<jats:p>Several independent gene clusters containing varying lengths of type I polyketide synthase genes were isolated from <jats:italic>‘Streptomyces nanchangensis’</jats:italic> 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) <jats:italic>Streptomyces</jats:italic> phage ϕC31 and its derived vectors can infect and lysogenize this strain. (2) pSET152, an <jats:italic>Escherichia coli</jats:italic> plasmid with ϕC31 <jats:italic>attP</jats:italic> site, and pHZ1358, a <jats:italic>Streptomyces–Escherichia coli</jats:italic> shuttle cosmid vector, both carrying <jats:italic>oriT</jats:italic> from RP4, can be mobilized from <jats:italic>E. coli</jats:italic> 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 <jats:italic>Streptomyces</jats:italic> 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 [<jats:italic>aac3(IV)</jats:italic>] gave rise to nanchangmycin non-producing mutants.