Heterologous expression in Escherichia coli of an intact multienzyme component of the erythromycin‐producing polyketide synthase

Abstract

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 biosynthesis of erythromycin A by Saccharopolyspora erythraea. The gene encoding DEBS 3 has previously been cloned and sequenced, and the deduced product is predicted to house nine fatty acid synthase‐like activities 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 the recombinant E. coli to apparent homogeneity, and was found not to be modified by covalent attachment of the prosthetic group 4′‐phosphopantetheine. Incubation with (R,S)‐methylmalonyl‐CoA, the presumed source of extension units for polyketide chain assembly, led to hydrolysis of the thioester, implying that the methylmalonyl‐CoA:ACP acyltransferase domains in DEBS 3 are correctly folded and able to catalyse this side‐reaction. During this reaction, DEBS 3 became transiently radiolabelled, consistent with the intermediacy of an acylenzyme. The native molecular mass of the protein by gel filtration chromatography was 668 kDa which corresponds either to a dimer or to a highly asymmetric monomer.