The cytochrome P450 enzymes MycCI and MycG are encoded within the mycinamicin biosynthetic gene cluster and are involved in the biosynthesis of mycinamicin II (a 16-membered macrolide antibiotic produced by Micromonospora griseorubida). Based on recent enzymatic studies, MycCI is characterized as the C-21 methyl hydroxylase of mycinamicin VIII, while MycG is designated multifunctional P450, which catalyzes hydroxylation and also epoxidation at C-14 and C-12/13 on the macrolactone ring of mycinamicin. Here, we confirm the functions of MycCI and MycG in M. griseorubida. Protomycinolide IV and mycinamicin VIII accumulated in the culture broth of the mycCI disruption mutant; moreover, the mycCI gene fragment complemented the production of mycinamicin I and mycinamicin II, which are produced as major mycinamicins by the wild strain M. griseorubida A11725. The mycG disruption mutant did not produce mycinamicin I and mycinamicin II; however, mycinamicin IV accumulated in the culture broth. The mycG gene was located immediately downstream of the self-resistance gene myrB. The mycG gene under the control of mycGp complemented the production of mycinamicin I and mycinamicin II. Furthermore, the amount of mycinamicin II produced by the strain complemented with the mycG gene under the control of myrBp was approximately 2-fold higher than that produced by the wild strain. In M. griseorubida, MycG recognized mycinamicin IV, mycinamicin V, and also mycinamicin III as the substrates. Moreover, it catalyzed hydroxylation and also epoxidation at C-14 and C-12/13 on these intermediates. However, C-14 on mycinamicin I was not hydroxylated. The cytochrome P450 enzymes (P450s) form a very large family of oxidative heme proteins, which are responsible for a diverse range of oxidative transformations across most life forms (12, 13). These reactions typically involve the modification of physiological and xenobiotic compounds and include the biosynthesis of various bioactive compounds (e.g., steroids, antibiotics, and signaling molecules). Approximately 40% of all known bacterial P450s are found in various species of the industrially important genus Streptomyces, which is the largest genus of the actinomycetes. Recent genome sequencing of the actinomycetes, particularly Streptomyces, has revealed an unexpectedly large number of genes encoding P450s (9,16,18,24,25). In secondary metabolic pathways, P450 genes are typically integrated within the biosynthetic cluster, where their products catalyze regiospecific and stereospecific oxidation of precursors. This results in structural diversity and also improved bioactivities of these molecules (21, 27). The P450s EryF (2) and EryK (30) are encoded within the erythromycin biosynthetic gene cluster and are involved in the biosynthesis of erythromycin A. Specifically, EryF catalyzes the hydroxylation of the macrolactone precursor 6-deoxyerthronolide B, while EryK catalyzes the formation of erythromycin D. As prototypic P450 hydroxylases involved in secondary metabolism, EryF and EryK exhibit stri...

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...

Some polyketide-derived bioactive compounds contain sugars attached to the aglycone core, and these sugars often enhance or impart specific biological activity to the molecule. 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. We previously engineered an expression plasmid pSETmycinose containing the D-mycinose biosynthesis genes from M. griseorubida A11725. This plasmid was introduced into Micromonospora sp. FERM BP-1076 cells, which produce the 16-membered macrolide antibiotic izenamicin. The resulting engineered strain TPMA0041 produced 23-O-mycinosyl-20-deoxy-izenamicin B(1) and 22-O-mycinosyl-izenamicin B(2). 23-O-mycinosyl-20-deoxy-izenamicin B(1) has been produced by the engineered strain M. rosaria TPMA0001 containing pSETmycinose as 23-O-mycinosyl-20-deoxo-20-dihydro-12,13-deepoxyrosamicin (=IZI) in our recent study, and 22-O-mycinosyl-izenamicin B(2) has previously been synthesized as a macrolide antibiotic TMC-016 with strong antibacterial activity. The production of 22-O-mycinosyl-izenamicin B(2) (=TMC-016) was increased when propionate, a precursor of methylmalonyl-CoA, was added to the culture broth.

Isolation and Characterization of 23-O-Mycinosyl-20-dihydro-rosamicin: A New Rosamicin Analogue Derived from Engineered Micromonospora rosaria. -The title compound IZIII (I) is isolated from Micromonospora rosaria TPMA0001. The antibacterial activity of (I) is lower than that of rosamicin. -(ANZAI*, Y.; SAKAI, A.; LI, W.; IIZAKA, Y.; KOIKE, K.; KINOSHITA, K.; KATO, F.; J.