牛樟芝萜類生合成基因之選殖、特性界定與表現

Cloning, characterization and expression of terpenoid biosynthetic genes from Antrodia cinnamomea

張逸軒(Yi-Hsuan Chang)

指導教授 ： 曾顯雄

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10541

摘要

牛樟芝（Antrodia cinnamomea）是台灣特有真菌，棲息於台灣特有牛樟樹(Cinnamomum kanehirai Hay)中心空洞處，為弱病原菌，但其子實體在民間被應用於醫療用途，如抗氧化、降低膽固醇、消炎鎮痛、抗癌以及抗B型肝炎等。目前許多研究主要聚焦於探討牛樟芝的次級代謝物之生理機能。如萜類、多醣類及固醇類等代謝產物，其中萜類化合物（terpenoid）對於抑癌或是保護神經似具顯著效果。然而，由於野外或人工培養之牛樟芝取得不易、生長緩慢，而限縮其萜類的萃取、分離、純化、生理活性暨機制等之探討以及實際層面的應用。本研究經由選殖牛樟芝萜類生合成基因，於E. coli中表現，蛋白質純化，活性分析，證實基因功能；另一方面多基因轉殖表現於Kluyveromyces lactis，發酵量產萜類化合物或其他有效成分，以供生理活性檢測。藉由本實驗室之牛樟芝cDNA library，選殖出萜類生合成途徑中之geranyl-trans-transferase (AcGTT) 及squalene synthase (AcSQS)二基因，彼可分別催化萜類前驅物farnesyl diphosphate與固醇類前驅物squalene的生成，將其建構於不同表現載體後，對E. coli (JM109)作轉型，並以SDS-PAGE分析表現，篩選較佳之轉型株進行後續純化。經由親和性管柱純化出AcGTT，並經過MALDI-TOF MS/MS進行蛋白質鑑定 (protein identification)，並對此酵素作活性測試與分析。此外，也將此二基因分別建構於穿梭載體 (shuttle vector) pKLAC1中，先從E. coli (DH5α) 轉型株確認基因序列無誤，之後對K. lactis (GG799) 作同源重組 (homologous recombination)，篩選出同時具有二個基因的轉型株，以YPGal培養基誘導異源蛋白的表現，未來將以HPLC分析產物組成分。

關鍵字

牛樟芝 ； 萜類化合物 ； 香葉醇轉移酶角鯊烯合成酶異源蛋白表現

並列摘要

Antrodia cinnamomea, an endemic basidiomycetes fungus of Taiwan, inhabited in the heart wood inner wall of endemic tree species Cinnamomum kanehirai Hay. The fruiting body it produced has been widely used as folk medicine, i.e. antioxidation, anti-inflammation, anticancer and anti-hepatitis B, etc. The major secondary metabolites, particularly terpenoids, were suspected attributed to such biological activity, and immensely studied in the past. Nevertheless, due to the difficulties to produce adequate quantity of fruiting body in vivo and in vitro, which together notably deter the the further clinic tests. In order to circumvent such bottleneck, we have cloned two of the intermediate genes attributed to the biosynthesis of terpenoids, i.e. geranyl-trans-transferase (AcGTT) and squalene synthase (AcSQS) by blasting the putative genes from our previously constructed A. cinnamomea cDNA library. The genes can catalyze the production of terpenoid precursor (farnesyl diphosphate), and steroid, respectively. The AcGTT was expressed in Escherichia coli and the expressed protein finally purified to homogenity via Ni-NTA affinity column, identity confirmed by MALDI-TOF MS/MS, and its catalytic activity verified. We also constructed these AcGTT and AcSQS in shuttle vector, pKLAC1, and transformed them to Kluyveromyces lactis expression host. The transformants harboring these two genes were selected. The analysis of the fermented products by the transformants is ongoing.

並列關鍵字

Antrodia cinnamomea ； terpenoid ； geranyl-trans-transferase ； squalene synthase ； heterologous protein expression

參考文獻

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