Function of cytochrome P450 enzymes RosC and RosD in the biosynthesis of rosamicin macrolide antibiotic produced by Micromonospora rosaria

Abstract

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.

Fig 1

The proposed biosynthetic pathway of rosamicin in M. rosaria IFO13697. The bold arrows represent the main pathway investigated in this study. The dashed arrow indicates the reaction with E. coli TPMB0003 cells expressing RosD. RosAI-V, putative polyketide synthase; RosB, putative glycosyltransferase; protorosanolide, putative macrolactone of rosamicin (tylactone) (15).

Fig 2

Physical maps of the region that includes rosC, rosD, and the flanking genes of the wild strain Micromonospora rosaria IFO13697, rosC disruption mutant TPMA0050, rosC complementation strain TPMA0053, rosD disruption mutant TPMA0055, rosD complementation strain TPMA0066, and rosC rosD double-disruption mutant TPMA0063. The strains, plasmids, and PCR primers used in this study are shown in Tables S1 and S2 in the supplemental material. The major products in the culture broth of the wild strain, disruption mutants, and complementation were detected by HPLC (see Fig. S2 in the supplemental material). Rosamicin and its biosynthetic intermediates in the ethyl acetate extracts from the culture broth were analyzed by HPLC. Disruption of the target gene and introduction of the complementation gene were confirmed by Southern blotting and PCR, as described in our previous report (; data not shown). The attB site lies within the ORF of the pirin homolog on the chromosome of M. rosaria IFO13697 (2). The gene names and their products or functions and promoter abbreviations are as follows: orf1, nucleosidase; orf2, ABC transporter; orf3, type II thioesterase; orf4, aminotransferase; rosC, cytochrome P450; rosD, cytochrome P450; rosAI, type I polyketide synthase; neo, neomycin resistance gene; aac(3)IV, apramycin resistance gene; oriT, origin of transfer from plasmid RP4 (12); rosCp, promoter of rosC; rosDp, promoter of rosD; rosAIp, promoter of rosAI.