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研究生:安馬瑞
研究生(外文):Anandharaj Marimuthu
論文名稱:建構酵母菌細胞表面纖維素分解酶複合體系統
論文名稱(外文):Constructing a yeast system to express the cellulosome complex on the cell surface
指導教授:李文雄李文雄引用關係
指導教授(外文):Wen-Hsiung Li
口試委員:李宗璘黃介辰何孟樵蔡怡陞梁博煌
口試委員(外文):Tsung-Lin LiChieh-Chen HuangMeng-Chiao HoIsheng Jason TsaiPo-Huang Liang
口試日期:2019-11-15
學位類別:博士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2019
畢業學年度:108
語文別:英文
論文頁數:92
中文關鍵詞:纖維素分解酶複合體支架蛋白锚定蛋白纤维素结合模块.
外文關鍵詞:Cellulosomescaffoldin proteinanchoring proteincellulose-binding module.
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厭氧菌所產生的纖維素酵素複合體(Cellulosomes)被認為是自然界中最好的纖維素分解機器,因此科學家一直持續嘗試在工業酵母菌中建構人工纖維素酵素複合體。由於纖維素酵素複合體的基因數量龐大且複雜,因此非常具有挑戰性。我們以先進的合成生物學技術去設計與合成Clostridium thermocellum的骨架蛋白(CipA)與錨定蛋白(OlpB)成功克服此困難,經過改造的益生菌酵母菌(Kluyveromyces marxianus)可生產一系列來自真菌的複合纖維素分解酶,包括內切葡聚醣酶(TrEgIII),外切葡聚醣酶(CBHII),β-葡萄糖苷酶(NpaBGS)和纖維素酶增強劑(TaLPMO和MtCDH)。共軛焦顯微鏡的結果證實細胞表面有OlpB-ScGPI的表現,且透過螢光活化細胞分類計分析顯示幾乎有81%的酵母細胞表面具有OlpB-ScGPI。同時我們也進一步利用非變性聚丙烯酰胺凝膠電泳與質譜儀分析的結果驗證了纖維素酵素複合體蛋白確實形成複合體。相較於前人以質體構築的纖維素酵素複合體的研究只能容納12種酵素,本實驗透過重組染色體的基因工程可生產容納多達63種酵素,是目前已知構築纖維素酵素複合體中最多的。更特別的是,與其他改造後的CipA相比,本實驗所使用的CipA2B9C (具有兩個纖維素連接模塊,CBM)所釋放的還原糖量明顯提昇,藉此結果也證實了纖維素結合結構模塊的數量對纖維素酵素複合體的重要性。經過改造的酵母菌可有效分解纖維素,並分別從微晶纖維素(Avicel)和磷酸膨脹纖維素(PASC)中生產3.09 g / L和8.61 g / L的乙醇,其生產量高於前人所構建的酵母菌纖維素酵素複合體。
Cellulosomes from anaerobic bacteria are considered nature’s finest cellulolytic machinery. Thus, constructing a cellulosome in an industrial yeast has long been a goal pursued by scientists. However, it remains highly challenging due to the size and complexity of cellulosomal genes. Here, we overcame the difficulties by designing and synthesizing the Clostridium thermocellum scaffoldin gene (CipA) and the anchoring protein gene (OlpB) using advanced synthetic biology techniques. The engineered Kluyveromyces marxianus, a probiotic yeast, secreted a cocktail of dockerin fused fungal cellulases, including an endoglucanase (TrEgIII), exoglucanase (CBHII), β-glucosidase (NpaBGS) and cellulase boosters (TaLPMO and MtCDH). The confocal microscopy results confirmed the cell surface display of OlpB-ScGPI and Fluorescence-Activated Cell Sorting analysis results revealed that almost 81% of yeast cells displayed OlpB-ScGPI. We have also demonstrated the cellulosome complex formation using purified and crude cellulosomal proteins. Native PAGE and mass spectrometric analysis further confirmed the cellulosome complex formation. Our engineered cellulosome can accommodate up to 63 enzymes, whereas the largest engineered cellulosome reported thus far could accommodate only 12 enzymes and was expressed by a plasmid instead of chromosomal integration. Interestingly, CipA 2B9C (with two cellulose binding module, CBM) released significantly higher quantities of reducing sugars compared with other CipA variants, thus confirming the importance of cohesin numbers and CBM domain on cellulosome complex. The engineered yeast host efficiently degraded cellulosic substrates and released 3.09 g/L and 8.61 g/L of ethanol from avicel and phosphoric acid swollen cellulose (PASC), respectively, which are higher than any previously constructed yeast cellulosome.
Table of Contents
Acknowledgements i
中文摘要 iii
Abstract iv
List of Tables vii
List of Figures viii
List of Appendix x
1. Introduction 1
1.1. Lignocellulosic biomasses as an alternative 1
1.2. Enzyme synergism: a key factor 1
1.3. ‘Cellulosome’: a superior enzyme complex 2
1.4. Cellulosome on yeast 5
1.5. Cellulolytic biorefinery concept 6
1.6. Consolidated bioprocessing (CBP): an ideal approach 6
1.7. Kluyveromyces marxianus: a potential host for CBP 7
1.8. Synthetic biology tool for K. marxianus 7
2. Materials and Methods 9
2.1. Strains and Media 9
2.2. Cellulosomal construct design and synthesis 9
2.3. Yeast transformation and clone screening 10
2.4. Expression and purification of cellulosomal proteins 11
2.5. Cellulosome complex assembly and purification 12
2.6. Immunofluorescence microscopy and FACS 13
2.7. Cellulosome assembly on cell surface 13
2.8. Real-time quantitative PCR 14
2.9. Enzyme assays 14
2.10. Fermentation and ethanol production 15
2.11. Homology modeling of dockerin-fused enzymes 15
2.12. Statistical analysis 15
3. Results 16
3.1. Design and synthesis of cellulosomal scaffoldins 16
3.2. To study the cellulolytic synergism with cellulase boosters 18
3.3. Conversion of free cellulases into cellulosomal mode 22
3.4. Chromosomal integration of cellulosomal genes 27
3.5. Demonstration of cell-surface display using immunofluorescence analysis 30
3.6. Confirmation of cellulosome complex assembly 34
3.7. Constructing enzyme hosts 40
3.8. Boosting effects of cellulase boosters 44
3.9. Effects of cohesin and CBM numbers on cellulosome efficiency 49
3.10. Demonstration of consolidated bioprocessing 51
3.11. Future direction: Designing designer Scaffoldin 54
3.12. Chromosomal integration of Designer cellulosomal genes 57
4. Discussion 59
5. References 65
6. Appendixes 73
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