Biosynthesis Of Geldanamycin And Cycloheximide

Natural products are the best resources for drug discovery. The studies on the biosynthesis of natural products would accelerate the process of developing novel compounds with better pharmaceutical properties from natural products. To understand the biosynthetic mechanisms of natural products in Streptomycetes and to obtain novel compounds through genetic engineering, the biosynthesis of geldanamycin and cycloheximide, which were discovered from Streptomyces autolyticus CGMCC0516and Streptomyces sp. YIM56141, respectively, was studied, their biosynthetic mechanism was partially elucidated, and an important anti-cancer compound and several compounds with possible novel structures were obtained through constructing mutants in their biosynthetic pathways.In order to further elucidate the biosynthetic mechanism of geldanamycin and to obtain new geldanamycin analogs through genetic manipulation, we constructed a genomic library of S. autolyticus, cloned the gene cluster for geldanamycin biosynthesis, studied its biosynthetic pathway through constructing knock-out mutants, complementation, overexpressing related enzymes and studying their properties. A methyltransferase gene (gdmMT), which located17kb upstream of GdmAI, was found to catalyze the C-17methyltransfer of geldanamycin. By knocking out gdmMT, a large amount of17-O-demethyl-geldanamycin and traces of17-amino-17-demethoxyl-geldanamycin (17-AG) were obtained, the latter is one of the most promising anti-tumor compound in development. This is the first report of obtaining17-AG by knocking out the methyltransferase gene. It is more economical and environment-friendly to generated17-AG by this way than traditional chemical modification.In order to obtain novel bioactive analogs of cyclothximide (CHX) by combinatorial biosynthesis approaches, the genomic library of Streptomyces sp. YIM56141was constructed, the gene cluster for CHX was cloned, and a45.5kb DNA fragment covering the cluster was sequenced, facilitated by a genome scanning method. Through bioinformatics analysis, gene knock-out, complementation, and protein overexpression,10genes (36.5kb) were confirmed to be involved in the CHX biosynthesis. Knock-out mutants of genes chxG, chxH, chxl, and chxJ were constructed, and several new compounds were isolated from their ferments. The structure of one of the compounds was illustrated to be8,9-dehydro-cycloheximide (8,9-dehydro-CHX), which is a new CHX analog that has not been reported before. The CHX gene cluster was also responsible for antiphenol biosynthesis. This finding provided solid foundation for generating novel CHX analogs by combinatorial biosynthesis approaches. Furthermore, the PKS of CHX gene cluster belongs to a unique AT-less type â…  PKS, which could be used as a model for studying the biosynthetic mechanism of AT-Less Type â…  PKS, as well as a suitable material to unveil the details of glutarimide formation.In conclusion, the biosynthetic pathways of geldanamycin and cycloheximide were studied and their mechanisms were partially elucidated in this work, which provided theoretical basis for generating novel analogs of these compounds with better pharmaceutical properties through combinatorial biosynthesis.