Establishment Of A High-Throughput Heterologous Expression Approach And Its Application In Study Of Secondary Metabolism In Streptomyces

Antibiotic biosynthesis in streptomycetes is controlled by both pathway-specific and global regulators. To discover global regulators of Streptomyces clavuligerus, we constructed a cosmid library of 2400 clones (7 genome equivalents) in Escherichia coli harboring a conjugation helper plasmid pUZ8002. The cosmids were then transferred by efficient integrated conjugation to Streptomyces lividans which contains near silent antibiotic biosynthesis gene clusters. Several of the cosmids greatly increased the production of both actinorhodin and the calcium-dependent antibiotic in S. lividans. A S. clavuligerus homologue of the global regulatory gene afsR was responsible for the promotion of antibiotic biosynthesis. Re-introducing this gene into S. clavuligerus, and thus increasing its copy number, increased clavulanic acid production and also activated the biosynthesis of holomycin which is encoded by a normally silent gene cluster. The efficient procedures for cloning and genetic manuipulation in E. coli, and for the transfer of an entire genomic library to S. lividans for the expression, can be applied for discovering regulatory genes from many actinomcyetes.