Production of hybrid macrolide antibiotics by exploiting the specific substrate recognition characteristics of multifunctional cytochrome P450 enzyme MycG.

Macrolide antibiotics are biosynthesized via enzymatic modifications, including glycosylation, methylation, and oxidation, after the core macro-lactone ring is generated by a polyketide synthase system. This study explored the diversification of macrolides by combining biosynthetic enzymes and reports an approach to produce unnatural hybrid macrolide antibiotics. The cytochrome (CYP) P450 monooxygenase MycG exhibits bifunctional activity, catalyzing late-stage hydroxylation at C-14 followed by epoxidation at C-12/13 during mycinamicin biosynthesis. The mycinose sugar of mycinamicin serves as a key molecular recognition element for binding to MycG. Thus, we subjected the hybrid macrolide antibiotic 23-O-mycinosyl-20-deoxo-20-dihydro-12,13-deepoxyrosamicin (IZI) to MycG, and confirmed that MycG catalyzed hydroxylation at C-22 and epoxidation at C-12/13 in IZI. In addition, the introduction of mycinose biosynthesis-related genes and mycG into rosamicin-producing Micromonospora rosaria enabled the fermentative production of 22-hydroxylated and 12,13-epoxidized forms of IZI. Interestingly, MycG catalyzed the sequential oxidation of hydroxylation and epoxidation in mycinamicin biosynthesis, but only single reactions in IZI. These findings highlight the potential for expanding the application of the multifunctional P450 monooxygenase MycG for the production of unnatural compounds.