請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/98845完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李昆達 | zh_TW |
| dc.contributor.advisor | Kung-Ta Lee | en |
| dc.contributor.author | 趙亭 | zh_TW |
| dc.contributor.author | Ting Chao | en |
| dc.date.accessioned | 2025-08-19T16:25:31Z | - |
| dc.date.available | 2025-08-20 | - |
| dc.date.copyright | 2025-08-19 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-08-12 | - |
| dc.identifier.citation | Black, W. B., Aspacio, D., Bever, D., King, E., Zhang, L., & Li, H. (2020). Metabolic engineering of Escherichia coli for optimized biosynthesis of nicotinamide mononucleotide, a noncanonical redox cofactor. Microbial Cell Factories, 19(1), 150. https://doi.org/10.1186/s12934-020-01415-z
Camacho-Pereira, J., Tarragó, M. G., Chini, C. C. S., Nin, V., Escande, C., Warner, G. M., Puranik, A. S., Schoon, R. A., Reid, J. M., Galina, A., & Chini, E. N. (2016). CD38 Dictates age-related NAD decline and mitochondrial dysfunction through an SIRT3-dependent mechanism. Cell Metabolism, 23(6), 1127–1139. https://doi.org/10.1016/j.cmet.2016.05.006 Cantó, C., Menzies, K. J., & Auwerx, J. (2015). NAD+ metabolism and the control of energy homeostasis: a balancing act between mitochondria and the nucleus. Cell Metabolism, 22(1), 31–53. https://doi.org/10.1016/j.cmet.2015.05.023 Cheng, F., Li, K. X., Wu, S. S., Liu, H. Y., Li, H., Shen, Q., Xue, Y. P., & Zheng, Y. G. (2024). Biosynthesis of nicotinamide mononucleotide: synthesis method, enzyme, and biocatalytic system. Journal of Agricultural and Food Chemistry, 72(7), 3302–3313. https://doi.org/10.1021/acs.jafc.3c09217 Covarrubias, A. J., Perrone, R., Grozio, A., & Verdin, E. (2021). NAD+ metabolism and its roles in cellular processes during ageing. Nature Reviews. Molecular Cell Biology, 22(2), 119–141. https://doi.org/10.1038/s41580-020-00313-x Datsenko, K. A., & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences of the United States of America, 97(12), 6640–6645. https://doi.org/10.1073/pnas.120163297 Evans, C., Bogan, K. L., Song, P., Burant, C. F., Kennedy, R. T., & Brenner, C. (2010). NAD+ metabolite levels as a function of vitamins and calorie restriction: evidence for different mechanisms of longevity. BMC Chemical Biology, 10, 2. https://doi.org/10.1186/1472-6769-10-2 Fang, E. F., Lautrup, S., Hou, Y., Demarest, T. G., Croteau, D. L., Mattson, M. P., & Bohr, V. A. (2017). NAD+ in aging: molecular mechanisms and translational implications. Trends in Molecular Medicine, 23(10), 899–916. https://doi.org/10.1016/j.molmed.2017.08.001 Galeazzi, L., Bocci, P., Amici, A., Brunetti, L., Ruggieri, S., Romine, M., Reed, S., Osterman, A. L., Rodionov, D. A., Sorci, L., & Raffaelli, N. (2011). Identification of nicotinamide mononucleotide deamidase of the bacterial pyridine nucleotide cycle reveals a novel broadly conserved amidohydrolase family. The Journal of Biological Chemistry, 286(46), 40365–40375. https://doi.org/10.1074/jbc.M111.275818 Hall, J. A., Dominy, J. E., Lee, Y., & Puigserver, P. (2013). The sirtuin family's role in aging and age-associated pathologies. The Journal of Clinical Investigation, 123(3), 973–979. https://doi.org/10.1172/JCI64094 He, Z., Yang, X., Tian, X., Li, L., & Liu, M. (2022). Yeast cell surface engineering of a nicotinamide riboside kinase for the production of β-nicotinamide mononucleotide via whole-cell catalysis. ACS Synthetic Biology, 11(10), 3451–3459. https://doi.org/10.1021/acssynbio.2c00350 Hillyard, D., Rechsteiner, M., Manlapaz-Ramos, P., Imperial, J. S., Cruz, L. J., & Olivera, B. M. (1981). The pyridine nucleotide cycle. Studies in Escherichia coli and the human cell line D98/AH2. The Journal of Biological Chemistry, 256(16), 8491–8497. Huang, Z., Li, N., Yu, S., Zhang, W., Zhang, T., & Zhou, J. (2022). Systematic Engineering of Escherichia coli for efficient production of nicotinamide mononucleotide from nicotinamide. ACS Synthetic Biology, 11(9), 2979–2988. https://doi.org/10.1021/acssynbio.2c00100 Kurnasov, O. V., Polanuyer, B. M., Ananta, S., Sloutsky, R., Tam, A., Gerdes, S. Y., & Osterman, A. L. (2002). Ribosylnicotinamide kinase domain of NadR protein: identification and implications in NAD biosynthesis. Journal of Bacteriology, 184(24), 6906–6917. https://doi.org/10.1128/JB.184.24.6906-6917.2002 Liu, L., Su, X., Quinn, W. J., 3rd, Hui, S., Krukenberg, K., Frederick, D. W., Redpath, P., Zhan, L., Chellappa, K., White, E., Migaud, M., Mitchison, T. J., Baur, J. A., & Rabinowitz, J. D. (2018). Quantitative analysis of NAD synthesis-breakdown fluxes. Cell Metabolism, 27(5), 1067–1080.e5. https://doi.org/10.1016/j.cmet.2018.03.018 Liu, Y., Yasawong, M., & Yu, B. (2021). Metabolic engineering of Escherichia coli for biosynthesis of β-nicotinamide mononucleotide from Nicotinamide. Microbial Biotechnology, 14(6), 2581–2591. https://doi.org/10.1111/1751-7915.13901 Luo, S., Zhao, J., Zheng, Y., Chen, T., & Wang, Z. (2023). Biosynthesis of nicotinamide mononucleotide: current metabolic engineering strategies, challenges, and prospects. Fermentation, 9(7), 594–608. https://doi.org/10.3390/fermentation9070594 Maharjan, A., Singhvi, M., & Kim, B. S. (2021). Biosynthesis of a therapeutically important nicotinamide mononucleotide through a phosphoribosyl pyrophosphate synthetase 1 and 2 engineered strain of Escherichia coli. ACS Synthetic Biology, 10(11), 3055–3065. https://doi.org/10.1021/acssynbio.1c00333 Marinescu, G. C., Popescu, R. G., Stoian, G., & Dinischiotu, A. (2018). β-nicotinamide mononucleotide (NMN) production in Escherichia coli. Scientific Reports, 8(1), 12278. https://doi.org/10.1038/s41598-018-30792-0 Mills, K. F., Yoshida, S., Stein, L. R., Grozio, A., Kubota, S., Sasaki, Y., Redpath, P., Migaud, M. E., Apte, R. S., Uchida, K., Yoshino, J., & Imai, S. I. (2016). Long-term administration of nicotinamide mononucleotide mitigates age-associated physiological decline in mice. Cell Metabolism, 24(6), 795–806. https://doi.org/10.1016/j.cmet.2016.09.013 Ngivprom, U., Lasin, P., Khunnonkwao, P., Worakaensai, S., Jantama, K., Kamkaew, A., & Lai, R. Y. (2022). Synthesis of nicotinamide mononucleotide from xylose via coupling engineered Escherichia coli and a biocatalytic cascade. Chembiochem: a European Journal of Chemical Biology, 23(11), e202200071. https://doi.org/10.1002/cbic.202200071 Nunn, D., Vick, J. E., & Salas-Santiago, B. (2021). Productions of NMN and its derivatives via microbial processes. WO 2021/226044 A1. Nunn, D., Vick, J. E., & Salas-Santiago, B. (2024). Productions of NMN and Its Derivatives via Microbial Processes. US 2024/0043894 A1 Pabarcus, M. K., & Casida, J. E. (2002). Kynurenine formamidase: determination of primary structure and modeling-based prediction of tertiary structure and catalytic triad. Biochimica et Biophysica Acta, 1596(2), 201–211. https://doi.org/10.1016/s0167-4838(02)00232-7 de Picciotto, N. E., Gano, L. B., Johnson, L. C., Martens, C. R., Sindler, A. L., Mills, K. F., Imai, S., & Seals, D. R. (2016). Nicotinamide mononucleotide supplementation reverses vascular dysfunction and oxidative stress with aging in mice. Aging Cell, 15(3), 522–530. https://doi.org/10.1111/acel.12461 Rizzi, M., Nessi, C., Mattevi, A., Coda, A., Bolognesi, M., & Galizzi, A. (1996). Crystal structure of NH3-dependent NAD+ synthetase from Bacillus subtilis. The EMBO Journal, 15(19), 5125–5134. Shoji, S., Yamaji, T., Makino, H., Ishii, J., & Kondo, A. (2021). Metabolic design for selective production of nicotinamide mononucleotide from glucose and nicotinamide. Metabolic Engineering, 65, 167–177. https://doi.org/10.1016/j.ymben.2020.11.008 Sinclair, D. A., & Ear, P. H. (2015). Biological production of NAD precursors and analogs. WO 2015/069860 A1. Sorci, L., Brunetti, L., Cialabrini, L., Mazzola, F., Kazanov, M. D., D'Auria, S., Ruggieri, S., & Raffaelli, N. (2014). Characterization of bacterial NMN deamidase as a Ser/Lys hydrolase expands diversity of serine amidohydrolases. FEBS Letters, 588(6), 1016–1023. https://doi.org/10.1016/j.febslet.2014.01.063 Sorci, L., Martynowski, D., Rodionov, D. A., Eyobo, Y., Zogaj, X., Klose, K. E., Nikolaev, E. V., Magni, G., Zhang, H., & Osterman, A. L. (2009). Nicotinamide mononucleotide synthetase is the key enzyme for an alternative route of NAD biosynthesis in Francisella tularensis. Proceedings of the National Academy of Sciences of the United States of America, 106(9), 3083–3088. https://doi.org/10.1073/pnas.0811718106 Sugiyama, K., Iijima, K., Yoshino, M., Dohra, H., Tokimoto, Y., Nishikawa, K., Idogaki, H., & Yoshida, N. (2021). Nicotinamide mononucleotide production by fructophilic lactic acid bacteria. Scientific Reports, 11(1), 7662. https://doi.org/10.1038/s41598-021-87361-1 Wang, L., Zhou, Y. J., Ji, D., Lin, X., Liu, Y., Zhang, Y., Liu, W., & Zhao, Z. K. (2014). Identification of UshA as a major enzyme for NAD degradation in Escherichia coli. Enzyme and Microbial Technology, 58-59, 75–79. https://doi.org/10.1016/j.enzmictec.2014.03.003 Yoshino, J., Mills, K. F., Yoon, M. J., & Imai, S. (2011). Nicotinamide mononucleotide, a key NAD+ intermediate, treats the pathophysiology of diet- and age-induced diabetes in mice. Cell Metabolism, 14(4), 528–536. https://doi.org/10.1016/j.cmet.2011.08.014 Zhu, X. H., Lu, M., Lee, B. Y., Ugurbil, K., & Chen, W. (2015). In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences. Proceedings of the National Academy of Sciences of the United States of America, 112(9), 2876–2881. https://doi.org/10.1073/pnas.1417921112 | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/98845 | - |
| dc.description.abstract | β-菸鹼醯胺單核苷酸 (NMN) 為煙醯胺腺嘌呤二核苷酸 (NAD+) 的前驅物，且先前研究顯示其可增強NAD+生合成並改善糖尿病和血管功能障礙。NMN因此成為一種廣受歡迎的營養保健品。然而，NMN生產的限制包含：(1) 使用化學或酵素合成方式生產NMN會需要昂貴的基質和催化劑；(2) 以微生物發酵方法生產 NMN 的效率和實用性低。因此，開發有效率且經濟的NMN生產方式是重要的。本研究以菸鹼酸 (NA) 為基質，大腸桿菌JM109為宿主生產NMN。本研究首先將大腸桿菌的pncC基因剔除，以防止其水解NMN上的醯胺基。 此pncC缺失的JM109菌株便為後續異源基因表現的宿主。為了生合成NMN，本研究也異源表現了來自土拉弗朗西斯菌 (Francisella tularensis) 的NMN合成酶ftNadE。ftNadE 催化菸鹼酸D-核糖核苷酸 (NaMN) 的醯胺化形成NMN。因此，當大腸桿菌吸收NA，其菸鹼酸磷酸核糖基轉移酶PncB將催化NaMN的合成。接著ftNadE便將合成的NaMN醯胺化，合成NMN。結果顯示，ftNadE 在異丙基-β-D-硫代半乳糖苷 (IPTG) 誘導後成功表現，且 pncC 也成功在大腸桿菌的基因組中剔除。經過搖瓶培養並使用高效液相層析法 (HPLC) 分析 NMN 的產量後，NMN 很可能在表現 ftNadE 的野生型菌株 (34.79 mg/L)、在表現ftNadE的pncC剔除菌株 (44.96 mg/L)、和在表現His-ftNadE的pncC剔除菌株中產生 (46.83 mg/L) 。未來將會需使用液相層析質譜儀確認NMN分子存在，並在生物反應器中培養，以進一步提高NMN 產量。 | zh_TW |
| dc.description.abstract | As a key precursor of nicotinamide adenine dinucleotide (NAD+), β-Nicotinamide mononucleotide (NMN) has been shown to enhance NAD+ synthesis and improve diabetes and vascular dysfunction. NMN has subsequently become a popular nutraceutical. Despite its popularity, NMN production has limitations which include: (a) the use of expensive substrates and catalysts in chemical or enzymatic synthesis and (b) low productivity and practicality in microbial fermentative methods. A cost-effective NMN production method is therefore welcome and worth exploring. This study aims to produce NMN using nicotinic acid (NA) as substrate and Escherichia coli JM109 (E. coli JM109) as host. E. coli’s pncC gene has been deleted to prevent it from hydrolyzing NMN’s amide group. The pncC-deleted JM109 strain (E. coli JM109 ΔpncC) then served as the heterologous gene expression host. To synthesize NMN, ftNadE, an NMN synthetase from Francisella tularensis (F. tularensis) that amidates nicotinate D-ribonucleotide (NaMN) to form NMN, has been heterologously expressed. So once E. coli cells take up NA, its nicotinate phosphoribosyltransferase, PncB, catalyzes the synthesis of NaMN. NMN synthetase ftNadE then amidates the synthesized NaMN to yield NMN. Results showed that ftNadE was successfully expressed after Isopropyl β-D-1 thiogalactopyranoside (IPTG) induction and that pncC was deleted from the E. coli genome. After shake-flask culturing and analyzing NMN production using high performance liquid chromatography (HPLC), NMN was likely produced in wild-type E. coli strains expressing ftNadE (34.79 mg/L), in E. coliΔpncC strains expressing ftNadE (44.96 mg/L) and in E. coli ΔpncC strains expressing His-ftNadE (46.83 mg/L). Future work would involve verifying NMN production using liquid chromatography-mass spectrometry (LC-MS) and maximizing NMN production in bioreactors. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2025-08-19T16:25:31Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2025-08-19T16:25:31Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 口試委員審定書 i
中文摘要 iv Abstract vi Abbreviations viii 中英文專有名詞對照表 x Contents xii Contents of figures xvi Chapter 1. Introduction 1 1.1 Nicotinamide mononucleotide (NMN) 3 1.2 Anti-aging potential of NMN 3 1.3 Nicotinamide mononucleotide synthesis 5 1.4 Nicotinamide mononucleotide synthetase ftNadE 6 1.5 Nicotinamide mononucleotide deamidase PncC 7 1.6 Aim of study 8 Chapter 2. Materials and Methods 9 2.1 Microorganisms and vectors 11 2.2 Plasmid construction 11 2.3 Plasmid extraction 11 2.4 Plasmid transformation into commercial competent cells 12 2.5 E. coli strain construction 13 2.5.1 Deletion of pncC using the Lambda Red recombineering method 13 2.5.1.1 Preparation of recombination cassette 13 2.5.1.2 Transformation of pKD46 plasmid 14 2.5.1.3 Electroporation of recombination cassette into E. coli JM109 cells 14 2.5.1.4 pCP20 plasmid transformation into E. coli JM109 ΔpncC 15 2.5.1.5 Transformation of constructed plasmids into E. coli JM109 ΔpncC 16 2.6 Shake-flask culturing and sample preparation 16 2.6.1 Growth curve 16 2.6.2 Shake-flask culturing 17 2.6.3 Sample preparation for further analyses 18 2.7 Protein expression analysis 18 2.8 NMN production analysis (high performance liquid chromatography) 18 Chapter 3. Results 21 3.1 Cloning of ftnadE 23 3.2 Verification of constructed E. coli strains 24 3.3 Growth curves of E. coli JM109 with pQE-30-Xa-ftnadE and E. coli JM109 ΔpncC with pQE-30-Xa-ftnadE 25 3.4 Heterologous protein expression analysis of ftnadE in constructed E. coli strains 26 3.5 HPLC standard curves of NMN and NA 27 3.6 NMN production analysis of constructed E. coli strains 27 Chapter 4. Conclusion and Discussion 29 4.1 Conclusion 31 4.2 Discussion 31 Figures 35 References 63 | - |
| dc.language.iso | en | - |
| dc.subject | 菸鹼醯胺單核苷酸 | zh_TW |
| dc.subject | 大腸桿菌 | zh_TW |
| dc.subject | 異源表現 | zh_TW |
| dc.subject | 基因剔除 | zh_TW |
| dc.subject | 搖瓶培養 | zh_TW |
| dc.subject | 高效液相層析法 | zh_TW |
| dc.subject | heterologous expression | en |
| dc.subject | nicotinamide mononucleotide | en |
| dc.subject | Escherichia coli | en |
| dc.subject | high performance liquid chromatography | en |
| dc.subject | shake-flask culturing | en |
| dc.subject | gene deletion | en |
| dc.title | 利用基因工程大腸桿菌生產菸鹼醯胺單核苷酸 | zh_TW |
| dc.title | Engineering Escherichia coli to Produce Nicotinamide Mononucleotide | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 蘇敬岳;蔡黛華 | zh_TW |
| dc.contributor.oralexamcommittee | Ching-Yueh Su;Dai-Hua Tsai | en |
| dc.subject.keyword | 菸鹼醯胺單核苷酸,大腸桿菌,異源表現,基因剔除,搖瓶培養,高效液相層析法, | zh_TW |
| dc.subject.keyword | nicotinamide mononucleotide,Escherichia coli,heterologous expression,gene deletion,shake-flask culturing,high performance liquid chromatography, | en |
| dc.relation.page | 70 | - |
| dc.identifier.doi | 10.6342/NTU202504049 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2025-08-14 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生化科技學系 | - |
| dc.date.embargo-lift | N/A | - |
| 顯示於系所單位： | 生化科技學系 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-113-2.pdf 未授權公開取用 | 3.07 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。