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研究生： 鮑越霞
Priskila Adjani Diankristanti
論文名稱： 透過基改大腸桿菌全細胞催化生產高產量衣康酸
Towards high-level itaconic acid (IA) production using engineered Escherichia coli by whole-cell bioconversion
指導教授： 吳意珣
Ng, I-Son
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程學系
Department of Chemical Engineering
論文出版年： 2023
畢業學年度： 111
語文別： 英文
論文頁數： 67
中文關鍵詞： 衣康酸 、全細胞催化 、分子伴侶蛋白 、大腸桿菌 、順烏頭酸酶 、冷激
外文關鍵詞： Itaconic acid, Whole-cell bioconversion, Escherichia coli, aconitase, cold treatment, cis-aconitate decarboxylase
ORCID： 0000-0002-2006-5747
ResearchGate： https://www.researchgate.net/profile/Priskila-Diankristanti
相關次數： 點閱：91  下載：5 
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    • 隨著工業和科技的進步，石油衍生化學品對海洋、水資源、土壤以及空氣等生態環境造成嚴重的危害。相較於某些石油衍生化學品，由生物製造的化學品通常具有較低的毒性和較少的有害副產物，有助於降低對土壤、水源和生物的污染風險。衣康酸(Itaconic acid, IA) 作為生物化學品原料，被應用於生成多種工業材料，如塑膠纖維、生物燃料與淨化水資源的去除劑。衣康酸可於生物體內藉由順烏頭酸酶(Acn, EC 4.2.1.3)和烏頭酸脫羧酶(CadA, EC 4.1.1.6)進行催化合成。然而，通過微生物發酵生產衣康酸會伴隨其他的生物反應，造成副產物的累積和過長的培養時間。為克服這些限制，本研究利用經基因工程改造的大腸桿菌進行全細胞生物催化反應，嘗試在短時內高效地生產體外(in vitro)衣康酸。
    本研究利用以甘油為基礎的限制營養培養基(M9培養基)，在含有pLemo輔助質體和GroELS基因簇的大腸桿菌BL21(DE3)中實現72.44 g/L的衣康酸產量。同時通過全面的保種與接種策略，使BD::7G和L21的穩定催化活性長達30天。為了進一步的提高產量，本研究在反應前進行了24小時的-80°C冷處理，使衣康酸的產量與轉化率分別達到了81.58 g/L和92.22%。此外，在含有高濃度底物的大規模衣康酸催化實驗中，成功實現了生物催化劑的再利用，提高了商業經濟的可行性。我們在3升的生物反應器中進行全細胞催化，通過精確的溶氧和pH值監測，在一次的批次培養中獲得大量的生物催化劑。最後，在50 mL的反應體系中獲得了105.68 g/L的衣康酸最高產量，並在四次的重複使用後，依舊保持相對穩定的活性。
    本研究透過大規模的全細胞催化生產大量的衣康酸，並成功實現生物催化劑的再利用。在提出一種對環境友善的生物催化法的同時，提高了生產衣康酸的經濟效益。本研究提供了高效生產衣康酸的全細胞催化法，為生物催化在生產化學品的應用提出新的可行性。未來，期許這些研究成果有助於推動生產可持續的生物化學品，為實現綠色和環保的化學工業盡一份心力。

    In the emerging advancement of biobased chemical production, itaconic acid (IA) is known as an essential building block for high-value chemicals in a wide range of application. IA is mainly synthesized through native fermentation in Aspergillus and Ustilago, highly relying on aconitase (Acn, EC 4.2.1.3) and cis-aconitic acid decarboxylase (CadA, EC 4.1.1.6). However, IA production via microbial fermentation suffers from undesired reactions, side products accumulation, and long cultivation time. Employing whole-cell biocatalyst, which is engineered to attain high productivity in a short cultivation period, to carry out in vitro IA production is a novel approach to mitigate the limitations. This study achieved 72.44 g/L IA using E. coli BL21(DE3) assisted by pLemo and GroELS in a glycerol-based minimal medium. Comprehensive seeding strategy was developed to maintain the biocatalysts stability up to 30 days, successfully conserving original activity of E. coli BD::7G and L21. Productivity was further enhanced by cold treatment in -80°C for 24 h prior to reaction, reaching 81.58 g/L and 92.22% conversion rate. Finally, large-scale IA bioconversion using high substrate concentration was conducted, along with the biocatalysts reutilization to achieve economic viability. The highest IA titer of 105.68 g/L was attained in a 50-mL reaction system, with relatively-steady activity after four times of repeated usages. These results pave the way of wider application of whole-cell biocatalysts for value-added chemicals.

    Summary i 摘要 ii List of Figures vi List of Tables viii Abbreviation ix Acknowledgements x Chapter 1 Introduction 1 1.1. Research background 1 1.2. Research framework 3 Chapter 2 Literature Review 5 2.1. Development of IA bioconversion 5 2.2. IA bioconversion in vivo vs in vitro 6 2.3. Whole-cell bioconversion 7 2.3.1. Genetic design 8 2.3.2. Biocatalyst cultivation 10 2.3.3. Productivity and stability enhancement 11 Chapter 3 Material and Methods 12 3.1. Materials and chemicals 12 3.2. Strains, plasmids, and primers 14 3.3. Experimental instruments 16 3.4. Engineered strains transformation and cultivation 17 3.4.1. Polymerase chain reaction (PCR) 17 3.4.4. Plasmid DNA extraction 19 3.4.5. Competent cell preparation 20 3.4.3. Transformation into E. coli via heat-shock 21 3.4.4. Cell cultivation and induction 22 3.4.5. Large-scale cultivation in 2.8 L bioreactor 23 3.5. Whole-cell in vitro IA bioconversion 24 3.6. Cold treatment 24 3.7. Determination of enzymatic kinetic properties 25 3.8. Seed preparation and storage 25 3.9. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 26 3.10. Zeta potential measurement 28 3.11. High Performance Liquid Chromatography (HPLC) 28 Chapter 4 Result and Discussion 30 4.1. Gene selection for in vitro bioconversion 30 4.2. Kinetic study of AcnA and CadA 32 4.3. TrxA effect on improving CadA 34 4.4. Optimization of IA bioconversion from citric acid 37 4.5. Host exploration of IA bioconversion chassis 38 4.6. Optimization of culture medium 39 4.7. Enhancing stability: Seeding strategy 42 4.8. Enhancing productivity: Cold treatment 43 4.9. Enhancing economic viability: High-level production and recycle 45 4.10. Large-scale production and bioconversion 47 Chapter 5 Conclusions and Future Prospects 52 5.1 Conclusions 52 5.2 Future prospects 53 Publications 54 References 55 Supplementary Data 65

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