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...

Some of the polyketide-derived bioactive compounds contain sugars attached to the aglycone core, and these sugars often impart specific biological activity to the molecule or enhance this activity. Mycinamicin II, a 16-member macrolide antibiotic produced by Micromonospora griseorubida A11725, contains a branched lactone and two different deoxyhexose sugars, D-desosamine and D-mycinose, at the C-5 and C-21 positions, respectively. The D-mycinose biosynthesis genes, mycCI, mycCII, mycD, mycE, mycF, mydH, and mydI, present in the M. griseorubida A11725 chromosome were introduced into pSET152 under the regulation of the promoter of the apramycin-resistance gene aac(3)IV. The resulting plasmid pSETmycinose was introduced into Micromonospora rosaria IFO13697 cells, which produce the 16-membered macrolide antibiotic rosamicin containing a branched lactone and D-desosamine at the C-5 position. Although the M. rosaria TPMA0001 transconjugant exhibited low rosamicin productivity, two new compounds, IZI and IZII, were detected in the ethylacetate extract from the culture broth. IZI was identified as a mycinosyl rosamicin derivative, 23-O-mycinosyl-20-deoxo-20-dihydro-12,13-deepoxyrosamicin (MW 741), which has previously been synthesized by a bioconversion technique. This is the first report on production of mycinosyl rosamicin-derivatives by a engineered biosynthesis approach. The integration site PhiC31attB was identified on M. rosaria IFO13697 chromosome, and the site lay within an ORF coding a pirin homolog protein. The pSETmycinose could be useful for stimulating the production of "unnatural" natural mycinosyl compounds by various actinomycete strains using the bacteriophage PhiC31 att/int system.

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Macrolides, including some of the most important antibiotics clinically used, contain deoxysugars attached to an aglycone core. These sugars often impart specific biological activity to the molecule or enhance this activity. Many genes involved in the biosynthesis of macrolide antibiotics have been cloned and sequenced; in addition, the functions of many proteins encoded by macrolide biosynthetic genes have been elucidated. With this information and experimental results, manipulation of the polyketide synthase and deoxysugar biosynthetic pathways to create novel macrolide antibiotics has become possible. 1 Therefore, a combined approach that uses genes involved in the biosynthesis of macrolactone rings and deoxysugars, and in the glycosylation of macrolactone rings has been used to modify the macrolide structure. 2 Rosamicin (that is rosaramicin; Figure 1) is a 16-membered macrolide antibiotic produced by Micromonospora rosaria IFO13697 (that is NRRL 3718). 3 It contains a branched lactone and deoxyhexose sugar D-desosamine at the C-5 position. The engineered strain M. rosaria TPMA0001 carries genes involved in the D-mycinose biosynthetic pathway of Micromonospora griseorubida A11725, namely, mycCI, mycCII, mycD, mycE, mycF, mydH and mydI; this engineered strain was found to produce a mycinosyl rosamicin derivative IZI. 4 M. griseorubida A11725 produces the 16-membered macrolide antibiotic mycinamicin II, which comprises a branched lactone and two different deoxyhexose sugars-D-desosamine and D-mycinose-at the C-5 and C-21 positions, respectively. All the genes involved in D-mycinose biosynthesis lie on the mycinamicin biosynthetic gene cluster. 5 The functions of these gene products have been elucidated through chemical, genetic and enzymatic analyses. The genes mycCI and mycCII encode a cytochrome P450 enzyme and ferredoxin, respectively, which function in combination with ferredoxin reductase to mediate the hydroxylation of mycinamicin VIII at the C-21 methyl group. On completion of this hydroxylation reaction, MycD transfers 6-deoxy-D-allose to the C-21 hydroxyl group by using dTDP-6-deoxy-D-allose as a substrate; dTDP-6-deoxy-D-allose is synthesized from dTDP-4-keto and 6-deoxy-D-glucose by MydH and MydI. The methyltransferases MycE and MycF convert the resulting compound mycinamicin VI to mycinamicin IV, which has D-mycinose attached at the C-21 position. In particular, we have recently elucidated the biochemical functions of MycCI, MycCII, MycE and MycF by using the purified form of these proteins, which were overexpressed in Escherichia coli. 6,7 In our earlier study, when EtOAc extracts obtained from culture broths of the wild-strain M. rosaria IFO13697 and the engineered strain M. rosaria TPMA0001 were compared using HPLC, two additional peaks-IZI and IZII-appeared in the chromatogram (at 285 nm) of the extract from the engineered strain. IZI was identified as a mycinosyl rosamicin derivative, 23-O-mycinosyl-20-deoxo-20-dihydro-12,13-deepoxyrosamicin. 4 Moreover, our detailed studies showed that anoth...

MycG is a multifunctional P450 monooxygenase that catalyzes sequential hydroxylation and epoxidation or a single epoxidation in mycinamicin biosynthesis. In the mycinamicin-producing strain Micromonospora griseorubida A11725, very low level accumulation of mycinamicin V generated by the initial C-14 allylic hydroxylation of MycG is observed due to its subsequent epoxidation to generate mycinamicin II, the terminal metabolite in this pathway. Herein, we investigated whether MycG can be engineered for production of the mycinamicin II intermediate as the predominant metabolite. Thus, mycG was subject to random mutagenesis and screening was conducted in Escherichia coli whole-cell assays. This enabled efficient identification of amino acid residues involved in reaction profile alterations, which included MycG R111Q/V358L, W44R, and V135G/E355K with enhanced monohydroxylation to accumulate mycinamicin V. The MycG V135G/E355K mutant generated 40-fold higher levels of mycinamicin V compared to wild-type M. griseorubida A11725. In addition, the E355K mutation showed improved ability to catalyze sequential hydroxylation and epoxidation with minimal mono-epoxidation product mycinamicin I compared to the wild-type enzyme. These approaches demonstrate the ability to selectively coordinate the catalytic activity of multifunctional P450s and efficiently produce the desired compounds.