MCAT is not required for in vitro polyketide synthesis in a minimal actinorhodin polyketide synthase from Streptomyces coelicolor

Background: It has been proposed that Streptomyces malonyl CoA:holo acyl ca rrier protein transacylases (MCATs) provide a link between fatty acid and p olyketide biosynthesis. Two recent studies have provided evidence that the presence of MCAT is essential for polyketide synthesis to proceed in recons tituted minimal polyketide synthases (PKSs). In contrast to this, we previo usly showed that the hole acyl carrier proteins (ACPs) from type II PKSs ar e capable of catalytic self-malonylation in the presence of malonyl CoA, wh ich suggests that MCAT might not be necessary for polyketide biosynthesis. Results: We reconstituted a homologous actinorhodin (act) type II minimal P KS in vitro, When act holo-ACP is present in limiting concentrations, MCAT is required by the synthase complex in order for polyketide biosynthesis to proceed, When holo-ACP is present in excess, however, efficient polyketide synthesis proceeds without MCAT. The rate of polyketide production increas es with holo-ACP concentration, but at low ACP concentration or equimolar A CP:KS:CLF (KS, ketosynthase; CLF, chain length determining factor) concentr ations this rate is significantly lower than expected, indicating that free holo-ACP is sequestered by the KS/CLF complex. Conclusions: The rate of polyketide biosynthesis is dictated by the ratio o f holo-ACP to KS and CLF, as well as by the total protein concentration. Th ere is no absolute requirement for MCAT in polyketide biosynthesis in vitro , although the role of MCAT during polyketide synthesis in vivo remains an open question. MCAT might be responsible for the rate enhancement of malony l transfer at very low free holo-ACP concentrations or it could be required to catalyse the transfer of malonyl groups from malonyl CoA to sequestered holo-ACP.