Biotechnology Research Center, The University of Tokyo , Tokyo 113-8657, Japan.
Graduate School of Agricultural Science, Tohoku University , Sendai 981-8555, Japan.
J Am Chem Soc. 2015 Sep 16;137(36):11846-53. doi: 10.1021/jacs.5b08319. Epub 2015 Sep 4.
Genome mining is a promising method to discover novel secondary metabolites in the postgenomic era. We applied the Aspergillus oryzae heterologous expression system to functionally characterize cryptic bifunctional terpene synthase genes found in fungal genomes and identified the sesterfisherol synthase gene (NfSS) from Neosartorya fischeri. Sesterfisherol contains a characteristic 5-6-8-5 tetracyclic ring system and is modified by cytochrome P450 monooxygenase (NfP450) to sesterfisheric acid. The cyclization mechanism was proposed on the basis of the analysis of in vivo and in vitro enzymatic reactions with isotopically labeled precursors. The mechanism involves C1 cation-olefin IV-olefin V cyclization followed by five hydride shifts, allowing us to propose a unified biogenesis for sesterterpenes branching from bicyclic (5-15), tricyclic (5-12-5), and tetracyclic (5-6-8-5) cation intermediates. Furthermore, the mechanism is distinct from that of a separate class of di/sesterterpenes including fusicoccins and ophiobolins. The difference between mechanisms is consistent with phylogenetic analysis of bifunctional terpene synthases, suggesting that the amino acid sequence reflects the initial cyclization mode, which is most likely related to the initial conformation of a linear prenyl diphosphate.
基因组挖掘是在后基因组时代发现新型次生代谢物的一种很有前途的方法。我们应用米曲霉异源表达系统对真菌基因组中发现的隐藏双功能萜烯合酶基因进行功能表征，并从 Neosartorya fischeri 中鉴定出了鲨烯合酶基因（NfSS）。鲨烯含有一个特征的 5-6-8-5 四环系统，并被细胞色素 P450 单加氧酶（NfP450）修饰为鲨烯酸。根据对带有同位素标记前体的体内和体外酶促反应的分析，提出了环化机制。该机制涉及 C1 阳离子-烯烃 IV-烯烃 V 环化，然后进行五次氢化物转移，使我们能够提出从双环（5-15）、三环（5-12-5）和四环（5-6-8-5）阳离子中间体分支的甾体萜烯的统一生物发生。此外，该机制与包括 fusicoccins 和 ophiobolins 在内的另一类二/甾体萜烯不同。机制上的差异与双功能萜烯合酶的系统发育分析一致，表明氨基酸序列反映了初始环化模式，这很可能与线性异戊二烯二磷酸的初始构象有关。
