紅檜葉部倍半萜合成酶及細胞色素P450的基因選殖和功能鑑定

Abstract

紅檜 (Chamaecyparis formosensis Matsum.)為臺灣特有的珍貴樹種，其精油成分以萜類化合物 (Terpenoids) 為主，並已被證實有多種優異的生物活性，故對紅檜的萜類合成酶 (Terpene synthase, TPS) 及細胞色素P450 (Cytochrome P450) 的功能研究更顯重要，未來可以應用於重要成分的半化學合成。且柏科於P450的研究亦較闕如，若能鑑定P450功能亦能協助裸子植物演化途徑的解析。本研究自紅檜葉部轉錄體的NGS (Next generation sequence) 資料庫取得4個倍半萜合成酶：Cf_6163、Cf_17788_1、Cf_17788_2及Cf_30044；以及3個P450的序列：Cf_791、Cf_9283及Cf_12832。其中Cf_6163、Cf_17788_1及Cf_17788_2各與FPP受質反應出Germacrene A、新化合物以及Isogermacrene A，其中新化合物被鑑定為1,2,3,5,6,7,8,8a-Octahydro-8a-methyl-1-methylene-7-(1-methylethyl) naphthalene，Cf_30044則未成功完成催化反應。Cf_17788_1/2的產物，可能具有特殊的環化機制，未來透過點突變試驗可對其環化機制有更進一步的了解。3個P450中，Cf_791屬於CYP706M亞族、Cf_9283及Cf_12832各屬於CYP76AA亞族。P450與TPS在大腸桿菌 (Escherichia coli) 的共表現系統僅有Cf_6163與Cf_12832有出現新的產物。未來可採用其他表現系統，並將其他TPS及P450納入試驗，以得到準確的P450功能鑑定。

Chamaecyparis formosensis Matsum.is a precious tree species unique to Taiwan. Its essential oil components are mainly terpenoids. It has been confirmed to have a variety of excellent biological activities. Therefore, it is important on the research of functional characterization of terpene synthase (TPS) and CYP450 (Cytochrome P450). The research can be applied to the semi-chemical synthesis of important components in the future. On the other hand, the study of P450 from Cupressaceae is also lacking. Once the function of P450 can be identified, it can also help the analysis of evolutionary pathway of gymnosperms. In this study, four sesquiterpene synthases were obtained from the NGS (Next generation sequence) database of Chamaecyparis formosensis Matsum leaf transcriptome: Cf_6163, Cf_17788_1, Cf_17788_2 and Cf_30044; and three P450 sequences: Cf_791, Cf_9283 and Cf_12832. Among them, Cf_6163, Cf_17788_1 and Cf_17788_2 reacted with FPP substrate to produce Germacrene A, a new compound and Isogermacrene A. The new compound was identified as 1,2,3,5,6,7,8,8a-Octahydro-8a-methyl- 1-methylene-7-(1-methylethyl) Naphthalene. Cf_30044 failed to complete the catalytic reaction. The product of Cf_17788_1/2 may have a special cyclization mechanism. In the future, a point mutation test can be used to further understand their cyclization mechanism. Among the three P450s, Cf_791 belongs to the CYP706M subfamily, while Cf_9283 and Cf_12832 belong to the CYP76AA subfamily. In the co-expression system of P450 and TPS in Escherichia coli, only Cf_6163 and Cf_12832 had a new product. In the future, other expression systems can be used, and other TPSs and P450s can be included in the test to obtain accurate P450 functional identification.