Cloning of Separate Meilingmycin Biosynthesis Gene Clusters by Use of Acyltransferase-Ketoreductase Didomain PCR Amplification

Abstract

Five meilingmycins, A to E, with A as the major component, were isolated from Streptomyces nanchangensis NS3226. Through nuclear magnetic resonance (NMR) characterization, meilingmycins A to E proved to be identical to reported milbemycins ␣11, ␣13, ␣14, ␤1, and ␤9, respectively. Sequencing of a previously cloned 103-kb region identified three modular type I polyketide synthase genes putatively encoding the last 11 elongation steps, three modification proteins, and one transcriptional regulatory protein for meilingmycin biosynthesis. However, the expected loading module and the first two elongation modules were missing. In meilingmycin, the presence of a methyl group at C-24 and a hydroxyl group at C-25 suggests that the elongation module 1 contains a methylmalonyl-coenzyme A (CoA)-specific acyltransferase (ATp) domain and a ketoreductase (KR) domain. Based on the conserved motifs of the ATp and KR domains, a pair of primers was designed for PCR amplification, and a 1.40-kb expected fragment was amplified, whose sequence shows significant homology with the elongation module 1 of the aveA1-encoded enzyme AVES1. A polyketide synthase (PKS) gene encoding one loading and two elongation modules, with a downstream C-5-O-methyltransferase gene, meiD, was subsequently localized 55 kb apart from the previously sequenced region, and its deletion abolishes meilingmycin production. A series of deletions within the 55-kb intercluster region rules out its involvement in meilingmycin biosynthesis. Furthermore, gene deletion of meiD eliminates meilingmycins D and E, with methyls at C-5. Our work provides a more specific strategy for the cloning of modular type I PKS gene clusters. The cloning of the meilingmycin gene clusters paves the way for its pathway engineering.Backbones of macrolides (e.g., erythromycin), polyenes (e.g., candicidin), polyethers (e.g., nanchangmycin), ansamycins (e.g., ansamitocin), and the polyketide portion of peptidepolyketide hybrids (e.g., oxazolomycin) are usually biosynthesized from simple carboxylic acids by the modular polyketide synthases (PKSs; type I PKSs) (6,8,30,37,38). Type I PKSs are multifunctional polypeptides and divided into modules, and each module is responsible for the incorporation of one carboxylic acid into the polyketide backbone. A minimal module is usually an ordered array of ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP) domains, with the ␤-carbonyl group unreduced in its thioester products (16). KS catalyzes the carbon-carbon bond formation between the incoming extender unit and polyketide intermediate by the Claisen condensation, whereas ACP serves as the carrier for both the incoming extender units and the extended chains using a covalently bound phosphopantetheine arm. Selection and loading of extender acyl units is executed by acyltransferase (AT) domains, which contributes to the structural diversity of polyketides by recruiting different extender units such as acetate-derived malonyl coenzyme A (CoA), propionate-derived methylmalonyl-CoA, and g...

Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function

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Enhancement of the Diversity of Polyoxins by a Thymine-7-Hydroxylase Homolog outside the Polyoxin Biosynthesis Gene Cluster

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Characterization and analysis of an industrial strain ofStreptomyces bingchenggensisby genome sequencing and gene microarray

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