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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 羅翊禎 | |
| dc.contributor.author | Wan-Jhen Chu | en |
| dc.contributor.author | 儲皖貞 | zh_TW |
| dc.date.accessioned | 2021-06-17T04:39:23Z | - |
| dc.date.available | 2021-08-16 | |
| dc.date.copyright | 2018-08-16 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-08-07 | |
| dc.identifier.citation | 許曉雙; 張福生; 秦雪梅, 三萜皂苷生物合成途徑及關鍵酶的研究進展. 世界科學技術: 中醫藥現代化 2014, 2440-2448.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70811 | - |
| dc.description.abstract | 三萜皂苷(triterpenoid saponins)是植物的次級代謝物之一。在植物中佔有的比例較低、結構複雜，且不易從植物中進行萃取或以化學合成，因此許多研究以生物合成方式產生三萜皂苷。三萜皂苷的結構由皂苷元(aglycon)及糖基以醣苷鍵鍵結組成，總類繁多，羅漢果皂苷(mogrosides)是一種從羅漢果(Siraitia grosvenirii)果實中分離出來的四環三萜皂苷(tetracyclic triterpene saponins)。三萜皂苷的生合成路徑前端為甲羥戊酸途徑(Mevalonate pathway)，此路徑為所有真核細胞生成固醇或三萜化合物均須具備的路徑，因此希望藉由基因工程來改良Saccharomyces cerevisiae原生的麥角固醇(ergosterol)生成路徑，建構出可生合成三萜皂苷的菌株。首先，我們嘗試置換特定基因(HMG1、ERG1、ERG9)的啟動子來調節三萜皂苷非環狀前驅物合成相關的蛋白表現。實驗結果顯示置換HMG1、ERG1的啟動子確實能使細胞內的squalene累積，相較於WT上升了約12倍，且以點試驗證實啟動子置換不會影響到細胞生長，但在該階段的所有菌株中均未看到2,3-oxidosqualene、2,3,22,23-dioxidosqualene累積。推測可能是因為lanosterol synthase活性太強，將整個代謝路徑快速帶往生成麥角固醇。因此後續實驗會建構ERG27基因剔除及upc2-1突變株來阻礙麥角固醇生成路徑，希望最後能如期看到2,3-oxidosqualene、2,3,22,23-dioxidosqualene累積。此實驗產生的菌株亦可做為未來三萜皂苷生成的基本平台。 | zh_TW |
| dc.description.abstract | Triterpenoid saponins are a type of secondary metabolites in plants. Due to the structure complexity and low levels of triterpenoid saponins in plants, it’s difficult extract or synthesize chemically. Recently, biosynthesis of triterpenoid saponins has been applied using biotechnology engineering in microbes. The complexity of triterpenoid saponins depend on the variation of lipophilic aglycon structure and the position and number of glycosides combining to the aglycon. Mogrosides are tetracyclic triterpenoid saponins isolated from the fruits of Siraitia grosvenirii. The biosynthetic pathway of mogrosides is derived from mevalonate pathway presenting in all eukaryotic cells to synthesize sterols or triterpene compounds. The goal of this study is to modify Saccharomyces cerevisiae ergosterol pathway by genetic engineering and to construct the strains that have ability to synthesize triterpenoid saponins. Firstly, the replacement of promoters of HMG1, ERG1 and ERG9 was opted to mediate protein expression related to triterpenoid saponin noncyclic precursors’ synthetic pathway. The data showed that HMG1 and ERG1 promoter replacement would accumulated the amounts of squalene, up to 12-fold increase compared to those in WT. No significant effect on cell growth of the genetic engineered strains. However, the accumulation of 2,3-oxidosqualene or 2,3,22,23-dioxidosqualene could not be observed in all genetic engineered strains. This may be due to that the activity of lanosterol synthase was intense, and lead to the synthesis of lanosterol and ergosteorl, but not the accumulation of 2,3-oxidosqualene or 2,3,22,23-dioxidosqualene. Currently, attempts to establish erg27 knockout and upc2-1 mutant strains may block the metabolic flux to ergosterol pathway. The established platform could possibly promote the biosynthesis of triterpenoids in the future. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T04:39:23Z (GMT). No. of bitstreams: 1 ntu-107-R05641015-1.pdf: 3726546 bytes, checksum: 919702f0731d0347217e96a70f1b7708 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 壹、 前言 1
貳、 文獻回顧 2 第一節、 植物的三萜皂苷(triterpenoid saponins)合成途徑 2 一、 甲羥戊酸途徑(Mevalonate pathway, MVA pathway) 2 二、 皂苷元骨架生成 4 三、 皂苷之修飾 4 第二節、 酵母菌的三萜皂苷合成 6 一、 酵母菌的麥角固醇(ergosterol)合成路徑 6 第三節、 羅漢果 7 一、 羅漢果皂苷(Mogorsides) 7 二、 皂苷萃取與純化 8 三、 羅漢果皂苷合成路徑與麥角固醇合成路徑 10 第四節、 提高酵母菌三萜皂苷前驅物含量之方法 13 一、 調控特定基因表現 13 1. HMG1 13 2. ERG9 14 3. ERG1 14 二、 減少麥角固醇合成 15 1. ERG27基因剔除 15 2. UPC2點突變 16 參、 研究目的與實驗架構 17 肆、 材料與方法 18 第一節、 實驗材料 18 一、 化學藥品 18 二、 標準品 18 三、 菌株 19 四、 質體 21 五、 培養材料 22 第二節、 儀器設備 22 一、 一般儀器設備 22 二、 應用軟體 23 第三節、 實驗方法 24 一、 菌株特定基因之啟動子(promoter)置換 24 二、 蛋白表現 28 三、 點試驗(spotting assay) 29 四、 upc2-1突變株 29 五、 ERG27基因剔除 32 六、 非皂化脂質代謝物分析 33 伍、 結果 35 第一節、 特定基因之啟動子置換 35 一、 HMG1 36 1. 菌株建構 36 2. 蛋白表現與菌株生長狀況 38 二、 ERG1、ERG9 40 1. 菌株建構 40 2. 蛋白表現與菌株生長狀況 42 三、 HMG1+ERG1 44 1. 菌株建構 44 2. 蛋白表現與菌株生長狀況 46 四、 loxP-Cre調控的篩選基因kanMX4剔除 48 五、 HMG1+ERG1+ERG9 52 1. 菌珠建構 52 2. 蛋白表現與菌株生長狀況 54 第二節、 基因工程對酵母菌生成非皂化脂質代謝物的影響 56 一、 非皂化脂質的定性與定量 56 二、 非皂化脂質標準品之檢量線 58 三、 啟動子置換菌株的非皂化脂質代謝物分析 59 陸、 討論 62 柒、 結論與展望 66 捌、 參考文獻 67 玖、 附錄 75 APPENDIX 84 | |
| dc.language.iso | zh-TW | |
| dc.subject | 固醇代謝突變株 | zh_TW |
| dc.subject | 三?皂? | zh_TW |
| dc.subject | 啟動子置換 | zh_TW |
| dc.subject | Saccharomyces cerevisiae | zh_TW |
| dc.subject | triterpenoid saponins | en |
| dc.subject | Saccharomyce cerevisiae | en |
| dc.subject | sterol auxotroph | en |
| dc.subject | promoter replacement | en |
| dc.title | 以酵母菌基因工程增加三萜皂苷前驅物 | zh_TW |
| dc.title | Increase the precursors of triterpenoid saponins in the genetic engineered yeast | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 呂廷璋,陳勁初,王如邦,謝淑貞,陳宏彰 | |
| dc.subject.keyword | 三?皂?,啟動子置換,固醇代謝突變株,Saccharomyces cerevisiae, | zh_TW |
| dc.subject.keyword | triterpenoid saponins,promoter replacement,sterol auxotroph,Saccharomyce cerevisiae, | en |
| dc.relation.page | 101 | |
| dc.identifier.doi | 10.6342/NTU201802664 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-08-07 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 食品科技研究所 | zh_TW |
| 顯示於系所單位： | 食品科技研究所 | |
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