The Studies On Geldanamycin Biosynthetic Gene Cluster In Streptomyces Hygroscopicus 17997

Geldanamycin (GDM), produced by the Streptomyces hygroscopicus 17997 which was isolated from China soil by our institute reseacher. GDM, belonging to the benzenenoid ansamycin, possess antitumor and antiprotozoon activity and recently, was discovered that it shows antivirus bioactivity. Extensive research have vertified that GDM can bind specifically to the ATP/ADP domain of heat shock protein 90 (Hsp90), thus down-regulate target proteins including tyrosine kinases, steroid receptors, transcription factors et al. As a specific inhibitor of Hsp90, GDM became a potential promising antitumor and antivirus medicine. To overcome the hypatotoxicity and poor solubility of GDM, the main research work was focused on finding its less toxic and more soluble derivatives. One of its derivatives, 17-AAG, is now in clinical trial (phase II) in United States. It was known that the biosynthesis of ansamycins involves the assembly 3-amino-5-hydroxybenzoic acid (AHBA) as starter unit, followed by the sequential addition of extender units such as acetate, malonate, and methoxymalonate to form a polyketide backbone catalyzed by type I polyketide synthsase (PKS), which then undergoes further downstream processing.During studies on the geldanamycin biosynthetic genes from Streptomyces hygroscopicus 17997, two possible separate AHBA biosynthetic gene clusters and two sets of PKS genes were found in our lab. But the involvement of these genes in the GDM biosynthesis was not identified, and the post-PKS modification genes were absent yet. In this study conserved sequences of one of possible post-PKS modification gene, the carbamoyltransferase gene (gdmN) in GDM biosynthesis were used to design the primers, by which a 732bp gene fragment was amplified from the Streptomyces hygroscopicus 17997 genome DNA. This gene fragment was sequenced and found it has identity of 94% with the gdmN of Streptomyces hygroscopicus NRRL 3602. Through the colony-PCR by the primers screening, six positive cosmid clones were identified from the Streptomyces hygroscopicus 17997 genomic library. The CT-4 cosmid was sub-cloned and sequenced. The 28.356kb foreign gene sequence of the CT-4 cosmid shows high identity with the known GDM biosynthesis gene cluster of Streptomyces hygroscopicus NRRL 3602 based on BLAST analysis. 12 possible ORFs were identified from the CT-4 cosmid including PKS gene...