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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 羅翊禎(Yi-Chen Lo) | |
| dc.contributor.author | Sook Yin Chong | en |
| dc.contributor.author | 張淑穎 | zh_TW |
| dc.date.accessioned | 2022-11-24T03:29:03Z | - |
| dc.date.available | 2021-08-23 | |
| dc.date.available | 2022-11-24T03:29:03Z | - |
| dc.date.copyright | 2021-08-23 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-08-19 | |
| dc.identifier.citation | Abdel-Banat, B. M.; Nonklang, S.; Hoshida, H.; Akada, R., Random and targeted gene integrations through the control of non-homologous end joining in the yeast Kluyveromyces marxianus. Yeast 2010, 27 (1), 29-39. Alagoz, M.; Kherad, N., Advance genome editing technologies in the treatment of human diseases: CRISPR therapy. Int. J. Mol. Med. 2020, 46 (2), 521-534. Arias, C. A. D.; Marques, D. d. A. V.; Malpiedi, L. P.; Maranhão, A. Q.; Parra, D. A. S.; Converti, A.; Junior, A. P., Cultivation of Pichia pastoris carrying the scFv anti LDL (−) antibody fragment. Effect of preculture carbon source. Braz. J. Microbiol. 2017, 48 (3), 419-426. Banat, I. M.; Nigam, P.; Marchant, R., Isolation of thermotolerant, fermentative yeasts growing at 52 C and producing ethanol at 45 C and 50 C. World J. Microbiol. Biotechnol. 1992, 8 (3), 259-263. Bendrioua, L.; Smedh, M.; Almquist, J.; Cvijovic, M.; Jirstrand, M.; Goksor, M.; Adiels, C. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/81070 | - |
| dc.description.abstract | 近年來非模式生物酵母菌如 Kluyveromyces marxianus，因具有高分泌蛋白的特性（high secretory capacity）、耐高溫及利用各種不同碳源的能力而被廣泛用於發酵工業上。然而，K. marxianus 之同源重組效率低，所以在利用傳統基因工程技術上有一定的困難。因此本實驗以 CRISPR/Cas9 基因編輯技術進行 K. marxianus 菌株之蛋白質表現系統的建立，應用於羅漢果皂苷的轉換。首先，將 K. marxianus 菌株中指定的 β-glucosidase基因（EXG1）剔除並嵌入目標基因（DbEXG1），使其菌株在發酵時具有能力將羅漢果皂苷 mogroside V水解成具有高甜度的 siamenoside I 。結果顯示，菌株在 KmEXG1基因剔除後喪失了水解羅漢果皂苷的能力而嵌入目標基因的菌株則能夠穩定表現 DbExg1 蛋白。為增加 DbExg1蛋白的產量，分別利用不同啟動子序列評估 K. marxianus 及 S. cerevisiae 菌株在大量表現 DbExg1蛋白的能力。出乎預料的是，當菌株利用 S. cerevisiae 的GAP 啟動子序列時（ScGAP），只需要發酵 24小時就能夠將 mogroside V 完全轉換成 siamenoside I。然而，當利用 K. marxianus 的 GAP 啟動子序列（KmGAP）則需要發酵超過72小時。這顯示 S. cerevisiae 菌株在 siamenoside I 皂苷轉換的效率相較基因改造後的 K. marxianus 較好。雖然 K. marxianus 較 S. cerevisiae 具有外泌大量總蛋白的能力，分別在使用 ScGAP 啟動子序列為 （0.68 ± 0.02 mg/mL 及 0.51 ± 0.01 mg/mL，p ˂ 0.05）而在使用 KmGAP 啟動子序列分別為（0.55 ± 0.02 mg/mL 及 0.27 ± 0.02 mg/mL，p ˂ 0.05）。但以 Western Blotting 分析發現 K. marxianus 對 DbExg1的外泌量卻較 S. cerevisiae 少，而 S. cerevisiae 在使用任一種類的 GAP 啟動子相較 K. marxianus 都具有能力外泌較多的 DbExg1 蛋白。因此，S. cerevisiae BY4741 系列在 siamenoside I 皂苷轉換有較好的能力。 未來可利用不同種類的 K. marxianus 進一步測試並評估其菌株在建立大量表現外源性蛋白之系統的可行性。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-24T03:29:03Z (GMT). No. of bitstreams: 1 U0001-1908202117372500.pdf: 5976258 bytes, checksum: f2ff59d9776148d2e3037a0b4118aaf3 (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | Acknowledgements i 摘要 ii Abstract iii Table of Contents v List of Figures vii List of Tables ix Supplementary x Appendix x CHAPTER 1 INTRODUCTION 1 CHAPTER 2 LITERATURE REVIEW 2 2.1 Introduction to yeast 2 2.1.1 Saccharomyces cerevisiae 2 2.1.2 Kluyveromyces marxianus 3 2.2 Mogrosides 4 2.2.1 Biotransformation of mogrosides 6 2.3 Genetic engineering 7 2.3.1 Homologous recombination 7 2.3.2 Non-homologous end joining 9 2.3.3 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) 11 CHAPTER 3 RESEARCH OBJECTIVES AND EXPERIMENTAL DESIGN 16 CHAPTER 4 MATERIALS AND METHODS 18 4.1 Materials 18 4.1.1 Yeast strains 18 4.1.2 Plasmids 18 4.1.3 Primers 18 4.1.4 Medium 25 4.1.5 Reagents 27 4.1.6 Device and instrument 30 4.1.7 Software 32 4.2 Methods 33 4.2.1 Characteristics of yeast strains 33 4.2.2 Genetic engineering 34 4.2.3 Biotransformation 42 4.2.4 Protein analysis 45 4.2.5 Determination of protein concentration and enzyme activity 48 CHAPTER 5 RESULTS AND DISCUSSION 49 5.1 Growth comparison of K. marxianus and S. cerevisiae 49 5.1.1 Growth curve 49 5.1.2 Cell viability 53 5.2 Genetic engineering in Kluyveromyces marxianus 56 5.2.1 Construction of auxotrophic mutants of K. marxianus 56 5.2.2 β-glucosidase gene disruption 65 5.2.3 Cell viability of mutant strains 67 5.3 Mogrosides bioconversion using genetic modified K. marxianus 71 5.4 DbExg1 protein expression in S. cerevisiae and K. marxianus 73 5.4.1 Plasmid construction 73 5.4.2 Yeast integration for the expression of DbExg1 84 5.4.3 Determination of DbExg1 expression 93 5.5 Evaluation of mogroside bioconversion in different yeast 97 5.5.1 Cell survival of yeast strains in medium containing mogroside extract 97 5.5.2 Mogroside bioconversion at different time points 100 5.5.3 Relative quantification in mogroside levels 111 5.6 Medium effect on protein production and enzyme activity 116 5.6.1 Glucose effect on protein production and enzyme activity 116 5.6.2 High fructose corn syrup and glycerol effect on protein production and enzyme activity 119 5.7 GAP promoter effect on DbExg1 protein expression 131 CHAPTER 6 CONCLUSION AND FUTURE PERSPECTIVE 135 CHAPTER 7 REFERENCES 137 CHAPTER 8 SUPPLEMENTARY 142 CHAPTER 9 APPENDIX 149 | |
| dc.language.iso | en | |
| dc.subject | Kluyveromyces marxianus | zh_TW |
| dc.subject | CRISPR/Cas9 | zh_TW |
| dc.subject | 生物轉換 | zh_TW |
| dc.subject | β-glucosidase基因 | zh_TW |
| dc.subject | GAP 啟動子 | zh_TW |
| dc.subject | β-glucosidase gene | en |
| dc.subject | GAP promoter | en |
| dc.subject | bioconversion | en |
| dc.subject | CRISPR/Cas9 | en |
| dc.subject | Kluyveromyces marxianus | en |
| dc.title | 利用Kluyveromyces marxianus建立蛋白質表現系統應用於siamenoside I 之轉換 | zh_TW |
| dc.title | Establishing Kluyveromyces marxianus as a robust platform for siamenoside I bioconversion | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 高承福(Hsin-Tsai Liu),呂廷璋(Chih-Yang Tseng),陳宏彰,王如邦 | |
| dc.subject.keyword | Kluyveromyces marxianus,CRISPR/Cas9,生物轉換,β-glucosidase基因,GAP 啟動子, | zh_TW |
| dc.subject.keyword | Kluyveromyces marxianus,CRISPR/Cas9,bioconversion,β-glucosidase gene,GAP promoter, | en |
| dc.relation.page | 157 | |
| dc.identifier.doi | 10.6342/NTU202102521 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2021-08-20 | |
| 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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