應用表觀遺傳學及遺傳學策略於真菌二次代謝產物之調控

Application of Epigenetic and Genetic Strategies on Modulation of Secondary Metabolites from Fungi

李其穎(Chi-Ying Li)

指導教授 ： 張芳榮

共同指導教授 ： 吳永昌(Yang-Chang Wu)

高雄醫學大學/藥學院/天然藥物研究所博士班/博士(2019年)

摘要

微生物二次代謝物在藥物開發扮演著相當重要之角色。臨床上有諸多藥物源自微生物二次代謝物或其衍生物，如cyclosporine、lovastatin及penicillin等。本研究應用表觀遺傳學 (epigenetics) 及遺傳學 (genetic) 之策略，針對真菌之二次代謝物進行研究探討，並搭配各式生物活性篩選 (細胞毒殺、抗發炎、抗血管新生、抗過敏、抗菌、抗線蟲活性等等) 找尋具有生物活性之天然物。
本研究以此方法作為核心進行探討，發現兩株菌種Lecanicillium antillanum、 Arthrobotrys foliicola，在其培養液中加入組蛋白去乙醯基酶抑制劑 (histone deacetylase inhibitor, HDAC) suberoyl bis-hydroxamic acid (SBHA) 會受到表觀遺傳學方法調控。經由各式管柱層析分離純化及結構鑑定，自L. antillanum之乙酸乙酯粗萃取物分離純化得一新骨架polyketide類化合物，11-norbetaenone (1)。此為首次自該屬發現之betaenone類化合物，並具有抑制血管新生之活性 (IC50 value : 34.1  1.7 µg/mL)。另外，由A. foliicola之乙酸乙酯粗萃取物，分離純化出12個化合物，包括一個首次自天然界中分離之coumarin類化合物4-ethyl-7-hydroxy-8-methyl-2H-chromen-2-one (2)、一個benzaldehyde類化合物 (3) 及十個2,5-diketopiperazine類化合物 (4‒13)。化合物 2 為首次自Arthrobotrys genus及Orbiliaceae family分離得到。然而，所送測之化合物 2‒13 於細胞毒殺、抗線蟲、抗發炎、抗過敏及抗血管新生等試驗中，未具有顯著之活性。
於遺傳學 (genetic) 研究中，使用doxycycline-dependent tet-on系統，應用於Aspergillus terreus 之基因簇探討，搭配生物資訊學推測，針對特定之基因或基因簇研究。藉由透過此系統啟動基因簇之promotor，開啟原先沉默之基因，再搭配基因剔除之技術，針對此基因簇之兩個PKS基因分別進行剔除實驗，探討其對生合成路徑之影響。結果發現此基因簇會產生一系列之azaphilone類二次代謝物，並從中分離得到一新化合物 azaterrilone A (14)，與先前生物資訊學所推測之結果吻合，進一步證實藉由表現此基因簇可調控一系列之azaphilone類化合物生成。
綜合上述之研究策略，利用表觀遺傳學及遺傳學方法，調控真菌之二次代謝物，能使其產生較為新穎之二次代謝物。

關鍵字

表觀遺傳學 ； 遺傳學 ； Lecanicillium antillanum ； Arthrobotrys foliicola ； Aspergillus terreus

並列摘要

Natural products derived from microorganisms play a key role in developing drug candidates. Several drugs are originated from secondary metabolites or its derivatives from microorganisms in clinical therapy, such as cyclosporine, lovastatin, and penicillin. This study applies epigenetic and genetic strategies on modulation of the secondary metabolites that generate from fungi as well as evaluating their biological activities, including cytotoxic, anti-inflammatory, anti-angiogenesis, anti-allergy, anti-microbial, and nematicidal activities to hit potential lead compounds.
This research utilized these strategies and found that Lecanicillium antillanum and Arthrobotrys foliicola can be modulated by the epigenetic approach through the addition of the histone deacetylase inhibitor, suberoyl bis-hydroxamic acid (SBHA), to the culture broth of these two fungi. After a series of column chromatography and structural elucidation, the EtOAc crude extract obtained from L. antillanum yielded one novel polyketide, and named as 11-norbetaenone (1), the first betaenone-type secondary metabolite reported from this genus, as well as possessing the anti-angiogenesis activity (IC50 value: 34.1  1.7g/mL) for the first time. Additionally, twelve compounds were isolated from the EtOAc crude extract of A. foliicola, include one coumarin type compound, 4-ethyl-7-hydroxy-8-methyl-2H-chromen-2-one 2 was isolated from nature for the first time, one benzaldehyde 3, and ten 2,5-diketopiperazine compounds 4‒13. Compound 2, induced by epigenetic stimulation, was found for the first time in the Arthrobotrys genus and the family Orbiliaceae. However, the tested compounds 2‒13 did not exhibit significant activities in cytotoxic, nematicide, anti-inflammatory, anti-allergic and anti-angiogenic assays.
The investigation in genetic research, which employed the doxycycline-dependent tet-on system for investigating the gene clusters in Aspergillus terreus and combined with the bioinformatics speculation to study specific genes or gene clusters. By utilizing this system to activate the promotor of gene cluster for turning on the silent genes as well as the gene knock out technique that was used to perform the experiments. Therefore, two specific PKS genes were knocked out respectively and investigate their effect in the biosynthesis pathway.
The results suggest that this gene cluster would generate a series of azaphilone type secondary metabolites and discover a new azaphilone analog named as azaterrilone A (14). These results match the bioinformatics speculation and proposed that expressing this gene cluster can regulate the production of a series of azaphilone type compounds.
According to the above strategies, it would be able to modulate the secondary metabolites from fungi by employing epigenetic and genetic approaches and make them to generate several novel compounds.

並列關鍵字

epigenetic ； genetic ； Lecanicillium antillanum ； Arthrobotrys foliicola ； Aspergillus terreus

參考文獻

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