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Research Paper |
Bio-X Life Science Research Center, Shanghai Jiaotong University, Shanghai 200030, China1
Huazhong Agricultural University, Wuhan 430070, China2
Jiangxi Agricultural University, Nanchang 330045, China3
John Innes Centre, Colney, Norwich NR4 7UH, UK4
Author for correspondence: Zixin Deng. Tel: +86 21 62933404. Fax: +86 21 62933404. e-mail: zxdeng{at}mail.sjtu.edu.cn
Several independent gene clusters containing varying lengths of type I polyketide synthase genes were isolated from ‘Streptomyces nanchangensis’ NS3226, a producer of nanchangmycin and meilingmycin. The former is a polyether compound similar to dianemycin and the latter is a macrolide compound similar to milbemycin, which shares the same macrolide ring as avermectin but has different side groups. Clusters A–H spanned about 133, 132, 104, 174, 122, 54, 37 and 59 kb, respectively. Two systems were developed for functional analysis of the gene clusters by gene disruption or replacement. (1) Streptomyces phage 
C31 and its derived vectors can infect and lysogenize this strain. (2) pSET152, an Escherichia coli plasmid with C31 attP site, and pHZ1358, a Streptomyces–Escherichia coli shuttle cosmid vector, both carrying oriT from RP4, can be mobilized from E. coli into NS3226 by conjugation. pHZ1358 was shown to be generally useful for generating mutant strains by gene disruption and replacement in NS3226 as well as in several other Streptomyces strains. A region in cluster A (
133 kb) seemed to be involved in nanchangmycin production because replacement of several DNA fragments in this region by an apramycin resistance gene [aac3(IV)] gave rise to nanchangmycin non-producing mutants.
Keywords: antibiotic biosynthetic genes, dianemycin, avermectin, polyketide synthase, gene replacement in Streptomyces
Abbreviations: PKS, polyketide synthase; DEBS, 6-deoxyerythronolide B synthase; ACP, acyl carrier protein; KS, ketosynthase; KR, ketoreductase; AT, acyltransferase; TE, thioesterase
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