Precursor-directed biosynthesis of 6-deoxyerythronolide B analogs in Streptomyces coelicolor: Understanding precursor effects

A fermentation process employing precursor-directed biosynthesis is being d eveloped for the manufacture of 6-deoxyerythronolide B (6-dEB) analogues. T hrough a plasmid-based system in Streptomyces coelicolor, 6-dEB synthesis i s catalyzed by 6-dEB synthase (DEBS). 6-dEB synthesis is abolished by inact ivation of the ketosynthase (KS) 1 domain of DEBS but can be restored by pr oviding synthetic activated diketides. Because of its inherent catalytic fl exibility, the KS1-deficient DEBS is capable of utilizing unnatural diketid es to form various 13-substituted 6-dEBs. Here we characterize process vari ables associated with diketide feeding in shake-flask experiments. 13-R-6-d EB production was found to depend strongly on diketide feed concentrations, on the growth phase of cultures at feeding time, and on the R-group presen t in the diketide moiety. In all cases a major portion of the fed diketides was degraded by the cells.