I plan to study the enzymology of formation of isopentenyl diphosphate, the universal precursor of all isoprenoids, from deoxyxylulose phosphate in Streptomyces UC5319, the producer of the sesquiterpene antibiotic pentalenolactone. This prokaryotic organism has been shown to use the non-classical pyruvate/triose phosphate pathway to isoprenoids recently discovered by Prof. Michel Rohmer of the Universit? Louis Pasteur in Strasbourg, France. Based on our own long-standing interest in the biosynthesis of terpenoids in general, and the biochemistry and molecular genetics of pentalenene biosynthesis in particular, we plan to collaborate with Prof. Rohmer to study the pathway linking the key intermediate, 1-deoxyxylulose phosphate, to isopentenyl diphosphate in Streptomyces UC5319. The specific goals during the proposed visit to Strasbourg will be: 1) Cloned 1-deoxyxylulose phosphate synthase from E. coli will be used to prepare isotopically labeled 1-deoxyxylulose from appropriately labeled samples of pyruvate and glyceraldehyde-3- phosphate; 2) Labeled 1-deoxyxylulose phosphate will be incubated with cell-free extracts of Streptomyces UC5319 and the products analyzed to detect derived metabolites such as 2-C-methyl-D-erythrose phosphate and other intermediates leading to isopentenyl diphosphate; and 3) The sequence of the E. coli deoxyxylulose phosphate synthase gene will be used to design PCR primers for amplification of specific probes intended to isolate the corresponding gene from a genomic library of Streptomyces UC5319. Period of visit: February 1, 1999 - June 30, 1999
