Abstract
Pyripyropenes are potent inhibitors of acyl-CoA:cholesterol acyltransferase, which were initially discovered to be produced by Aspergillus fumigatus. Recently, Penicillium coprobium PF1169 has also found to produce pyripyropene A (PyA), which exhibits insecticidal properties. Pyripyropenes are natural hybrid products of both terpenoid and polyketide origin. In our research, based on data generated using the Genome Sequencer FLX for P. coprobium PF1169, we predicted the biosynthetic gene cluster of PyA by blast analysis comparing with polyketide synthase and prenyltransferase of other species. By screening the genomic fosmid library, nine open reading frames (ppb1 to ppb9) related to the biosynthesis of PyA were deduced. Among them, two cytochrome P450 monooxygenase genes (ppb3 and ppb4) were separately introduced into the model fungus A. oryzae. Bioconversion of certain predicted intermediates in the transformants has elucidated the manner of hydroxylation in the biosynthetic pathway by the expressed products of these two genes (P450-1 and P450-2). That is, P450-1 exhibits monooxygenase activity and plays the hydroxylation role at C-11 of pyripyropene E. While P450-2 plays an active role in the hydroxylation of C-7 and C-13 of pyripyropene O.
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Acknowledgements
We wish to express our thanks to Hakutsuru Sake Brewing company for providing the A. oryzae HL-1105 and HL-1034. We also wish to thank Dr Yamada of the National Research Institute of Brewing for providing the plasmid vector pUSA; Dr Yamashita for the documents on transformation; Ms Tsuchida for providing assistance in our LC-MS and LC-NMR analysis; and Dr Pam Kazmierczak and Dr Furutani for reviewing this paper.
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Hu, J., Okawa, H., Yamamoto, K. et al. Characterization of two cytochrome P450 monooxygenase genes of the pyripyropene biosynthetic gene cluster from Penicillium coprobium. J Antibiot 64, 221–227 (2011). https://doi.org/10.1038/ja.2010.162
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DOI: https://doi.org/10.1038/ja.2010.162
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