Gene targeting for O-methyltransferase genes, mycE and mycF, on the chromosome of Micromonospora griseorubida producing mycinamicin with a disruption cassette containing the bacteriophage phi C31 attB attachment site.

Mycinamicin, a 16-membered macrolide antibiotic produced by Micromonospora griseorubida, comprises a macrolactone and two deoxysugars: desosamine and mycinose. Mycinose is synthesized through two modification steps: the methylation of 6-deoxyallose in mycinamicin VI and of javose in mycinamicin III. 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 FRT-neo-oriT-FRT-attB for the integration of phiC31-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.