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ABSTRACTThe cytochrome P450 enzymes MycCI and MycG are encoded within the mycinamicin biosynthetic gene cluster and are involved in the biosynthesis of mycinamicin II (a 16-membered macrolide antibiotic produced by Micromonospora griseorubida). Based on recent enzymatic studies, MycCI is characterized as the C-21 methyl hydroxylase of mycinamicin VIII, while MycG is designated multifunctional P450, which catalyzes hydroxylation and also epoxidation at C-14 and C-12/13 on the macrolactone ring of mycinamicin. Here, we confirm the functions of MycCI and MycG in M. griseorubida. Protomycinolide IV and mycinamicin VIII accumulated in the culture broth of the mycCIdisruption mutant; moreover, the mycCIgene fragment complemented the production of mycinamicin I and mycinamicin II, which are produced as major mycinamicins by the wild strain M. griseorubidaA11725. The mycGdisruption mutant did not produce mycinamicin I and mycinamicin II; however, mycinamicin IV accumulated in the culture broth. The mycGgene was located immediately downstream of the self-resistance gene myrB. The mycGgene under the control of mycGpcomplemented the production of mycinamicin I and mycinamicin II. Furthermore, the amount of mycinamicin II produced by the strain complemented with the mycGgene under the control of myrBpwas approximately 2-fold higher than that produced by the wild strain. In M. griseorubida, MycG recognized mycinamicin IV, mycinamicin V, and also mycinamicin III as the substrates. Moreover, it catalyzed hydroxylation and also epoxidation at C-14 and C-12/13 on these intermediates. However, C-14 on mycinamicin I was not hydroxylated.