牛樟芝之倍半萜Antrocin的生合成途徑解析與異源表達

Abstract

牛樟芝為台灣特有的藥用菇類，許多研究報導牛樟芝具有廣泛的生物活性，例如: 抗癌症、抗發炎、抗氧化以及抑制腫瘤的活姓。Antrocin為倍半萜 (sesquiteropenoid)之化合物，擁有草椎烷(drimane)以及內酯環(lactone ring)骨架，已知由牛樟芝的子實體分離而得，其對於許多癌細胞具有細胞毒性，能促使癌細胞凋亡或抑制生長。然而，antrocin化合物的取得主要來自於牛樟芝子實體的化學分離以及純化，供應產量有限，因此使antrocin的藥物發展受到限制。目前已有antrocin全合成方法的報導，然而，antrocin在牛樟芝的生合成路徑依然未知。在本研究中，我們利用異源表達與化學實驗鑑定出具有合成antrocin結構骨架功能的萜烯環化酶AncC (terpene cyclase)，此外，我們也發現在此基因簇中有三個氧化酶參與antrocin生合成路徑，分別為AncB (P450 monooxygenase)、AncD (P450 monooxygenase)與AncE (dehydrogenase)，並在異源表達系統重建並分離出四個化合物(2~5)，且均為新化合物。最後我們成功鑑定出在牛樟芝基因組中參與antrocin生合成的基因以及生合成路徑，中間產物之結構經由質譜與核磁共振鑑定，本研究成果未來將可能促進antrocin的藥物發展。

Antrodia cinnamomea is a unique mushroom and indigenous to Taiwan. Many studies have reported a diverse range of biological activities of A. cinnamomea including anticancer, anti-inflammatory, antioxidative, antitumor activity. Antrocin is a drimane-type sesquiterpenoid containing a lactone scaffold isolated from A. cinnamomea. It exhibits potent cytotoxic activity against several cancer cell lines. The supply of antrocin was mainly isolated from the fruiting body of 2-3 years-old A. cinnamomeam, which may limit the development of antrocin as a potential medicine. While the total chemical synthestic strategies were developed to synthesize antrocin molecule, the genetic and enzymatic basis of antrocin remains unknown.Here we reported the identification and characterization of a terpene synthase (AncC) from A. cinnamomea that synthesizes the antrocin skeleton with heterologous reconstitution and biochemical characterization. Besides, we have identified three oxygenases (AncB, AncD and AncE) that catalyzes oxidative modifications to afford novel compounds in the antrocin biosynthetic pathway with heterologous expression and in vivo feeding experiments. The structure of these compounds were characterized with MS and NMR spectroscopic methods. Our study unveiled the mystery of antrocin biosynthesis in the medicinal fungus and may facilitate the development of antrocin as a potential therapeutic agent.