? The long-range goal of our research is to develop economical methods for producing multigram quantities of experimental antitumor drugs from marine sources. Our approach is based on cloning the biosynthetic genes from the producing organism followed by expression of these genes in a bacterial host that can be grown in large-scale fermentations. Mechanistic and preclinical investigations of bryostatins, discodermolide, halichondrins, laulimalide and other marine cytotoxins, which are made by pathways involving polyketide synthases, have been hampered by inadequate supplies of the marine polyketides because they are available only by isolation or total synthesis at a very high cost. The goal of Phase I research is to identify the genetic locus that contains the biosynthesis genes for laulimalide by the following specific aims: (1) Obtain fresh sponge tissue from a reliable source, and show that it (or its symbionts) contains laulimalide. (2) Detect modular polyketide synthase (PKS), CYP450, """"""""hydroxymalonate (HM)"""""""" and hydroxymethylglutaryI-Coenzyme A synthase (HMGS) gene homologs by PCR analysis of DNA from cells or cell extracts of the marine sponge (and/or their microbial symbionts) that produce laulimalide. (3) Prepare fosmid and BAC libraries using DNA isolated from the sponge/symbiont complex that produces laulimalide or from a putative microbial symbiont, and by PCR and DNA hybridization analyses obtain clones that contain the PKS, CYP450, HM and HMGS genes. (4) Sequence the region that contains the PKS, CYP450, HM and HMGS genes to the extent required to justify their involvement in laulimalide biosynthesis. Our experimental design is built on work in progress in which we are attempting to clone the discodermolide biosynthesis genes. Success will lead to development of a fermentation-based process to make large amounts of laulimalide or one of its analogs for preclinical studies as an antitumor drug. ? ? ?
