrocins is not yet clear.In this study,we cloned a 92.677 kb region of the ATCC29253 genome containing the entire hygrocins biosynthetic gene cluster.This cluster is composed of five PKS genes,five genes responsible for AHBA biosynthesis,four oxygenase genes,five regulator genes and many genes of unknown function.The biosynthetic pathway of hygrocins was proposed through bioinformatics analyses of organization of the active domains in PKS,substrate specificity of AT domains and active sites of the reductive domains.We constructed many gene disruption or gene knockout mutant strains of key genes in the hygrocins biosynthetic gene cluster.A PKS gene was disrupted by single crossover for identification of the cluster.Two genes in each flanking region of the cluster were selected to be disrupted.So far,we achieved two gene disruption mutant strains of them.We also constructed plasmids for gene knockout of three oxygenase genes and four regulator genes.So far,the gene knockout mutant strain of orf23 was obtained and selection of the others from their single crossover mutants is in progress.Hygrocins have the similar structure of rifamycin B.The oxygen atoms in their macrolides are supposed to be catalyzed by oxygenase.We optimized electroporation of Amycolatopsis mediterranei S699,the producing strain of rifamycin B.In order to investigate the oxidative cleavage of the hygrocins macrolide,the gene knockout mutant strains of two oxygenase genes,rif-orf5 and rif-orf16,in the rifamycin B biosynthetic gene cluster were achieved for heterologous complementation experiments by using the optimized genetic manipulation system.