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

Abstract

-EJB 92 1720/2 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.Erythromycin A is a clinically important macrolide antibiotic produced by the Gram-positive soil-borne bacterium, Saccharopolyspora erythraea [l]. The biosynthetic pathway to erythromycin A begins with the processive assembly of a polyketide chain from one molecule of propionyl-CoA and six molecules of methylmalonyl-CoA by a mechanism directly analogous to that of fatty acid biosynthesis [2]. Following six rounds of condensation and reduction the polyketide chain is cyclised to give the 14-membered macrolactone, 6-deoxyerythronolide B, the earliest enzyme-free intermediate [3] so far identified. The lactone is subsequently modified by specific hydroxylases, glycosyltransferases and a methyltransferase to give erythromycin A [4].The erythromycin biosynthetic genes are clustered on the chromosome together with the resistance determinant ermE. The genes encoding 6-deoxyerythronolide B synthase (DEBS) are located within the eryA region of the biosynthetic gene cluster and together span over 30 kbp [ 5 ] , representing more than 50% of the total size of the cluster. Partial nucleotide sequencing of this region revealed multiple copies of sequences encoding fatty acid synthase-like activi-

Pristinamycin I biosynthesis in Streptomyces pristinaespiralis: molecular characterization of the first two structural peptide synthetase genes

“…This is the first time, to our knowledge, that a PPS protein containing two modules has been overexpressed in E. coli by using the T7 RNA polymerase system, and the failure of the overexpressed SnbC protein to catalyze the adenylation reactions could be due to a misfolding of the enzyme in the absence of the phosphopantetheinyl arm. It seems now certain that E. coli is not the ideal host for overexpression of heterologous PPS or polyketide synthetase genes (8,34), as the products are not correctly posttranslationally modified. Direct purification from S. pristinaespiralis gives yields too low for performance of mechanistic studies.…”

Expression of a functional fungal polyketide synthase in the bacterium Streptomyces coelicolor A3(2)

“…Functional expression of a heterologous PKS in a similar expression plasmid in S. coelicolor CH999 has been demonstrated for the erythromycin PKS genes from Saccharopolyspora erythraea (21). In contrast, the erythromycin PKS proteins isolated from E. coli could not direct chain extension in vitro (although acyl transfer occurred) because of the lack of 4Ј-phosphopantetheine group addition (25,35). The type II Streptomyces glaucescens PKS ACP component from the tetracenomycin C antibiotic gene cluster also lacked significant addition of the 4Ј-phosphopantetheine group when it was expressed in E. coli (36).…”

Analysis of core sequences in the D-Phe activating domain of the multifunctional peptide synthetase TycA by site-directed mutagenesis

“…A comparison of the amino acidracemizing domains of peptide synthetases revealed four homologous motifs at the C-terminal end of each domain (12), which were not the subject of this investigation. The racemization reaction of GrsA is supposed to occur on the thioesterified phenylalanine, and a base group has been suggested to function as a proton donor and acceptor during racemization (21 (50), whereas 6-deoxyerythronolide B synthase 3 of S. erythraea, which is involved in the synthesis of the polyketide erythromycin A, could not be labeled in E. coli even though it shows the 4'-phosphopantetheine-binding motif of ACPs (45). We suggest that TycA is a poor substrate for the ACP synthase of E. coli and that therefore modification of TycA with 4'-phosphopantetheine is incomplete.…”