HETEROLOGOUS EXPRESSION IN ESCHERICHIA-COLI OF AN INTACT MULTIENZYME COMPONENT OF THE ERYTHROMYCIN-PRODUCING POLYKETIDE SYNTHASE

6-Deoxyerythronolide B synthase 3 (DEBS 3) is proposed to catalyse the fifth and sixth condensation cycles in the assembly of the polyketide 6-deoxyerythronolide B, the first isolatable intermediate in the bios ynthesis of erythromycin A by Saccharopolyspora erythraea. The gene en coding DEBS 3 has previously been cloned and sequenced, and the deduce d product is predicted to house nine fatty acid synthase-like activiti es on a 330-kDa polypeptide chain. The gene has been engineered into a pT-7-based expression system for over-expression in Escherichia coli. Recombinant DEBS 3 was found to constitute, after induction, 1-2% of soluble intracellular protein. DEBS 3 was purified from extracts of th e recombinant E. coli to apparent homogeneity, and was found not to be modified by covalent attachment of the prosthetic group 4'-phosphopan tetheine. Incubation with (R,S)-methylmalonyl-CoA, the presumed source of extension units for polyketide chain assembly, led to hydrolysis o f the thioester, implying that the methylmalonyl-CoA:ACP acyltransfera se domains in DEBS 3 are correctly folded and able to catalyse this si de-reaction. During this reaction, DEBS 3 became transiently radiolabe lled, consistent with the intermediacy of an acyl-enzyme. The native m olecular mass of the protein by gel filtration chromatography was 668 kDa which corresponds either to a dimer or to a highly asymmetric mono mer.