表觀遺傳調控方法應用於真菌功能性物質開發

微生物在臨床藥物來源持續扮演著一個重要角色。本研究室追求更有效率的天然物開發方法趨勢下，結合表觀遺傳學(epigenetics)之概念，開發具生物活性之微生物二次代謝產物，力求產生更新穎性的化合物。以此方法篩選三十三種不同來源的真菌，最後發現共有五種的絲狀真菌與未加抑制劑的控制組比較下，具有明顯的新二次代謝物生成，此五種真菌分別為Cordyceps indigotica、Gibellula formosana、Isaria tenuipes、Aspergillus sydowii、Beauveria felina。由這五種真菌經放大培養後，一共獲得42個二次代謝物，其中有17個新化合物，並對分離出之化合物做抗糖尿病與抗發炎試驗。 Cordyceps indigotica經DNMT抑制劑5-AZ加入PDB培養基調控後，總共分離得到8個化合物(CI-1~8)，其中CI-1和CI-2為新polyketide類型化合物，且CI-2只出現在具抑制劑處理之實驗組；Aspergillus sydowii經DNMT抑制劑5-AZ加入PDB培養基調控後，總共分離得到12個化合物(AS-1~12)，其中AS-1~3為bisabolane-type倍半萜類之新化合物，AS-4為dihydroxanthone類型之新化合物；Beauveria felina經HDAC抑制劑SAHA加入YM培養基調控後，一共分離得到8個環胜肽類化合物(BF-1~8)，其中BF-1~3為新環胜肽類化合物，且BF-3帶有一個在環胜肽類型中少有的N-methyl-butyric acid之部分結構；Gibellula formosana經合併加入HDAC抑制劑SBHA與DNMT抑制劑RG-108在YM培養基條件下培養，一共獲得13個化合物，包括2個新型ergosterol化合物並含有一獨特的cis-22,23-epoxide 側鏈(GF-1~2)，及5個polyketide類型新化合物(GF-4~8)。基於這樣的發現，我們推論如果不同之菌株可以產生相似之二次代謝產物，必含有相似的gene clusters，並且可受DNMT或HDAC抑制劑所影響。因此經搜尋文獻，尋找是否有其他與G. formosana產生相同polyketides類型之菌種，結果發現，isariotin衍生物曾經從真菌Isaria tenuipes被分離得到，於是我們同樣合併使用相同濃度的RG-108與SBHA與I. tenuipes共同培養，結果發現，此菌株確實可被酵素抑制劑所調控，並且從此菌株分離得到一個新骨架polyketide化合物IT-1及2個已知C10 lactone 環之化合物(IT-2~3)。活性方面，從A. sydowii所分離得到之bisabolane-type倍半萜類型化合物具有促進葡萄糖利用率且不會促進脂肪累積之之活性，其中以化合物AS-6之活性為最佳；從B. felina所獲得之環胜肽類型化合物具有抑制FMLP/CB刺激嗜中性白血球所引發的超氧自由基釋放與elastase活性。由上述研究成果證實此方法在微生物新穎性結構的開發具可行性，未來將持續透過此方法尋找新穎之功能性化合物，並結合後續相關藥理實驗研究，發展具潛力之先導藥物，有助於現今藥物之改善及研發。

關鍵字

表觀遺傳學； DNA甲基轉移酶

並列摘要

Microbial natural products continue to play a vital role in modern drug-based therapy of various diseases. Currently, we are eager to combine the concept of epigenetics with an efficient method for production of novel compounds with biological activities from microorganisms. Application of this method to screen thirty-three fungi from different resources led to the findings of five strains, including Cordyceps indigotica、Gibellula formosana、Isaria tenuipes、Aspergillus sydowii and Beauveria felina, which changed significantly compared to untreated control. After scale-up cultivation, 42 secondary metabolites including 17 new compounds were obtained from the five fungal strains and some of them were evaluated for their anti-diabetic and anti-inflammatory activities. Treatment of the potato-dextrose broth (PDB) of C. indigotica with 5-azacytidine, a DNA methyltransferase inhibitor, led to the isolation of eight compounds (CI-1~8). Among them, CI-1 and CI-2 are new aromatic polyketide glycosides. The addition of a DNA methyltransferase inhibitor, 5-azacytidine, to PDB medium of Aspergillus sydowii significantly changed its secondary metabolites profile. It led to the isolation of three new bisabolane-type sesquiterpenoids (AS-1~3) and one new dihydroxanthone (AS-4), along with eight known compounds (AS-5~12). Beauveria felina cultivated in the presence of the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), to the yeast-malt (YM) medium induced the production of secondary metabolites and resulted in the isolation of eight compounds, including three new cyclodepsipeptides (BF-1~3), along with five known cyclodepsipeptides compounds (BF-4~8). Interestingly, BF-3 possessing a cyclodepsipeptide ring with N-methyl-butyric acid is rare in natural peptides. The concomitant addition of a histone deacetylase inhibitor, SBHA, and a DNA methyltransferase inhibitor, RG-108, to the YM medium of Gibellula formosana, an entomopathogenic fungus, induced a significant increase in diversity of secondary metabolites. The culture media were further isolated and yielded two new highly oxidized ergosterols (GF-1~2), and five new isariotin analogs (GF-4~8), together with six known compounds (GF-3, GF-9~13). The side chains of (GF-1~3) contained cis-22,23-epoxide, which is rarely present in naturally occurring sterols and triterpenes. Based on the aforementioned research, we propose that there may be conserved similar gene clusters in the different fungi as they are able to produce the similar secondary metabolites when affected by DNMT or/and HDAC inhibitors. Thus, we try to survey the related literature about fungi which reported the same isariotin derivatives as G. formosana. According to the literature, isariotin derivatives were obtained from an entomopathogenic fungus, Isaria tenuipes. Therefore, application of this approach to the YM culture medium of I. tenuipes in the concomitant presence of a histone deacetylase and a DNA methyltransferase inhibitor led to the isolation of a novel skeletal polyketide (IT-1), together with C10 lactone derivatives (IT-2~3). Regarding the biological activities of isolates, the bisabolane-type sesquiterpenoids from A. sydowii did not only potentiate insulin-stimulated glucose consumption but also prevented lipid accumulation in 3T3-L1 adipocytes, especially for AS-6. The cyclodepsipeptides obtained from B. felina showed moderate anti-inflammatory activity on inhibiting superoxide anion generation and elastase release by FMLP/CB-induced human neutrophils. In summary, epigenetic approach is an efficient and powerful tool for trigging silent gene clusters of fungus in discovering of new compounds. In the future, we will continue to search functional secondary metabolites using this method and combine it with pharmaceutical assays to develop potent lead compounds that are helpful for the improvement of current medication.

並列關鍵字

epigenetics ； DNMT

參考文獻

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表觀遺傳調控方法應用於真菌功能性物質開發

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