Cloning Of The Dithiolopyrrolone Antibiotics Biosynthetic Gene Cluster In Saccharothrix Algeriensis NRRL B-24137

Saccharothrix algeriensis NRRL B-24137(B-24137)produces various dithiolopyrrolone antibiotics,which exhibit obvious antimicrobial activities against Gram-Positive and Gram-Negative bacteria,yeast and filamentous fungi.The variety and yield of dithiolopyrrolones productions in B-24137 changed with culture media.To clone the dithiolopyrrolones biosynthetic gene cluster in B-24137,we constructed a genomic cosmid library of B-24137 and fished out 12 positive clones from the library by PCR screening with a pair of primers designed for amplification of the thioredoxin reductase.However,heterologous expression of the positive cosmids in Streptomyces lividans ZX1 and Streptomyces ablus failed.Meanwhile,the putative dithiolopyrrolones biosynthetic gene cluster was found in the genome sequence of B-24137.Bioinformatics analyses showed that this cluster had certain degree of similarities with the holomycin biosynthetic gene cluster in Streptomyces clavuligerus and the thiolutin biosynthetic gene cluster in Streptomyces thioluteus.Two genes encoding isolated Cy and TE domains are missing in the cluster of B-24137,which have been verified to be essential for the biosynthesis of dithiolopyrrolones.No genetic manipulation method for Saccharothrix species is reported.We tried to transfer the integrative plasmid pSET152 and the autoreplicative plasmid pKC1139 from Escherichia coli to B-24137 by intergeneric conjugation useing E.coli S17-1 and ET12567(pUZ8002)as the donor host strains.We tested the effects of conjugation media,recipient/donor ratio,heat-shock temperature,time point of overlay and supplemented MgCl2 concentration on conjugation frequency.But all attempts failed until we achieved conjugants by using the positive cosmids containing large homologous sequences with the B-24137 genome.Establishemnt of a genetic manipulation system for B-24137 will not only benefit functional studies of the dithiolopyrrolones biosynthetic pathway but also contribute to genetics study of Saccharothrix in the future.