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. 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...

Post-PKS Tailoring Steps of the Spiramycin Macrolactone Ring in Streptomyces ambofaciens

“…The acylation of spiramycin I to yield spiramycins II and III may be catalyzed by Srm2 (8). The timing of the post-PKS tailoring reactions proposed here is similar to what has been observed in the biosyntheses of two other 16-member macrolides, tylosin and rosamicin, biosyntheses in which the glycosylation of the C-5 hydroxyl group occurs prior to the oxidation of the C-20 methyl group (18,28). Moreover, in Streptomyces fradiae, the absence of TylI, the homologue of Srm13 involved in tylosin biosynthesis, is likely to result in the synthesis of C-20-methyl tylosin, an outcome similar to that observed for the srm13 deletion mutant, which produces C-19-methyl-spiramycins.…”

“…3D, peak 1), supporting the hypothesis that compound 9 is the C-19-hydroxylated, C-9-oxidized form of forocidin. The presence of a hydroxyl group on C-19 may suggest that oxidation of the C-19 methyl group into a formyl group by Srm13 goes through a hydroxyl intermediate, as has been proposed for rosamicin biosynthesis (18). However, the C-19 formyl, C-9 keto form of forocidin was not detected in the SPM515 culture supernatant, as would be expected in this case.…”

“…The rosamicin derivative devoid of the C-20 formyl group (RS-B) (equivalent to the spiramycin C-19 methyl group) is 10 to 60 times less active than rosamicin, suggesting that this group is important to the antibacterial activity of rosamicin (18). To determine whether the C-19-methyl-spiramycin derivatives possess some antibacterial activity, culture supernatants of strain SPM513 were tested by a bioassay.…”

“…A Bruker Daltonik Esquire HCT ion trap mass spectrometer (MS) equipped with an orthogonal atmospheric pressure interface-electrospray ionization (AP-ESI) source was coupled online to the HPLC system and was used for MS and tandem MS (MS-MS) analyses. Elution from the Atlantis dC 18 column was split into two flows: 1/10 was directed to an ESI mass spectrometer for MS and MS-MS measurements and the remaining 9/10 to a diode array UV detector. In the ESI process, nitrogen served as the drying and nebulizing gas, and helium gas was introduced into the ion trap both for efficient trapping and cooling of the ions generated by ESI and for fragmentation processes.…”

Targeting Reactive Carbonyls for Identifying Natural Products and Their Biosynthetic Origins

“…In the first, the aldehyde is formed through oxidation of a hydroxyl group by cytochrome P450-type enzymes, as in the polyketides tylosin and rosamicin (Figure 2). 22,23 The second pathway is unique to non-ribosomal peptides (NRPs) and results from reductive release from the non-ribosomal peptide synthetase (NRPS, e.g. flavopeptin, Figure 2).…”