牛樟芝三萜類生合成基因角鯊烯單氧合酶及羊毛甾醇合成酶之選殖、特性界定、表現及其功能性分析

Abstract

牛樟芝(Antrodia cinnamomea)為生長於台灣特有保育樹種牛樟樹之空胴心材內壁之台灣特有種擔子菌，其粗萃取物在諸多藥理活性測試中顯現了抗癌、抗發炎、抗氧化等生理活性而被認為具有醫藥應用之潛能，其中似與其獨特且多樣的三萜類有關，然而困於量微而難以進行活性檢驗與應用，因此利用基因代謝工程技術作為生產途徑，成為牛樟芝三萜類應用研究的新興策略。應用本實驗室建構的牛樟芝轉錄體資料庫，檢視出註解有三萜類生合成途徑相關的角鯊烯單氧合酶(squalene monooxygenase, SM)與羊毛甾醇合成酶(lanosterol synthase, LS)二酵素的部分基因片段。本研究利用RACE (rapid amplification of cDNA ends)及先前所建構之牛樟芝fosmid資料庫，取得二基因之cDNA及開放讀框 (open reading frame, ORF)的全長，並完成生化特性界定，也藉由酵母菌突變株的功能性互補試驗成功驗證此二酵素所具有之功能。此外，在檢視多種融合蛋白標籤(fusion protein tag)與表現宿主的表現測試後顯示，以Escherichia coli Rosetta表現二基因的GST重組蛋白的組合，能夠成功表現並可獲得較佳的重組蛋白表現量，然而卻未能檢測出E. coli異源表現之AcSM與AcLS蛋白的酵素活性，推測可能是蛋白摺疊構型的不正確所致。未來期待，利用AcLS在牛樟芝的表現尋找與其有高度正相關表現關係的cytochrome P450，將可逐步建構出牛樟芝完整的三萜類生合成途徑網絡。

Antrodia cinnamomea, a unique native basidiomycetes, inhabitats on the inner trunk of Taiwanese endemic tree species, Cinnamomum kanehirae. The crude extracts from A. cinnamomea have been shown medicinal potential, including anti-cancer, anti-inflammation, and anti-oxidation, etc. Triterpenoids, the major secondary metabolites of A. cinnamomea, are supposed to be correlated with these biological activities. However, clinic trials are always being impeded due to lacking adequate quantity of putative active gradients. To circumvent the obstacles, metabolic engineering of the genes of A. cinnamomea to biosynthesize the potent medicinal triterpenoids may be an alternative choice. We found two of the putative genes squalene monooxygenase (SM) and lanosterol synthase (LS) in triterpenoid biosynthesis pathway from constructed and annotated A. cinnamomea transcriptome database. Using approaches with RACE (rapid amplification of cDNA ends) and A. cinnamomea fosmid library, full-length cDNA of AcSM and AcLS have been worked out, and open reading frames (ORF) were defined. Additionally, these two genes were characterized by analysis of the high conserved domains, active sites, and phylogenetical relatedness, and also their functions were verified by complementation test in yeast knockout (YKO) strains. The recombinant fusion proteins of GST-AcSM and GST-AcLS were successfully expressed in Escherichia coli Rosetta, and have been purified to homogeneity. However, the enzymatic activities of AcSM and AcLS heterologously expressed in E. coli could not be confirmed. It is suspected that the recombinant AcSM and AcLS are non-functional due to the inappropriate folding. In the future, metabolic engineering of the AcSM and AcLS in combination with cytochrome P450 and heterologously expressing in a pertinent host to generate bioactive triterpenoids may be possible.