DISCODERMOLIDE BIOSYNTHESIS GENES

DESCRIPTION (provided by applicant): The long-range goal of our research is to develop an economical method for producing the experimental anti-tumor drug, (+) -discodermolide, by microbiological fermentation. Pre-clinical investigation of discodermolide has been hampered by inadequate supply of this marine polyketide. It currently is available by isolation or total synthesis, but the cost of the drug is very high. To enable development of a fermentation based process, the discodermolide biosynthetic genes will be cloned from the marine sponge from which discodermolide is isolated, followed by expression of these genes in a bacterial host. The immediate goal of Phase I research is to identify and clone many of the discodermolide biosynthesis genes by pursuing the following specific aims: (1) Detect modular polyketide synthase (PKS) and carbamoyl phosphate transferase (CPT) gene homologs by PCR analysis of cells or cell extracts of the marine sponge that produces discodermolide. (2) Prepare a fosmid library using DNA isolated from the sponge or its putative microbial symbiont and by PCR analysis identify clones that contain these PKS and CPT genes. (3) Sequence the PKS and CPT genes to the extent required to justify their involvement in discodermolide biosynthesis. Our research will also establish the technological base for a general approach to making many other currently scarce marine polyketides with anti-tumor activity available in large amounts for drug development.