Cloning and analysis of geldanamycin partial biosynthetic gene cluster of streptomyces hygroscopicus 17997

Abstract

A geldanamycin (GDM)-producing strain, Streptomyces hygroscopicus 17997, was isolated from the soil of Yunnan, China, by the researchers of the Institute of Medicinal Biotechnology, CAMS & PUMC. GDM is an ansamycin antibiotic, which has the ability to bind with Hsp90 (heat shock protein 90) and alter its function. Hsp90 plays a key role in regulating the physiology of cells exposed to environmental stress and in maintaining the malignant phenotype of tumor cells. As an inhibitor of Hsp90, GDM possesses potent antitumoral and antiviral bioactivity, but the hypatotoxicity and poor solubility in water limit its clinical use. To accomplish the structural modification of GDM by genetic means, an attempt to obtain the biosynthetic gene cluster of GDM from S. hygroscopicus 17997 was made. In this study, a pair of primers was designed according to a conserved sequence of one of the possible post-PKS (polyketides synthase) modification genes, the carbamoyltransferase (CT) gene ( gdmN) in GDM biosynthesis. The 732-bp PCR product was obtained from the S. hygroscopicus 17997 genomic DNA. Through the colony-PCR Binary Search Method, using the CT gene primers, six positive cosmid clones, CT1-6, were identified from the S. hygroscopicus 17997 cosmid genomic library. The CT-4 positive cosmid was then sub-cloned and sequenced. Approximately 28.356 kb of foreign gene sequence from CT-4 cosmid and by further PCR extension reaction was obtained. According to BLAST analysis, this sequence contains 13 possible ORFs, and they are believed to be involved in GDM production. The obtained possible GDM biosynthetic gene cluster in S. hygroscopicus 17997 will facilitate the further functional analysis of the genes and the modification of the structure of GDM through combinatorial biosynthesis.