2007 Annual Report 1a.Objectives (from AD-416) The objectives are to. 1)identify the genes responsible for the production of the diterpenoid precursors to prostratin and DPP from genetic libraries originating from Euphorbia esula,. 2)identify candidate genes from an existing expressed sequence tag (EST)-database and obtain full-length genes from the cDNA libraries developed from Euphorbia esula,. 3)engineer candidate genes into microbial hosts and test for their value in synthesis of diterpenoid precursors, and. 4)identify terpenoid products formed. 1b.Approach (from AD-416) This will be a joint effort between ARS, who will provide genetic material, sequence information, and expertise for Euphorbia esula, and the University of California, who will be responsible for expressing candidate genes in suitable microbial hosts and identifying any terpenoid products formed. Both partners will collaborate in identifying candidate genes for screening and will contribute to the identification and isolation of full-length cDNA clones for candidate genes. The approach being used for the production of these diterpenoid products is novel and would likely involve patenting applications if successful. 3.Progress Report This report documents a Non-Funded Cooperative Agreement between USDA-ARS and the University of California-Berkeley. Additional information can be found in the report of the parent CRIS 5442-21220-017-00D. The objectives of this collaboration are to identify the genes responsible for production of the diterpenoid precursors to prostratin (12-deoxyphorbol 13-acetate) and DPP (12-deoxyphorbol 13-phenylacetate) from genetic libraries originating from Euphorbia esula. These related compounds have recently shown great potential for the treatment of HIV. The supplies of prostratin and DPP are limited, as they both originate from uncommon Euphorbiaceae plant sources, and chemical synthesis routes have not been devised. The USDA-ARS was provided genetic material, sequence information, and expertise for Euphorbia esula and the University of California is using these resources and information to isolate full-length candidate genes which are then engineered for expression into suitable microbial hosts to test for their value in synthesis of diterpenoid precursors. So far, two full length genes encoding putative terpene synthases (TPS1 & TPS2) have been isolated from the E. esula cDNA library and cloned into both the Escherichia coli expression vector pTrc99A and the Saccharomyces cerevisiae expression vector pRS425-gal I. Initial testing suggests that TSP1 likely encodes a GGPP synthase while TSP2 likely encodes a casbene synthase. The ADODR actively monitors the project by providing technical advice, making site visits, and reviewing accomplishments with cooperating personnel.