Premature release of polyketide intermediates by hybrid polyketide synthase in Amycolatopsis mediterranei S699

The polyketide backbone of rifamycin B is assembled by the type I rifamycin polyketide synthase (PKS) encoded by the rifA-rifE genes. In order to produce novel analogs of rifamycin via engineering of the PKS genes, inactivation of the beta-ketoacyl:acyl carrier protein reductase (KR) domain in module 8 of rifD, by site-specific mutagenesis of the NADPH binding site. was attempted. Module 8 contains a nonfunctional dehydratase (DH) domain and a functional KR domain that is involved in the reduction of the beta-carbonyl group,resulting in the C-21 hydroxyl of rifamycin B. This mutant strain produced linear polyketides, from tetraketide to octaketide. which were also produced by a rifD-disruption mutant as a consequence of premature termination of the polyketide assembly. Another attempt to replace the DH domain of module 7, which has been considered nonfunctional, with a functional homolog derived from module 7 of rapamycin-producing PKS also resulted in the production of linear polyketides, including the heptaketide intermediate and its precursors. Premature release of the carbon chain assembly intermediates is an unusual property of the rifamycin PKS that is not seen in other PKSs such as the erythromycin PKS.