Format

Send to

Choose Destination
Appl Environ Microbiol. 2010 May;76(10):3283-92. doi: 10.1128/AEM.02262-09. Epub 2010 Mar 26.

Cloning of separate meilingmycin biosynthesis gene clusters by use of acyltransferase-ketoreductase didomain PCR amplification.

Author information

1
School of Life Science & Biotechnology and Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai 200030, China.

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 alpha11, alpha13, alpha14, beta1, and beta9, 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.

PMID:
20348291
PMCID:
PMC2869129
DOI:
10.1128/AEM.02262-09
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center