耐熱酵素於去氧核醣核苷三磷酸之合成與增強生物合成假尿嘧啶核苷的代謝工程策略

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研究生：	鄭乃瑋 Cheng, Nai-Wei
論文名稱：	耐熱酵素於去氧核醣核苷三磷酸之合成與增強生物合成假尿嘧啶核苷的代謝工程策略 Thermotolerant Enzymes in Deoxynucleoside Triphosphate Synthesis and Metabolic Engineering Strategies for Enhanced Pseudouridine Biosynthesis
指導教授：	張晃猷 Chang, Hwan-You
口試委員:	高茂傑 Kao, Mou-Chieh 張壯榮 Chang, Chuang-Rung 簡志青 Chien, Chih-Ching 賴怡琪 Lai, Yi-Chyi
學位類別：	博士 Doctor
系所名稱：	生命科學暨醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2025
畢業學年度：	113
語文別：	英文
論文頁數：	73
中文關鍵詞：	去氧核醣核苷三磷酸、假尿苷
外文關鍵詞：	Pseudouridine, thermus phage p23-45
相關次數：	點閱：3 下載：0
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核酸是一種大型生物分子，對所有生命體和病毒都很重要。它們由核苷酸組成，核苷酸是單體成分：一個五碳糖、一個磷酸基團和一個含氮鹼基。核酸的兩個主要類別是去氧核糖核酸 (DNA) 和核糖核酸 (RNA)。去氧核醣核苷三磷酸 (dNTP)，是DNA合成的基本原料，在許多分子生物技術例如DNA序列分析、基因定序、聚合酶連鎖反應(Polymerase-Chain-Reaction)、DNA標記等反應中都是不可或缺的生物有機分子；此外在醫藥、食品、香妝品等相關領域也有用途。在RNA方面，除了常見的NTP用於RNA合成上，假尿嘧啶核苷(Pseudouridine)作為細胞中最常見的修飾後核苷酸，在mRNA疫苗中有著重要的作用。隨著近年來生物科技產業的蓬勃發展，核苷酸的需求量也逐年上升，然而市面上所販售的核苷酸主要是以化學方式來合成，不但會用到許多的毒性有機溶劑，還有產量不高以及高成本等問題；如果要以生物合成的方法又有受質專一性過高、酵素不穩定這些問題。本研究目的在於建立一套以酵素合成dNTP的系統，此外也透過代謝工程生產假尿苷。
dNTP的生產過程分為兩步驟，首先以去氧核醣核甘酸單磷酸為起始反應物，藉由酵素催化生成去氧核醣核甘酸二磷酸，之後再進一步生成去氧核醣核苷三磷酸。我們從NCBI基因庫中找出幾個可能具有催化潛力的耐熱去氧核醣核苷酸單磷酸激酶、乙酸激酶，和丙酮酸激酶，然後分別在大腸桿菌中表現蛋白，在純化蛋白過後再對這些蛋白進行熱穩定以及功能性的分析。結果顯示這些激酶在pH 7.0到8.5，溫度在55度到75度的環境下都仍然具有相當的活性。我們也將這些酵素用來初步生產dNTP，並將產物以聚合酶連鎖反應進行檢測，就結果看來與市面上所販售的dNTP相比並無顯著差異，顯示這套系統可應在於生產乾淨的dNTP。在假尿嘧啶核苷方面，首先，我們通過在 Escherichia coli BW25113 中上調 rbsK、yeiN 和 PHM8 的表達，加強了假尿嘧啶核苷的主要合成途徑。其次，為了減少能量損失和副產物的形成，刪除 thrA 和 yeiC 有效地提高了假尿嘧啶核苷的積累，第三，我們識別並優化了與假尿嘧啶核苷相關的潛在運輸途徑。最後，由於磷酸戊糖途徑是核糖-5-磷酸(Ribose-5-phosphate)的主要合成路徑，我們通過增強該途徑的表達，將假尿嘧啶核苷產量提升至 1466 mg/L。總結來說，本研究成功建立了一株假尿嘧啶核苷的高產菌株，展示了其在工業規模應用中的潛力。

Nucleic acids are large biomolecules essential for all living organisms and viruses. They are composed of nucleotides, which are monomers consisting of a five-carbon sugar, a phosphate group, and a nitrogenous base. The two major types of nucleic acids are DNA and RNA. Deoxyribonucleoside triphosphates (dNTPs), the fundamental building blocks for DNA synthesis, are indispensable biomolecules in various molecular biotechnologies, such as DNA sequencing, polymerase chain reaction (PCR), and DNA labeling. Additionally, dNTPs have applications in pharmaceuticals, food, and cosmetics. In RNA synthesis, alongside the common four nucleoside triphosphates (NTPs), pseudouridine, the most abundant modified nucleotide in cells, plays a critical role in mRNA vaccines. With the rapid development of the biotechnology industry in recent years, the demand for nucleotides has been increasing annually. However, commercially available nucleotides are primarily synthesized chemically, which involves toxic organic solvents and suffers from low yield and high cost. While biosynthesis offers an alternative, it faces challenges such as high substrate specificity and enzyme instability. This study aims to establish an enzyme-based dNTP synthesis system and produce pseudouridine through metabolic engineering.
The dNTP production process involves two steps. First, deoxyribonucleoside monophosphates are used as starting substrates, and enzymes catalyze their conversion to deoxyribonucleoside diphosphates, which are subsequently converted into deoxyribonucleoside triphosphates. From GenBank, we identified several thermotolerant deoxyribonucleoside monophosphate kinases, acetate kinases, and pyruvate kinases with potential catalytic abilities. These enzymes were expressed in Escherichia coli, purified, and analyzed for thermal stability and functionality. The results showed that these kinases retained significant activity under pH 7.0–8.5 conditions and temperatures of 55–75°C. Using these enzymes, we preliminarily synthesized dNTPs and validated the products using PCR. The results showed no significant differences compared to commercially available dNTPs, demonstrating the system’s potential for producing high-purity dNTPs.
For pseudouridine production, we first enhanced its primary synthesis pathway by upregulating the expression of rbsK, yeiN, and PHM8 in Escherichia coli BW25113. Second, the deletion of thrA and yeiC effectively reduced energy loss and by-product formation, increasing pseudouridine accumulation. Third, we identified and optimized potential transport pathways associated with pseudouridine. Finally, as the pentose phosphate pathway (PPP) is the primary route for synthesizing ribose-5-phosphate, we enhanced its expression, further boosting pseudouridine yield to 1466 mg/L.
In summary, this study successfully established a high-yield strain, demonstrating its potential for industrial-scale applications in nucleotide production.

中文摘要    i
Abstract    iii
Acknowledgment    v
Table of contents    vi
List of Tables    xi
List of Figures    xii
Abbreviations    xiv
Chapter 1. Synthesis of deoxynucleoside triphosphates by a one-pot thermal stable system    1
1.1.    Introduction    2
1.1.1.    Deoxynucleoside triphosphate synthesis    2
1.1.2.    Phage Nucleoside monophosphate kinase (ϕNMK)    3
1.1.3.    Acetate kinase (ACK)    4
1.1.4.    Pyruvate kinase (PYK)    5
1.1.5.    Specific Aims    5
1.2.    Materials and Methods    7
1.2.1.    Cloning, overexpression, and purification of thermal stable enzymes used for nucleotide synthesis    7
1.2.2.    Characterization of the enzyme kinetic properties    8
1.2.3.    Sequence alignment and molecular structure modeling    9
1.2.4.    Site-directed mutagenesis    9
1.2.5. Crystallization and data collection.    9
1.2.6.    One-pot dNTP synthesis system    10
1.2.7. High-Performance Liquid Chromatography (HPLC) Analysis    10
1.2.    Results    11
1.3.1.    Overexpression and synthesis of ϕNMK and other thermostable enzymes used in the study    11
1.3.2.    Optimal conditions for ϕNMK catalytic activity    11
1.3.3.    Deoxynucleoside monophosphate conversion rates of ϕNMK at different temperatures    12
1.3.4.    Kinetic constant of ϕ NMK    12
1.3.5.    Sequence analysis and structure modeling of ϕNMK    13
1.3.6.    Identification of amino acid residues in ϕNMK critical for the catalysis    13
1.3.7.    Establishment of a one-pot dNTP synthesis system using ϕNMK    14
1.3.8.    Testing dNTPs by polymerase chain reaction (PCR)    15
1.3.    Discussion and Perspective    16
Chapter 2. Enzymatic and metabolic engineering for pseudouridine biosynthesis    18
2.1.    Introduction    19
2.2.    Materials and Methods    22
2.2.1    Cloning, overexpression, and purification of enzymes used in this study    22
2.2.2    Generation of E. coli knockout strains    23
2.2.3    Characterization of the enzyme kinetic properties    24
2.2.4    Two-step enzymatic pseudouridine synthesis    24
2.2.5    High-performance liquid chromatography analysis    24
2.2.6    Cultivation in shake flasks    25
2.3.    Results and Discussion    25
2.3.1.    Overexpression and purification of recombinant enzymes for pseudouridine synthesis    25
2.3.2.    Enzyme-based synthesis of pseudouridine    26
2.3.3.    Engineering an In Vivo Synthetic Pathway for Enhanced Pseudouridine Production    27
2.3.4.    Effects of modification of the uridine transport system on pseudouridine production    29
2.3.5.    Effects of overexpression of pentose phosphate pathway    31
2.4.    Conclusion and Perspective    32
Tables    34
Table 1. Sequences of the primers used in chapter 1    35
Table 2. The specific activity of the recombinant enzymes used in chapter 1    36
Table 3. Kinetic characteristics of ϕNMK    37
Table 4. Effects of different amino acid substitutions to the activity of ϕNMK    38
Table 5. Composition of charged amino acids in the thermostable enzymes used in chapter 1    39
Table 6. Bacterial strains and plasmids used in chapter 2.    40
Table 7. Primers used in chapter 2.    41
Table 8. Kinetic parameters of enzymatic reaction.    43
Figures    44
Figure 1. Challenges in current dNTP biosynthesis methods.    45
Figure 2. Analysis of purified recombinant proteins used in chapter 1 by SDS-PAGE.    46
Figure 3. The optimal temperature for ϕNMK catalysis.    47
Figure 4. Thermal stability of ϕNMK.    48
Figure 5. Effects of different metal ions on the activity of ϕNMK.    49
Figure 6. Optimal pH for ϕNMK activity.    50
Figure 7. Effects of different temperatures on the synthesis of dNDP by ϕNMK.    51
Figure 8. Alignment of Thermus phage p23-45 and T4 phage NMK sequences.    52
Figure 9. The structure of ϕNMK simulated by Swiss-Model.    53
Figure 10. Crystal structure of ϕNMK    54
Figure 11. The dNTP biosynthesis scheme used in this study.    55
Figure 12. Quantitative analysis of dNTP yield from a one-pot synthesis reaction using different ATP-recycled enzymes.    56
Figure 13. Kinetic analysis of dNTP biosynthesis in a one-pot reaction at 55°C.    57
Figure 14. Comparison of dNTP obtained from a commercial source and dNTP synthesized using the one-pot reaction with ϕNMK as a template in PCR.    58
Figure 15. Pseudouridine metabolic pathways and strain engineering.    59
Figure 16. Analysis of purified recombinant proteins used in chapter 2 by SDS-PAGE.    60
Figure 17. Kinetics of the two-step enzymatic pseudouridine synthesis.    61
Figure 18. Pseudouridine yield in different engineered strains grown in SMSF medium for 48 h.    62
Figure 19. The effects of yeiC and thrA knockout on cells.    63
Figure 20. Profiles of pseudouridine concentrations in cultures of recombinant strains and different transporters in SMSF medium after a 48-h fermentation period.    64
Figure 21. Profiles of pseudouridine concentrations in cultures of recombinant strains with varying pentose phosphate pathway genes grown in SMSF medium after 48 hours of fermentation.    65
Figure 22. The metabolic pathway of Ribulose-5-P in E. coli.    66
Figure 23. HPLC analysis of the fermentation product.    67
References    68


                                

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