Presentation Information
[P01-075]A metabolic engineering approach targeting the addition of mycinose to rosamicin
○Yohei Iizaka1, Ayaka Ikenoue1, Sakura Suzuki1, Rio Takayama1, Yojiro Anzai1 (1. Toho Univ. (Japan))
Keywords:
Biosynthetic engineering,Inducible expression,Macrolide,Cytochrome,Mycinose biosynthesis
Rosamicin, a 16-membered macrolide antibiotic, is expected to exhibit enhanced activity against drug-resistant bacteria through side-chain modification. We previously constructed a production system for mycinosyl rosamicin derivatives by heterologously introducing the mycinose biosynthetic genes into the rosamicin-producing strain Micromonospora rosaria IFO 13697. Although mycinose-containing intermediates were detected, production of the predicted most potent antibacterial compound, mycinosyl rosamicin, has not yet been confirmed, likely because the cytochrome P450 enzymes (P450s) RosC and RosD, which are involved in rosamicin biosynthesis, do not recognize the mycinose-containing intermediates as substrates. We therefore reasoned that, in order to produce mycinosyl rosamicin, mycinose attachment would need to occur after the oxidative reactions catalyzed by RosC and RosD. In this study, we investigated the construction of a production system for mycinosyl rosamicin by regulating expression of mycinose biosynthetic genes.
To test this strategy, we employed the thiostrepton-inducible tipA promoter, which enables regulated gene expression in actinomycetes. M. rosaria MyIF-5 was generated by introducing the mycinose biosynthetic genes under tipA promoter into M. rosaria IFO 13567, and secondary metabolite production was analyzed following thiostrepton induction. Induction resulted in the production of mycinose-containing intermediates of rosamicin, confirming successful expression of the introduced genes. Furthermore, even after rosamicin production was confirmed, induction with thiostrepton enabled mycinose attachment. However, mycinosyl rosamicin was not deteched.Further analysis suggested that the substrate specificity of P450 MycCI, which catalyzes hydroxylation at the C-23 position required for mycinose attachment, limits production of mycinosyl rosamicin. MycCI did not accept the RosD-generated C-12/13-epoxidized intermediates, indicating that the MycCI-catalyzed reaction must precede RosD-mediated epoxidation. However, because RosD does not recognize the mycinose-containing intermediate as a substrate, an alternative strategy was required to establish a mycinosyl rosamicin production system.
We therefore focused on MycG, a P450 responsible for C-12/13 epoxidation in mycinamicin biosynthesis. Given that MycG catalyzes epoxidation of mycinose-containing substrates, its catalytic activity toward mycinosyl rosamicin derivatives was evaluated, resulting in the successful generation of a putative mycinosyl rosamicin. These findings suggest that the RosD-mediated epoxidation can be functionally replaced by MycG. Accordingly, we plan to introduce the mycinose biosynthetic genes and mycG under the control of the tipA promoter into a rosD-disrupted strain of M. rosaria to establish a production system for mycinosyl rosamicin.
To test this strategy, we employed the thiostrepton-inducible tipA promoter, which enables regulated gene expression in actinomycetes. M. rosaria MyIF-5 was generated by introducing the mycinose biosynthetic genes under tipA promoter into M. rosaria IFO 13567, and secondary metabolite production was analyzed following thiostrepton induction. Induction resulted in the production of mycinose-containing intermediates of rosamicin, confirming successful expression of the introduced genes. Furthermore, even after rosamicin production was confirmed, induction with thiostrepton enabled mycinose attachment. However, mycinosyl rosamicin was not deteched.Further analysis suggested that the substrate specificity of P450 MycCI, which catalyzes hydroxylation at the C-23 position required for mycinose attachment, limits production of mycinosyl rosamicin. MycCI did not accept the RosD-generated C-12/13-epoxidized intermediates, indicating that the MycCI-catalyzed reaction must precede RosD-mediated epoxidation. However, because RosD does not recognize the mycinose-containing intermediate as a substrate, an alternative strategy was required to establish a mycinosyl rosamicin production system.
We therefore focused on MycG, a P450 responsible for C-12/13 epoxidation in mycinamicin biosynthesis. Given that MycG catalyzes epoxidation of mycinose-containing substrates, its catalytic activity toward mycinosyl rosamicin derivatives was evaluated, resulting in the successful generation of a putative mycinosyl rosamicin. These findings suggest that the RosD-mediated epoxidation can be functionally replaced by MycG. Accordingly, we plan to introduce the mycinose biosynthetic genes and mycG under the control of the tipA promoter into a rosD-disrupted strain of M. rosaria to establish a production system for mycinosyl rosamicin.
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