To confirm the role of mycE and mycF genes in mycinamicin biosynthesis in M. griseorubida, disruption mutants of mycE and mycF were constructed by disruption plasmids containing attB in the disruption cassette Selleck CX-5461 FRT-neo-oriT-FRT-attB for the integration of φC31-derivative vector plasmids; the disruption mutants were complemented through the integration of pSET152 derivatives containing intact mycE or mycF into the artificially inserted attB site. These disruption mutants did not produce mycinamicin II, but mainly accumulated mycinamicins VI and III, indicating that MycE and MycF methylated

the C2″-OH group of 6-deoxyallose in mycinamicin VI and the C3″-OH group of C2″-methylated 6-deoxyallose in mycinamicin III, respectively. The complemented strains of mycE and mycF recovered the mycinamicin II productivity. In general, to confirm the function of a gene in a microorganism, the mutant with a disrupted gene should LEE011 nmr be isolated, and genetic complementation studies for the mutant should be performed. Recently, a simple and highly efficient PCR-targeting method was developed with the phage λ-Red recombinase to disrupt chromosomal genes in Escherichia

coli in which PCR primers provide the homology to the targeted gene (Datsenko & Wanner, 2000), and a modified system was also developed for gene targeting of Streptomyces strains with a disruption cassette, which contained an oriT region with a selectable antibiotic resistance gene to efficiently transfer a targeted plasmid from E. coli to Streptomyces by intergeneric conjugation (Gust et al., 2003). In Streptomyces strains, genetic complementation studies could be performed with transconjugation vectors, possessing a φC31 int gene and an attP site, that were site specifically inserted into the φC31 attB attachment site of a host chromosome. The attB site is distributed widely throughout Streptomyces strains, but there are few reports regarding the attB site of non-Streptomyces actinomycetes (Anzai et al.,

2009). Saccharopolyspora erythraea, which produces erythromycin, does not possess the φC31 attB site on its chromosome; the site was artificially introduced into the chromosome for antibiotic production using a combinatorial biosynthesis technique (Rodriguez et al., 2003). Dichloromethane dehalogenase Mycinamicin, which is produced by Micromonospora griseorubida A11725, is a 16-membered macrolide antibiotic with strong antibacterial activity against gram-positive bacteria (Satoi et al., 1980). Mycinamicin consists of a macrolactone substituted with two different sugars: desosamine and mycinose. The nucleotide sequence of the complete mycinamicin biosynthetic gene cluster has been reported (Anzai et al., 2003), wherein two putative O-methyltransferase (OMT) genes mycE and mycF were identified. It was reported previously by Inouye et al. (1994) that mycinamicin III (M-III) was converted to mycinamicin IV (M-IV) by the crude E.