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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃慶璨(Ching-Tsan Huang) | |
| dc.contributor.author | Wen-Tsung Chiang | en |
| dc.contributor.author | 江玟璁 | zh_TW |
| dc.date.accessioned | 2021-06-17T09:05:43Z | - |
| dc.date.available | 2023-02-03 | |
| dc.date.copyright | 2021-02-22 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-02-05 | |
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Am J Med Genet C Semin Med Genet 2011, 157c(1):45-53. 黃雅惠: 厭氣性真菌Neocallimastix frontails木聚醣酶基因之選殖、表現與重組蛋白酵素特性分析 Cloning and expression of anaerobic fungus neocallimastix frontalis xylanase genes and characterization of the recombinant proteins 國立臺灣大學微生物與生化學研究所; 2005. Collins T, Gerday C, Feller G: Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiology Reviews 2005, 29(1):3-23. 呂承鴻: 利用密碼子最適化的α-factor提升P.pastoris異源蛋白質外泌表現量 The use of codon optimized alpha-factor to enhance the secretion of heterologous protein in Pichia pastoris: 國立臺灣大學生化科技學系; 2013. Georis J, Giannotta F, Lamotte-Brasseur J, Devreese B, Van Beeumen J, Granier B, Frère J: Sequence, overproduction and purification of the family 11 endo-beta-1,4-xylanase encoded by the xyl1 gene of Streptomyces sp. S38. Gene 1999, 3(1):122-133. Wang K, Luo H, Tian J, Turunen O, Huang H, Shi P, Hua H, Wang C, Wang S, Yao B: Thermostability improvement of a streptomyces xylanase by introducing proline and glutamic acid residues. Appl Environ Microbiol 2014, 80(7):2158-2165. 蔡昌廷: 利用嗜甲醇酵母菌進行重組蛋白質表現之醱酵生產及分子調控之研究 The study of recombinant protein expression by fermentation and molecular control in methyltrophic yeasts 國立臺灣大學微生物與生化學研究所; 2009. Ren HT, Yuan JQ, Bellgardt KH: Macrokinetic model for methylotrophic Pichia pastoris based on stoichiometric balance. J Biotechnol 2003, 106(1):53-68. Houard S, Heinderyckx M, Bollen A: Engineering of non-conventional yeasts for efficient synthesis of macromolecules: the methylotrophic genera. Biochimie 2002, 84(11):1089-1093. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74661 | - |
| dc.description.abstract | Pichia pastoris是嗜甲醇酵母菌的一種，為極具潛力的異源蛋白質表達系統，其優勢在於培養成本低、生長快速和產量高等，同時具有原核表達系統不具備的轉譯後修飾，因此廣泛應用生產重組蛋白質。然而AOX1啟動子 (alcohol oxidase I promoter) 調控嚴謹，使P. pastoris只能使用有毒、易燃的甲醇作為碳源時，才可誘導AOX1啟動子啟動，產出重組蛋白質。本研究希望發展非甲醇的誘導方式，來生產重組蛋白質，以改善甲醇誘導所帶來的缺點。本研究延續實驗室前人的研究，以額外表現MXR1之菌株進行實驗，並搭配AOX1啟動子表現木聚醣酶，結果確認額外表現MXR1菌株可在甲醇誘導時，提升重組蛋白質產量。以甘油受限誘導的方法進行無甲醇製程研究，額外表現MXR1菌株在醱酵槽的培養可成功誘導木聚醣酶的表現。以轉錄因子MXR1、甘油運輸蛋白GT1 (glycerol transporter I) 之基因表現量可以解釋甘油受限時可誘導目標蛋白質表達的原因；並以氨甲醯磷酸合成酶CPA1 (carbamoyl phosphate synthetase I) 之基因表現量解釋，在甘油受限下，缺乏碳源可由提高CPA1表現量，回收二氧化碳，合成尿素，作為額外的碳氮源來源。最後根據甘油碳流量的分析，解釋在甘油受限誘導時，提高甘油誘導量可以提高重組蛋白質產量。 | zh_TW |
| dc.description.abstract | Pichia pastoris is a kind of methylotrophic yeast, with a high potential in application of heterologous protein expression system. It has lots of advantages such as low culture cost, fast growth and high yield. Moreover, it has characteristics of post-translation modifications that are not available in prokaryotic expression systems. Thus, it is widely used to produce recombinant proteins. However, the AOX1 promoter (alcohol oxidase I promoter) is strictly regulated, and is induced to start and produce recombinant proteins only when toxic and flammable methanol is used as a carbon source. Therefore, the goal of this study is to develop a methanol-free induction method which is used to produce recombinant protein and improve the disadvantages. Based upon previous works, this study used strains expressing additional MXR1 are paired with the AOX1 promoter to express xylanase. The results confirmed that the MXR1-reprogrammed strains increased the production of recombinant protein under methanol induction. The methanol-free process was performanced by glycerol-starvation induction method, and successfully demonstrated the experiment of the fermentor cultures.The glycerol-starvation induction was attributed to the gene expression of transcription factor MXR1 and glycerol transporter I (glycerol transporter I, GT1), and the gene expression of carbamoyl phosphate synthetase I (carbamoyl phosphate synthetase I, CPA1). Under glycerol restriction, lack of carbon source results in the increase of CPA1 expression and recovering carbon dioxide as an additional source of carbon and nitrogen to synthesize urea. Finally, according to the analysis of glycerol carbon flow, it is explained that under the glycerol-starvation induction, increasing the amount of glycerol-starvation induction can increase the production of recombinant protein. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T09:05:43Z (GMT). No. of bitstreams: 1 U0001-0102202119161100.pdf: 4030138 bytes, checksum: b61b59cb731481655aba0c21853aa797 (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | 摘要 I Abstract II 目錄 III 圖目錄 V 表目錄 VII 第一章 前言 1 1.1 表現系統 1 1.2 嗜甲醇酵母菌 1 1.2.1 具有轉譯後修飾 3 1.2.2 具外泌異源蛋白質的能力 3 1.2.3 高表現的AOX啟動子 3 1.3 AOX1 啟動子的轉錄調控 3 1.4 非甲醇誘導策略 6 1.4.1 GT1 (Glycerol transporter 1) 7 1.4.2 CPA1 (Carbamoyl phosphate synthetase I) 7 1.5 瘤胃真菌Neocallimastix frontalis的木聚醣酶 11 1.6 研究動機 13 第二章 材料與方法 14 2.1實驗菌株與培養條件 14 2.1.1 細菌 14 2.1.2 真菌 14 2.1.3 菌株保存 14 2.2 培養基 15 2.3 表現載體 17 1 pPICZα-xyn11B’ 17 2. pAOX2KH-Mxr1 17 2.4 嗜甲醇酵母菌電穿孔轉形 17 2.4.1 P. pastoris勝任細胞製備 17 2.4.2 電穿孔轉形 17 2.5 轉形株培養與分析 18 2.5.1 試管誘導 18 2.5.2 搖瓶誘導 18 2.5.3 醱酵槽誘導 19 甘油饋料培養 (Fed-batch) 19 誘導 (Induction) 19 2.6 mRNA表現量分析 20 2.7 蛋白質分析 22 2.7.1 木聚醣酶活性測定 22 (I) 木聚醣基質溶液 22 (II) DNS試劑 22 2.7.2聚丙烯醯胺膠體電泳分析 23 第三章 結果 25 3.1 額外表現Mxr1對重組蛋白生產之產量影響 25 3.1.1 篩選適合用於甲醇誘導之菌株 25 3.1.2 額外表現Mxr1可增加重組蛋白產量 26 3.1.2.1搖瓶試驗 26 3.1.2.2醱酵槽試驗 26 3.2 額外表現Mxr1，賦予甘油受限誘導產出重組蛋白 42 3.2.1 篩選適合以甘油受限誘導之菌株 42 3.2.2 額外表現Mxr1，賦予甘油受限誘導產出重組蛋白 42 3.2.3 菌體於甘油受限誘導之mRNA 反應 43 3.2.4提高甘油添加量進行甘油受限誘導，可提升重組蛋白產出 44 第四章 討論 59 4.1 甲醇誘導產出目標蛋白參數 59 4.2 甘油受限誘導成因討論 59 4.3 甘油受限誘導產出目標蛋白參數 61 第五章 結論 66 第六章 未來展望 67 第七章 參考文獻 68 | |
| dc.language.iso | zh-TW | |
| dc.subject | CPA1 | zh_TW |
| dc.subject | methanol‑free | zh_TW |
| dc.subject | methanol-independent | zh_TW |
| dc.subject | MXR1 | zh_TW |
| dc.subject | GT1 | zh_TW |
| dc.subject | Pichia pastoris | zh_TW |
| dc.title | 以甘油受限誘導策略誘導Mxr1再程序化之嗜甲醇酵母菌生產木聚醣酶 | zh_TW |
| dc.title | Glycerol-starvation induction strategy for recombinant xylanase production in Mxr1-reprogrammed Komagataella phaffii (Pichia pastoris) | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 109-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 吳亘承(Hsuan-Chen Wu),傅煦媛(Hsu-Yuan Fu) | |
| dc.subject.keyword | Pichia pastoris,methanol‑free,methanol-independent,MXR1,GT1,CPA1, | zh_TW |
| dc.relation.page | 71 | |
| dc.identifier.doi | 10.6342/NTU202100342 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2021-02-07 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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