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

Yunlong He, Yuhui Sun, Tiangang Liu, Xiufen Zhou, Linquan Bai*, and Zixin Deng

Appliedand Environmental Microbiology 2010, 76(10):3283-3292

Epub Date: 26 March 2010

DOI: 10.1128/AEM.02262-09


Five meilingmycins, A to E, were isolated from Streptomyces nanchangensis NS3226. Through nuclear magnetic resonance characterization, meilingmycins A to E proved to be identical to reported milbemycins α11, α13, α14, β1, and β9, respectively. Sequencing of a previously cloned 103-kb region identified three modular type I polyketide synthase (PKS) 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-coenzymeA (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-encodedenzyme AVES1. A PKS gene encoding one loading and two elongation modules, with a downstream C-5-O-methyltransferasegene, 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 inter cluster 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.

In vitro reconstruction of tetronate RK-682 biosynthesis

Yuhui Sun*, Frank Hahn, Yuliya Demydchuk, James Chettle, Manuela Tosin, Hiroyuki Osada, and Peter F. Leadlay*

Nature Chemical Biology 2010, 6(2):99-101

Epub Date: 20 December 2009

DOI: 10.1038/nchembio.285


The protein phosphatase inhibitor RK-682 is one of a number of potentially valuable tetronate polyketide natural products. Understanding how the tetronate ring is formed has been frustrated by the inaccessibility of the putative substrates. We report the heterologous expression of rk genes in Saccharopolyspora erythraea and reconstitution of the RK-682 pathway using recombinant enzymes, and we show that RkD is the enzyme required for RK-682 formation from acyl carrier protein-boundsubstrates.