臺灣杉Tc-Lon、Tc-Ced/Ger、Tc-αCub/Ger、Tc-Ger1與Tc-Ger2萜類合成酶基因之選殖與特性分析

Gene Cloning and Characterization of Tc-Lon、Tc-Ced/Ger、Tc-αCub/Ger、Tc-Ger1 and Tc-Ger2 Terpene Synthases from Taiwania cryptomerioides

鄭仰兌(Yang-Tui Cheng)

指導教授 ： 曲芳華

國立臺灣大學/生物資源暨農學院/森林環境暨資源學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201702815

摘要

臺灣杉（Taiwania cryptomerioides Hayata）為臺灣原生種，含有豐富的萜類化合物，藉由研究萜類合成酶能更瞭解萜類化合物的生合成途徑。本研究利用聚合酶連鎖反應（polymerase chain reaction, PCR）與cDNA末端快速增幅（rapid amplification of cDNA ends, RACE）等技術，自臺灣杉選殖出Tc-Lon、Tc-Ced/Ger、Tc-αCub/Ger、Tc-Ger1和Tc-Ger2萜類合成酶基因。經功能分析，Tc-Lon可合成longifolene，Tc-Ced/Ger和Tc-αCub/Ger則具有雙功能性，能同時合成倍半萜類產物和單萜類產物。Tc-Ced/Ger之主要產物為倍半萜cedrol與單萜geraniol，Tc-αCub/Ger之主要產物為倍半萜α-cubebene與單萜geraniol。分析胺基酸序列的差異性，發現Tc-Ger1缺失了DDXXD功能區塊（motif），因此利用點突變實驗將Tc-Ger1的胺基酸轉換為典型的DDXXD motif序列，其兩個突變株（mutant）A359D和A359D/H363D也如野生型（wild type）的功能一樣能產生geraniol，但GC-MS分析偵測訊號減弱。Tc-Ger1和Tc-Ger2的產物geraniol構造簡單，因此推測可能是受到大腸桿菌中的磷酸酶影響而生成，因此設計實驗證實Tc-Ger1和Tc-Ger2的確具有催化產生geraniol的能力。本研究也成功利用代謝工程技術建立萜類化合物的生合成系統，期盼未來能提供大量生產。

關鍵字

代謝工程技術 ； 點突變 ； 臺灣杉 ； 萜類化合物 ； 萜類合成酶

並列摘要

Taiwania (Taiwania cryptomerioides Hayata) is a conifer species native to Taiwan, which is known for several bioactive secondary metabolites from it. Studying terpene synthases is the key way to understand the pathway of terpene biosynthesis. In this study, Tc-Lon, Tc-Ced/Ger, Tc-αCub/Ger, Tc-Ger1 and Tc-Ger2 were isolated from Taiwania by using polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) methods. Tc-Lon is a sequiterpene synthase and it can synthesize longifolene. Tc-Ced/Ger and Tc-αCub/Ger are bifunctional terpene synthases, which can produce monoterpenes and sesquiterpenes simultaneously. Tc-Ced/Ger can produce sesquiterpene cedrol and monoterpene geraniol. Tc-αCub/Ger can produce sesquiterpene α-cubebene and monoterpene geraniol. DDXXD motif is absent in Tc-Ger1, so the two mutants A359D and A359D/H363D were made to clarify these two residues how them work. Interestingly, A359D and A359D/H363D have the same function like the wild type of Tc-Ger1, and it was confirmed that Tc-Ger1 and Tc-Ger2 can produce geraniol. In this study, terpene synthesis pathways were built by using metabolic engineering. Using the strategy of the metabolic engineering for terpene overproduction should also work in other synthetic pathways in the future.

並列關鍵字

metabolic engineering ； site-directed mutagenesis ； Taiwania cryptomerioides ； terpenoid ； terpenoid synthase

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