Ethyl-substituted erythromycin derivatives produced by directed metabolic engineering

Abstract

A previously unknown chemical structure, 6-desmethyl-6-ethylerythromycin A (6-ethylErA), was produced through directed genetic manipulation of the erythromycin (Er)-producing organism Saccharopolyspora erythraea. In an attempt to replace the methyl side chain at the C-6 position of the Er polyketide backbone with an ethyl moiety, the methylmalonate-specific acyltransferase (AT) domain of the Er polyketide synthase was replaced with an ethylmalonate-specific AT domain from the polyketide synthase involved in the synthesis of the 16-member macrolide niddamycin. The genetically altered strain was found to produce ErA, however, and not the ethyl-substituted derivative. When the strain was provided with precursors of ethylmalonate, a small quantity of a macrolide with the mass of 6-ethylErA was produced in addition to ErA. Because substrate for the heterologous AT seemed to be limiting, crotonyl-CoA reductase, a primary metabolic enzyme involved in butyryl-CoA production in streptomycetes, was expressed in the strain. The primary macrolide produced by the reengineered strain was 6-ethylErA.

Biosynthesis of the Pseudomonas polyketide coronafacic acid requires monofunctional and multifunctional polyketide synthase proteins

“…Crotonyl-CoA reductase catalyzes one step in the conversion of acetoacetyl-CoA to butyryl-CoA, and the latter product, as ethylmalonyl-CoA, is used as a four-carbon extender unit in polyketide synthesis (36). A ccr gene is also part of the biosynthetic gene cluster encoding the polyketide tylosin (36), and the overexpression of ccr in Saccharopolyspora erythraea has been utilized to produce novel derivatives of erythromycin (37). The recruitment of a ccr gene into the CFA gene cluster may reflect the requirement of butyryl-CoA as a precursor for CFA synthesis.…”

Complete genome sequence of the erythromycin-producing bacterium Saccharopolyspora erythraea NRRL23338

“…There is a single cluster of the adenosylcobalamin-dependent methylmalonyl-CoA mutase genes (SACE_5638-5640). S. erythraea, unlike many streptomycetes, has no homolog of crotonyl-CoA reductase or of adenosylcobalamin-dependent isobutyryl-CoA mutase, explaining its inability to furnish butyrate units for polyketide biosynthesis 38,39 ; it also has no homolog of meaA in S. coelicolor, which has been implicated in provision of methylmalonyl-CoA from acetoacetyl-CoA 40 . The availability of the complete genome sequence now provides the basis for systematic approaches to identify and manipulate such feeder pathways with the aim of increasing polyketide production.…”

Carboxylases in Natural and Synthetic Microbial Pathways

“…This principle has been already successfully demonstrated. Introduction of the CCR gene into the erythromycin producer Saccharopolyspora erythraea resulted in the production of an ethyl-substituted erythromycin derivative (113). Similarly, CCR was shown to provide ethylmalonyl-CoA for an engineered carboxymethylproline synthase in vitro, yielding new precursors for carbapenem antibiotic synthesis (58).…”