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

Gareth Roberts, James Staunton, Peter F. Leadlay

Research output: Contribution to journalArticlepeer-review

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.
Original languageEnglish
Pages (from-to)305-311
Number of pages6
JournalEuropean Journal of Biochemistry
Volume214
Issue number1
DOIs
Publication statusPublished - 1 May 1993

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