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標題: | 解析棘孢素生物合成途徑中形成萜烯胺基酸之酶工具 Characterization of the Enzymatic Machineries for the Formation of Terpene-Amino acid Hybrid Compounds in Aculenes Biosynthesis |
作者: | Li-Xun Chen 陳立訓 |
指導教授: | 林曉青(Hsiao-Ching Lin) |
關鍵字: | 萜烯胺基酸,去甲基化,細胞色素 P450,水解酶,非核糖體肽合成酶, Terpene-amino acid hybrid,demethylation,cytochrome P450,hydrolase,non-ribosomal peptide synthetase, |
出版年 : | 2020 |
學位: | 碩士 |
摘要: | 在細菌與真菌中,非核糖體胜肽合成酶為催化生成非核糖體胜肽之酵素。棘孢素是由棘孢麴黴所生產的萜烯胺基酸天然雜合物。先前的研究中發現 ane 基因群負責棘孢素的生物合成。然而,對於下列兩個步驟中的重要酵素及其機制仍尚未了解:(1) 倍半萜類的骨架轉換為十四碳倍半萜類之去甲基化反應。(2) 參與在轉移脯胺酸形成萜烯胺基酸雜合物反應中,非核糖體肽合成酶 AneB和水解酶 AneE 之催化功能。 為了了解倍半萜類去甲基化反應,我們使用米麴菌作為異源表達系統,並重建三個細胞色素 P450s−AneD、AneF 以及 AneG之功能。結果發現這三個細胞色素 P450s 的接續催化能誘發十五碳倍半萜類的去甲基化,進而生成十四碳倍半萜產物。為了鑑定 AneB 的功能,我們以突變以及表達部分片段 AneB 以進行生物轉化試驗。實驗結果確認 AneB 催化功能的關鍵胺基酸位點,並藉此提出萜烯胺基酸的合成機轉。另外,藉由體外以及體內試驗,我們證明了水解酶 AneE 具有促進非核糖體肽合成酶催化功能的效果。此外,比起野生型的菌株,我們發現在 AneE 基因敲除的棘孢麴黴菌株中有兩種二次代謝物無法被生產,這表示 AneE 可能參與其生物合成步驟。我們成功分離並鑑定此兩種代謝物麥角黃酮酸 D 和 F,但水解酶在此途徑扮演的角色仍需進一步的探討。 在本次研究當中,我們發現十四碳倍半萜類的生成機制,並進一步鑑定了非核糖體胜肽合成酶和水解酶的催化特性。此研究擴增了棘孢素的生合成途徑,並使得生物體生產萜烯胺基酸之天然物的方式更被為了解。 Non-ribosomal peptide synthetases (NRPSs) catalyze the formation of backbone of non-ribosomal peptides (NRPs) in many bacteria and fungi. Aculenes are amino acid-terpene hybrid natural products produced by Aspergillus aculeatus. In the previous study, the ane gene cluster that is responsible for the biosynthesis of aculenes has been identified. However, the enzymes and their mechanisms involved in the two key steps to form aculenes remained unknown, (i) demethylation process to convert the sesquiterpene (C15) core skeleton to the norsesquiterpene (C14); (ii) the function of the hydrolase (AneE) and the NRPS (AneB) involved in proline transfer to form the terpene-amino acid hybrid products. To understand the demethylation process from the sesquiterpene skeleton, we have reconstituted three cytochrome P450s in the Aspergillus oryzae heterologous system. The results showed that three cytochrome P450s (AneD, AneF, and AneG) are required and catalyze a stepwise demethylation process. To characterize the function of AneB (NRPS), the truncated and mutated AneB have been generated. Based on the results of the biotransformation experiments, several key residues in AneB were discovered and the catalytic mechanism was proposed. In both of in vitro and in vivo experiments, the results supported that the role of AneE (hydrolase) to facilitate the formation of terpene-amino acid product catalyzed by AneB. On the other hand, we observed that AneE may take part in the biosynthesis of other two secondary metabolites from A. aculeatus due to their disappearance in the AneE deleted mutant compared to the wild type strain. Those two metabolites have been purified and elucidated as secalonic acids D and F. The role of AneE involved in the biosynthesis of secalonic acids will be further investigated. Our study elucidated the key steps in the formation of norsesquiterpenes and characterized the biochemical properties of the NRPS and hydrolase in aculenes biosynthesis. The study expanded the biosynthetic pathway of aculenes and led to understand how nature generates amino acid-terpene hybrid natural products |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/69071 |
DOI: | 10.6342/NTU202003636 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科學研究所 |
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