title: Characterization of two acetyltransferase genes in the pyripyropene biosynthetic gene cluster fromPenicillium coprobium.
abstract: Pyripyropenes potently and selectively inhibit acyl-CoA:cholesterol acyltransferase 2 (ACAT-2). Among multiple isomers of pyripyropene (A to R), pyripyropene A (PyA) has insecticidal properties in addition to its growth inhibition properties against human umbilical vein endothelial cells. Based on the predicted biosynthetic gene cluster of pyripyropene A, two genes (ppb8andppb9) encoding two acetyltransferases (ATs) were separately isolated and introduced into the model fungusAspergillus oryzae, using the protoplast-polyethylene glycol method. The bioconversion of certain predicted intermediates in the transformants revealed the manner by which acetylation occurred in the biosynthetic pathway by the products expressed by these two genes (AT-1 and AT-2). The acetylated products detected by high-performance liquid chromatography (HPLC) in the extracts from AT-1 and AT-2 transformant clones were not present in the extract from the transformant clone with an empty vector. The HLPC charts of each bioconversion study exhibited high peaks at 12, 10.5 and 9 min, respectively. Further ultraviolet absorption and mass spectrometry analyses identified the products as PyE, PyO and PyA, respectively. AT-1 acetylated the C-1 of deacetyl-pyripyropene E (deAc-PyE), while AT-2 played an active role in acetylating the C-11 of 11-deAc-PyO and C-7 of deAc-PyA at two different steps of the biosynthetic pathway.
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, Peking University , Beijing , China.
authors.forename: Jie
authors.initials: J
authors.lastname: Hu
authors.affiliation: Gene Research Center, Ibaraki University , Ibaraki , Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Furutani
authors.affiliation: Agricultural & Veterinary Research Laboratories, Meiji Seika Pharma Co., Ltd. , Kanagawa , Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Yamamoto
authors.affiliation: Agricultural & Veterinary Research Laboratories, Meiji Seika Pharma Co., Ltd. , Kanagawa , Japan.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Oyama
authors.affiliation: Agricultural & Veterinary Research Laboratories, Meiji Seika Pharma Co., Ltd. , Kanagawa , Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Mitomi
authors.affiliation: Gene Research Center, Ibaraki University , Ibaraki , Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Anzai
keywords: Penicillium coprobium; acetyltransferase; biosynthesis; pyripyropene; transformants