PURIFICATION AND IN-VITRO RECONSTITUTION OF THE ESSENTIAL PROTEIN-COMPONENTS OF AN AROMATIC POLYKETIDE SYNTHASE

A minimal set of proteins which catalyze the synthesis of aromatic pol yketides from malonyl CoA has been purified and partially characterize d. Plasmid-encoded actinorhodin (net) ketosynthase/chain-length factor (KS/CLF) complex was purified from Streptomyces coelicolor CH999/pSEK 38, and assayed with purified aromatic PKS holo-ACPs which were overpr oduced and purified from Escherichia coli and phosphopantetheinylated in vitro using purified E. coli holo-ACP synthase, When highly purifie d preparations of KS/CLF, and holo-ACP failed to catalyze polyketide b iosynthesis, a fourth protein was sought and purified from the S. coel icolor CH999 host on the basis of its ability to complement KS, CLF, a nd holo-ACP in polyketide synthesis. N-terminal sequencing identified this protein as the fatty acid synthase (fabD) malonyl CoA:ACP transac ylase (MAT), recruited from primary metabolism. A alpha(2) beta(2) str ucture was shown for the act KS/CLF complex, and three malonyl-enzyme biosynthetic intermediates were identified, defining an escorted path followed by malonyl groups en route from CoA to polyketide.