以半生合成方式生產之Aspergillus terreus 二次代謝物生物活性分析

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Title:	以半生合成方式生產之Aspergillus terreus 二次代謝物生物活性分析 Bioassay of Secondary Metabolites Produced in an Aspergillus terreus Semi-Biosynthesis System
Authors:	盧郁淳
Contributors:	化粧品應用與管理系劉坤湘
Keywords:	抗氧化抗腫瘤 acetylaszonalenin 半生合成土麴菌 antitumor acetylaszonalenin semi-biosynthesis Aspergillus terreus antioxidant
Date:	2016
Issue Date:	2016-12-21 15:29:40 (UTC+8)
Abstract:	絲狀真菌產生的二次代謝物 (secondary metabolites) 來源豐富、具有不同結構及廣泛生物活性，對於生技、食品、醫療上發揮相當大的作用。近年來，運用基因工程產生二次代謝物漸具應用潛力且可取代化學合成。本研究利用土麴菌 (Aspergillus terreus) 中ana基因叢集的anaPS進行基因剔除，於合成二次代謝物acetylaszonalenin途徑中，由人工合成的前驅物餵養突變株取代途徑前半段，後半段以 A. terreus 完成之半生合成方式，產生結構複雜的acetylaszonalenin。隨後調整環境因子如pH值、溫度、轉速等，優化目標產物acetylaszonalenin的產率，同時探討二次代謝物的抗癌活性及抗氧化能力。以前驅物benzodiazepinedione及其衍生物餵養A. terreus之anaPS基因剔除株，經由HPLC及LC-ESI-MS分析，可由此半生合成方式產生最終目標產物，並證明anaPS基因參與 acetylaszonalenin生合成路徑。針對提升目標產物的產量改變環境因子，發現以液態培養優於固態培養，且提高溫度、維持原本的pH值及降低轉速則可使acetylaszonalenin二次代謝物產率提高。經由RT-PCR實驗分析，改變環境因子因而增加目標產物的產量，有可能是因為ana基因叢集的基因表現增加，使轉譯出的酵素增加，而令生合成路徑進行更順暢所致。以30℃與37℃培養A. terreus NIH2624及基因剔除株之粗萃物，具顯著毒殺人類大腸直腸癌細胞之能力；純化之目標產物acetylaszonalenin於200 uM濃度下，具毒殺大腸直腸癌細胞能力。以30℃ 與37℃培養A. terreus基因剔除株及前驅物benzodiazepinedione餵養基因剔除株之粗萃物，皆具DPPH自由基清除能力，且37℃培養所產生之粗萃物，在50 ug/ml濃度以下，對於人類角質層細胞不具毒性。綜合上述實驗結果，我們不僅成功建立並優化半生物合成系統以生產真菌之二次代謝物外，此等二次代謝物並具有生物活性，未來可應用於藥粧的領域。 Filamentous fungi produce secondary metabolites with distinct structures and various biological activities for a wide range in biotechnology, food, and medical fields. In recent years, secondary metabolites produced by biosynthetic engineering are found to be greater potential and can replace synthetic chemicals. The precursor of acetylaszonalenin, benzodiazepinedione, catalyzed by anaPS of ana gene cluster in the biosynthetic pathway of acetylaszonalenin was chemically synthesized. The anaPS deletion mutant was cultured with benzodiazepinedione in the semi-biosynthesis of acetylaszonalenin from A. terreus. On the other hand, the semi-biosynthesis of acetylaszonalenin was studied in the feeding of precursor and its analogues, benzodiazepinedione, and adaption of environmental factors such as pH value, temperature, rotation speed. Secondary metabolites extracted from A. terreus were identified by HPLC and LC-ESI-MS. Acetylazonalenin was successfully obtained in A. terreus anaPS deletion mutant after feeding benzodiazepinedione.The yield of acetylaszonalenin in submerged fermentation is better than solid state fermentation. The original pH value, raising temperature and reducing the speed of the acetylaszonalenin could increase the yield of acetylazonalenin. The changes of the yield of the target product extracted in various environmental conditions were possibly due to the levels of gene expression in ana gene clusters evaluated by RT-PCR analysis. The results also indicated that there is cytotoxicity when primary colorectal adenocarcinoma SW480 cell and lymph node metastasis SW620 cell were treated with the crude extracts of NIH2624 or anaPS deletion mutant. In addition, crude extracts of anaPS deletion mutant, and benzodiazepinedione-feeding anaPS deletion mutant were shown the antioxidant capacity. The semi-biosynthetic system we successfully established and should be useful for the application of biological activity and the cosmeceutical of fungal secondary metabolites.
Relation:	網際網路公開：2021-09-08，學年度：104，152頁
Appears in Collections:	[Dept. of Cosmetic Science and institute of cosmetic science] Dissertations and Theses

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