臺灣博碩士論文加值系統

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.

中文摘要 I
Abstract III
誌謝 V
目錄 VII
圖目錄 XIV
英文縮寫對照表 XXI
第一章、緒論 1
1.1 麴菌屬 (Aspergillus sp. ) 簡介 1
1.2.真菌代謝物 1
1.2.1 生合成基因叢集biosynthetic gene clusters (BGCs) 5
1.2.2 二次代謝物生合成路徑 6
1.3 影響二次代謝物生產之環境因子 7
1.4 土麴菌 (Aspergillus terreus) 9
1.4.1 Aspergillus terreus之benzodiazepinedione 11
1.4.2 Aspergillus terreus之acetylaszonalenin 12
1.5 費式麴菌 (Neosartorya fischeri) 13
1.6 癌症 13
1.6.1 Aspergillus terreus二次代謝物之抑癌活性 15
1.7 抗氧化與抗老化 16
1.8 研究動機與目的 19
1.9 實驗流程圖 21
第二章、材料與方法 22
2.1 材料 22
2.1.1 藥品 22
2.1.2 儀器 24
2.1.3 軟體 25
2.1.4 真菌 26
2.1.5 哺乳動物細胞株 26
2.1.6 餵養真菌基因剔除株之前驅物及其衍生物 26
2.1.7 前驅物及衍生物合成方法 28
2.2 Aspergillus terreus之培養 32
2.2.1 Aspergillus terreus培養基 32
2.2.2 Aspergillus terreus固態培養 33
2.2.3 Aspergillus terreus液態培養 35
2.2.4 Aspergillus terreus繼代培養 35
2.3 餵養Aspergillus terreus前驅物及其衍生物之方法 35
2.3.1 固態培養 35
2.3.2 液態培養 35
2.4 液態培養1-14天目標產物acetylaszonalenin之分析 36
2.5 液態培養二次代謝物之優化條件測試 36
2.5.1 pH值 36
2.5.2 溫度 36
2.5.3 轉速 36
2.6 真菌二次代謝產物萃取方法 37
2.6.1 真菌固態培養之二次代謝物萃取 37
2.6.2 真菌液態培養之二次代謝物萃取 37
2.7 高效能液相層析 38
2.8 串聯式液相層析電灑游離質譜儀 40
2.9 Aspergillus terreus二次代謝物之細胞生物活性檢測 40
2.9.1 細胞培養 40
2.9.2 細胞冷凍保存 41
2.9.3細胞解凍 42
2.9.4 細胞計數 42
2.9.5 Aspergillus terreus粗萃物稀釋 42
2.9.6 目標產物acetylaszonalenin稀釋 43
2.9.7 細胞存活率分析 (MTT assay) 43
2.10 DPPH自由基清除能力試驗 (DPPH free radical scavenging activity) 43
2.11 anaPS、anaPT、anaAT基因表現之分析 45
2.11.1 Total RNA之萃取 45
2.11.2 cDNA合成 46
2.11.3 anaPS、anaPT、anaAT 引子設計與基因表現分析 46
2.11.4 瓊脂凝膠電泳 48
第三章、結果 49
3.1 Aspergillus terreus固態及液態培養之型態 49
3.2 Aspergillus terreus固態培養二次代謝產物分析 49
3.3 Aspergillus terreus液態培養二次代謝產物分析 50
3.4 Aspergillus terreus液態培養及餵養前驅物二次代謝物分析 50
3.5 目標產物acetylaszonalenin之質譜圖 52
3.6 Aspergillus terreus液態培養1-14天之二次代謝產物分析 52
3.7 Aspergillus terreus液態培養條件優化之二次代謝產物分析 52
3.8 Aspergillus terreus液態培養二次代謝產物對SW480細胞之影響 53
3.9 Aspergillus terreus液態培養二次代謝產物對SW620細胞之影響 55
3.10 Aspergillus terreus液態培養二次代謝產物對HaCaT細胞之影響 56
3.11目標產物acetylaszonalenin對SW620細胞之影響 58
3.12 Aspergillus terreus液態培養二次代謝產物之抗氧化活性評估 58
3.13 Aspergillus terreus液態培養菌絲體之anaPS、anaPT、anaAT基因表現 59
第四章、討論 60
4.1 Aspergillus terreus固態及液態培養之菌絲體型態 60
4.2 Aspergillus terreus不同餵養方式固態培養二次代謝產物之分析 60
4.3 Aspergillus terreus固態及液態培養二次代謝產物之比較分析 61
4.4 Aspergillus terreus液態培養及餵養前驅物培養二次代謝產物之分析 62
4.5 目標之二次代謝產物acetylaszonalenin之分析 63
4.6 Aspergillus terreus液態培養1-14天目標產物acetylaszonalenin之分析 64
4.7 Aspergillus terreus液態優化培養二次代謝產物之分析 64
4.8 Aspergillus terreus液態培養之二次代謝產物對細胞存活率與外觀型態之影響 65
4.9 Acetylaszonalenin二次代謝產物對細胞存活率與外觀型態之影響 70
4.10 Aspergillus terreus液態培養二次代謝產物之抗氧化活性評估 70
4.11 Aspergillus terreus之anaPS、anaPT、anaAT基因表現分析 72
第五章、結論 74
參考文獻 76

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