請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59307
標題: | 牛樟芝聚酮合成酶基因的功能界定及其相關生合成路徑探討 Functional Characterization of Polyketide Synthase-encoding Genes and the Related Biosynthetic Pathway in Antrodia cinnamomea |
作者: | Po-Wei Yu 余浡維 |
指導教授: | 曾顯雄,劉瑞芬,李宗徽 |
關鍵字: | 牛樟芝,酮合成?,生合成,苔蘚酸,安卓奎諾爾, Antrodia cinnamomea,polyketide synthase,biosynthesis,orsellinic acid,antroquinonol, |
出版年 : | 2017 |
學位: | 博士 |
摘要: | 牛樟芝(Antrodia cinnamomea)是台灣特有真菌,其子實體在民間被廣泛應用於醫療用途,如降低膽固醇、消炎鎮痛、抗癌以及保肝等。除了牛樟芝豐富的三萜類代謝物之外,其所生成的安卓奎諾爾(Antroquinonol)相關衍生物在抗癌及免疫方面的藥用潛力亦為人所熟知。然而,牛樟芝相關的研究多聚焦在其藥用活性及天然物化學,而對天然物生合成路徑及相關基因功能則較無著墨。由化學結構來看,安卓奎諾爾等產物的環己烯酮部分與芳香族聚酮化合物相似,本研究遂以聚酮合成酶(PKS)基因為標的進行後續探討。由已建構的牛樟芝基因體資料庫搜尋,我們界定了四個可能的聚酮合成酶基因,包括三個還原型聚酮合成酶和一個非還原性聚酮合成酶。我們利用原生質體的製備以及同源置換方式建立了適用於牛樟芝的基因操作平台,並成功剔除標的非還原型聚酮合成酶基因pks63787。實驗結果顯示,與野生株相比,不具pks63787的突變株(Δpks63787)無法產生苯環類代謝物,進而影響其抗氧化能力以及外觀菌落型態。另一方面,我們利用類緣關係的分析指出PKS63787應屬於苔蘚酸(orsellinic acid)合成酶,並藉由後續實驗探討苔蘚酸於牛樟芝代謝物生合成所扮演的角色。結果顯示,苔蘚酸的添加不僅可以恢復Δpks63787外部形態色素特徵的缺失,且補全,甚至提高其苯環類產物及安卓奎諾爾的生合成量,證實安卓奎諾爾的環己烯酮部分的確是由PKS63787所合成的聚酮化合物。另外,在分析PKS63787和苔蘚酸相關化合物組成的同時,我們發現了六個新化合物,包括兩種苯酚1、2,兩種安卓奎諾爾3、4和兩種馬來酰亞胺類化合物5、6。總結以上,我們的研究有助於擔子菌類聚酮合成酶的功能解析以及對牛樟芝中苔蘚酸衍生物和安卓奎諾爾生合成的了解,而所建立的基因操作平台亦有利於以牛樟芝及其他擔子菌的天然物相關基因研究。 Antrodia cinnamomea is a unique resupinate basidiomycete endemic to Taiwan. Besides the abundant triterpenoid metabolites, A. cinnamomea is known for producing antroquinonols, which were reported to have notable medicinal potential in oncology and immunology. However, neither the biosynthetic pathway of these compounds nor the corresponding genes are currently clear. To investigate the biosynthesis of antroquinonols in A. cinnamomea, we focused on the polyketide synthase (PKS) genes due to the similar structure of the cyclohexenone moiety of antroquinonols to the aromatic polyketide. Four putative PKS genes, including three reducing PKSs and one non-reducing PKS, pks63787, were characterized in A. cinnamomea based on the partially deciphered genome and the constructed fosmid library. For the first time, a gene disruption platform was established in A. cinnamomea via a protoplast-mediated transformation system. Our study showed that the pks63787 knock-out mutant of A. cinnamomea (∆pks63787) is deficient in the biosynthesis of several aromatic metabolites which are involved in the antioxidant activity and colony morphology. In the further study, we pointed out by phylogenetic analysis that pks63787 likely encodes an orsellinic acid synthase, whose function was double-confirmed with a complementary feeding test. The amendment of orsellinic acid not only restores the ability of ∆pks63787 in producing its deficient pigment, benzenoids and antroquinonols, but also enhances the productivity of several antroquinonols. These results provide direct evidence that the PKS63787 is involved in the biosynthesis of antroquinonols, and supported our hypothesis that the cyclohexenone moiety is a polyketide synthesized via the PKS63787-conducted polyketide pathway. Along with the identification of numerous PKS63787- and orsellinic acid-mediated components, six compounds, including two benzenoids 1, 2, two antroquinonols 3, 4 and two antrocinnanoates 5, 6, were reported for the first time. In conclusion, our study has contributed to the understanding of the PKS genes and the biosynthesis of antroquinonols in A. cinnamomea, and the adopted procedure may be conducive to genetics research focusing on natural products in A. cinnamomea and other basidiomycetes. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59307 |
DOI: | 10.6342/NTU201701259 |
全文授權: | 有償授權 |
顯示於系所單位: | 植物病理與微生物學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-106-1.pdf 目前未授權公開取用 | 33.67 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。