The cytochrome P450 enzyme-encoding genes rosC and rosD were cloned from the rosamicin biosynthetic gene cluster of Micromonospora rosaria IFO13697. The functions of RosC and RosD were demonstrated by gene disruption and complementation with M. rosaria and bioconversion of rosamicin biosynthetic intermediates with Escherichia coli expressing RosC and RosD. It is proposed that M. rosaria IFO13697 has two pathway branches that lead from the first desosaminyl rosamicin intermediate, 20-deoxo-20-dihydro-12,13-deepoxyrosamicin, to rosamicin. O xidation catalyzed by cytochrome P450 enzymes in postpolyketide synthase (post-PKS) modification of macrolide antibiotics contributes to structural diversification and modulates bioactivity. Rosamicin, which is a 16-member macrolide antibiotic produced by Micromonospora rosaria IFO13697 (1), contains an epoxide and a formyl group at the C-12/13 and C-20 positions, respectively, and it is expected that two different types of P450s generate these functional groups (Fig. 1). Recently, we reported that the mycinosyl rosamicin derivatives were produced by genetic engineering of M. rosaria TPMA0001 (2, 3). Here, we cloned the cytochrome P450 enzyme-encoding genes rosC and rosD from M. rosaria IFO13697 and demonstrated the functions of RosC and RosD in the rosamicin biosynthetic pathway.Six complete protein-coding regions (orf1 to orf4, rosC, and rosD) and a partial protein-coding region (rosAI) were contained in the 9,036-bp DNA fragment in the cosmid pRS85, which was isolated using a PCR product amplified with degenerate primers as a DNA probe for colony hybridization. The primers were designed from two conserved regions of deduced amino acid sequences of P450s implicated in formylation of 16-member macrolides (4-8). The complete nucleotide sequence of the rosamicin biosynthetic gene cluster in Micromonospora carbonacea subsp. aurantiaca NRRL2997 was determined by Farnet et al. (8). The deduced amino acid sequences of RosC and RosD were most similar to P450s encoded in ORF3 and ORF4 of M. carbonacea subsp. aurantiaca NRRL 2997 (87% and 83% identity, respectively) (see Fig. S1 in the supplemental material). In BLAST searches, RosC and RosD were similar to TylI (71% identity) in tylosin biosynthesis and OleP (48% identity) in oleandomycin biosynthesis, respectively (4, 9).To obtain the rosC and rosD disruption mutants of M. rosaria IFO13697, disruption plasmids pRS511 and pRS514 were constructed using a PCR-targeting method (10). These disruption plasmids were introduced into M. rosaria IFO13697 by conjugation using our previous procedure (2). The resulting disruption mutants, TPMA0050 and TPMA0055, did not produce rosamicin when the strains were cultured in 172F medium. However, an unknown compound, RS-B, accumulated in the TPMA0050 culture broth, and unknown peaks RS-C, RS-D, and RS-E were detected in ethyl acetate extract of the TPMA0055 culture broth by high-performance liquid chromatography (HPLC) analysis (Fig. 2; see Fig. S2 in the supplemental material). Furthermore, when ros...

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