臺灣杉萜類合成酶的演化與生化功能探討

Abstract

臺灣杉 (Taiwania cryptomerioides Hay.) 為柏科 (Cupressaceae) 臺灣杉屬 (Taiwania) 下的單種屬植物 (monotypic species)，木材抗腐朽性質良好，同時也是臺灣重要造林樹種之一。臺灣杉的萜類化合物是木材高耐久性 (durability) 的原因，特別是具有良好抗真菌與抗白蟻活性的倍半萜類化合物 cadinol，以及與雙萜類化合物ferruginol，被視為臺灣杉重要的防禦物質。萜類合成酶 (terpene synthases, TPSs) 是萜類生合成當中決定化學主要架構的關鍵酵素，且隨著演化推進，植物漸漸形成物種特有的萜類合成酶群，以應付環境中不同的逆境，然而許多柏科特有的萜類生合成途徑仍然未知。要探討柏科特有的萜類化合物的生合成途徑，以及萜類合成酶在裸子植物的功能演化歷程，必須結合轉錄體、蛋白質體與代謝體的研究，而臺灣杉具有特殊演化地位及豐富萜類化合物，能為柏科植物萜類生合成相關研究建立一個指標性的參考模板。本研究從臺灣杉的轉錄體中分離出萜類，從序列親緣性與生化功能都顯示了臺灣杉在演化上的獨特性。其中十二個酵素功能是尚未在裸子植物 (gymnosperms) 發現過的，包括兩個單功能的class II 雙萜合成酶 (labda-13-en-8-ol diphosphate 合成酶 (TcCPS2) 與(+)-copalyl diphosphate 合成酶 (TcCPS4))、六個class I雙萜合成酶 (manoyl oxide合成酶 (TcKSL1、TcKSL3～TcKSL7)，biformene 合成酶 (TcKSL1)，levopimaradiene合成酶 (TcKSL3) 與phyllocladanol 合成酶 (TcKSL5)) 以及四個倍半萜合成酶 (zingiberene合成酶 (TcTPS1)、germacrene-4-ol 合成酶 (TcTPS4與TcTPS12)與cedrol合成酶 (TcTPS6)。當中TcCPS4與TcKSL3藉由levopimaradiene這個中間產物參與abietatriene的生合成，並且相當可能參與臺灣杉防禦用雙萜化合物ferruginol生合成途徑。特別的是，裸子植物特有雙萜類化合物生合成，倚賴雙功能性的class I/II 雙萜類合成酶上兩個催化位置來完成兩步驟的催化作用，而臺灣杉則是由單功能的class II 與 class I 雙萜類合成酶，以接力的方式分開完成反應。加上臺灣杉特有的倍半萜合成酶基因分群的內隱子遺失現象 (intron loss)，顯示基因與蛋白質結構改變使得這些酵素擁有尚未在裸子植物發現的特質，並且造就臺灣杉獨特的萜類化合物組成。本研究為裸子植物萜類化合物代謝的分子演化與蛋白質功能性構象帶來更深刻的見解，更有助於了解萜類化合物在針葉樹化學防禦上所扮演的角色。

Taiwania cryptomerioides is a monotypic gymnosperm species, valued for the high decay resistance of its wood. This durability has been attributed to the abundance of terpenoids, especially the sesquiterpenoid cadinene and diterpenoid ferruginol with antifungal and antitermite activity. The important committed step in terpenoid biosynthesis is the backbone formation catalyzed by terpene synthases (TPSs), and to adapt to the environment, plants have evolved their own group of TPSs along with the species evolution. However, the biosynthesis pathways of many Cupressaceae-specific terpenoids remained unknown. To expand our knowleage of terpene biosyntehesis and evolution of TPSs in Cupressaceae, a research included transcriptome, proteome and metabolome is recquired, and T. cryptomerioides can provide a good model for terpenoid research in Cupressaceae species because of its unique evolutionary opposition and high numbers of terepnoids. In this study, we identified unrecognized groups of TPSs in T. cryptomerioides, including monofunctional diTPSs, which suggests a distinct evolutionary divergence of the TPS family in this species. Specifically, twelve TPS functions not previously observed in gymnosperms were characterized, including two monofunctional class II diTPSs ((labda-13-en-8-ol diphosphate synthase (TcCPS2) and (+)-copalyl diphosphate synthase (TcCPS4)), six class I diTPSs (manoyl oxide synthase (TcKSL1, TcKSL3~TcKSL7)，biformene synthase (TcKSL1)，levopimaradiene synthase (TcKSL3) and phyllocladanol synthase (TcKSL5)), and four sesquiterpene synthases (zingiberene synthase (TcTPS1)、germacrene-4-ol synthase (TcTPS4 and TcTPS12) and cedrol synthase (TcTPS6)). Furthermore, TcCPS4 and TcKSL3 likely contribute to abietatriene biosynthesis via levopimaradiene as an intermediate in ferruginol biosynthesis in Taiwania. Notably, diterpene biosynthesis in Pinaceae speciese denpends on bifunctional diterpene synthases to catalylyze two-step reaction on two catalytic sites of an enzyme, but Taiwania employs dominantly pairs of monofunctional diterpene synthases to complete the two-step reaction successively. In addition, the uncharacterized genome structures with intron loss were also observed in a phylogenetically distant group of Taiwania sesquiterpene synthases, suggesting the terpenoid biosynthesis of Taiwania is unique and diverse due to the genome and protein strucuture modification. This study provides deeper insight into the functional landscape and molecular evolution of specialized terpenoid metabolism in gymnosperms as a basis to better understand the role of these metabolites in tree chemical defenses.