Department of Applied Life Science, United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Tokyo, Japan.
J Antibiot (Tokyo). 2011 Mar;64(3):221-7. doi: 10.1038/ja.2010.162. Epub 2011 Jan 12.
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.
吡咯并吡咯二酮类化合物是酰基辅酶 A：胆固醇酰基转移酶的有效抑制剂，最初是从烟曲霉中发现的。最近，发现拟青霉 PF1169 也能产生吡咯并吡咯酮 A（PyA），具有杀虫特性。吡咯并吡咯二酮类化合物是萜烯和聚酮类天然杂合产物。在我们的研究中，基于使用基因组测序仪 FLX 为 P. coprobium PF1169 生成的数据，我们通过与其他物种的聚酮合酶和 prenyltransferase 进行 Blast 分析，预测了 PyA 的生物合成基因簇。通过筛选基因组 fosmid 文库，推断出与 PyA 生物合成相关的九个开放阅读框（ppb1 到 ppb9）。其中，两个细胞色素 P450 单加氧酶基因（ppb3 和 ppb4）分别被引入模式真菌 A. oryzae。在转化子中对某些预测中间体的生物转化阐明了这些两个基因（P450-1 和 P450-2）表达产物在生物合成途径中羟化的方式。即，P450-1 表现出单加氧酶活性，在吡咯并吡咯酮 E 的 C-11 位上发挥羟化作用。而 P450-2 在吡咯并吡咯酮 O 的 C-7 和 C-13 位的羟化中起积极作用。
