臺灣博碩士論文加值系統

葫蘆素B是葫蘆科植物的次級代謝物，具有廣泛的藥理特性。但該化合物在植物中含量低且結構複雜，難以利用化學合成或溶劑萃取獲得。因此，利用釀酒酵母生物合成葫蘆素B可做為一種替代方法。先前實驗室研究，已透過酵母菌成功將2,3-氧化角鯊烯導向葫蘆二烯醇的合成，作為葫蘆素B的前驅物，然需進一步進行羥基化等修飾。過去文獻顯示植物之細胞色素P450酶與細胞色素還原酶共同作用時，可修飾葫蘆二烯醇。因此，本研究選擇不同植物來源之酵素，並將相關基因整合到酵母中，希望藉由基因工程建構合成葫蘆素B前驅物之酵母菌。實驗中將羅漢果葫蘆二烯醇酶SgCS、苦瓜細胞色素還原酶McCPR及香瓜細胞色素P450酶Cm890基因整合至酵母菌中，以西方墨點法結果顯示酵母菌成功表現蛋白，而且插入外源基因並未影響菌株的生長，並且結果顯示酵母菌細胞可生成化合物11-carbonylcucurbitadienol和11-carbonyl-20β-hydroxycucurbitadienol。為提升這些化合物的含量，以利後續葫蘆素B的合成，研究進一步修改麥角固醇途徑中的HMG1基因表現量，以期增加關鍵前驅物2,3-氧化角鯊烯的含量，然而並未成功表達此基因於酵母菌中，同時會造成原先整合之基因被剔除。因此，未來研究可進一步選用誘導型啟動子調整基因之表現，以達成酵母菌中葫蘆素B的建構。

Cucurbitacin B is a secondary metabolite produced by Cucurbitaceae plants with various pharmacological properties. Due to the low contents and complexity of the structure of this compound in plants, it is difficult to be synthesized chemically or extracted by solvents. Therefore, biosynthesis using yeast, Saccharomyces cerevisiae, can be an alternative method to obtain cucurbitacin B. In our previous study, we modified the yeast and successfully produced cucurbitadienol, the precursor of cucurbitacin B. However, further hydroxylation is required for the synthesis of cucurbitacin B. According to studies, plant cytochrome P450s (CYPs) are often co-expressed with cytochrome P450s reductase (CPR) to chemically modify cucurbitadienol. Thus, in the study, enzymes will be selected from various plants, and the related genes will be integrated into yeast. This process is designed to enable the biosynthesis of cucurbitacin B precursors within the yeast. Our results showed that the recombinant yeast strains with cucurbitadienol synthase from Siraitia grosvenorii, CPR from Momordica charantia and Cm890 from C. melo successfully expressed these proteins. The spotting assay revealed that overexpressing these genes did not impact cell growth, and the yeast produced minor quantities of 11-carbonylcucurbitadienol and 11-carbonyl-20β-hydroxycucurbitadienol. To enhance the synthesis of these compounds, increasing the content of the precursor 2,3-oxidosqualene is essential. This was attempted through the modification of the HMG1 gene in the ergosterol pathway, but the data revealed that this gene was not successfully integrated into the yeast, leading to the loss of previously integrated genes. In the future, the selection of inducible promoters may be further explored to regulate the expression of these genes, with the aim of achieving the synthesis of cucurbitacin B in yeast.

謝誌 I
中文摘要 II
Abstract III
Graphical Abstract IV
總目錄 V
圖目錄 VIII
表目錄 X
附錄目錄 XI
第一章、前言 1
第二章、文獻回顧 2
第一節、植物三萜類皂苷合成路徑 2
一、甲羥戊酸途徑 2
二、皂苷元骨架生成 3
三、皂苷元修飾 3
第二節、酵母菌之三萜皂苷合成 4
一、酵母菌麥角固醇合成路徑 4
第三節、葫蘆素 5
一、葫蘆素B 5
二、葫蘆素B的取得 7
三、植物中葫蘆素B與酵母菌中麥角固醇合成路徑 8
第四節、建立及提高酵母菌葫蘆素B之方法 11
一、建立葫蘆素B生成路徑 11
二、提升特定基因tHMG1表現 12
第三章、研究目的與實驗架構 14
第四章、材料與方法 15
第一節、實驗材料 15
一、化學藥品 15
二、標準品 15
三、菌株 16
四、質體 17
五、限制酵素 19
六、培養材料 19
第二節、儀器設備與數據處理軟體 20
第三節、實驗方法 21
一、在酵母菌中建立葫蘆素B前驅物生合成路徑 21
1.苦瓜細胞色素還原酶McCPR終止子置換 21
2.香瓜細胞色素P450酶Cm890之建構 21
3.McCPR與Cm890之多基因建構 23
4.SgCS, McCPR及Cm890之多基因建構 23
二、提升tHMG1基因表現 24
1.tHMG1基因之建構 24
2.建構帶有同源臂及URA3 marker之tHMG1質體 25
三、酵母菌異源表達 30
四、蛋白表現 30
五、點試驗 31
六、非皂化脂質代謝物分析 31
第五章、實驗結果 33
第一節、在酵母菌建立葫蘆素B前驅物生合成路徑 33
一、酵母菌表達苦瓜細胞色素還原酶McCPR 34
1.質體建構之終止子置換 34
2.將建構完成之McCPR質體重組至酵母菌中 36
3.細胞色素還原酶McCPR蛋白表現與菌株生長 37
二、酵母菌表達香瓜細胞色素酶Cm890 39
1.Cm890質體之建構 40
2.將建構完成之Cm890質體重組至酵母菌中 43
3.細胞色素酶Cm890蛋白表現與菌株生長 44
三、McCPR與Cm890在酵母菌中共表達 46
1.質體建構序列片段McCPR+Cm890 46
2.將建構完成之McCPR+Cm890質體重組至酵母菌中 48
3.McCPR與Cm890蛋白表現與菌株生長 49
四、SgCS, McCPR, Cm890在酵母菌中共表達 51
1.質體建構序列片段SgCS+McCPR+Cm890 51
2.將建構完成之SgCS+McCPR+Cm890質體重組至酵母菌中 53
3.SgCS, McCPR, Cm890蛋白表現與菌株生長 54
第二節、基因工程酵母菌非皂化脂質代謝物分析 56
一、薄層層析法 56
二、ESI-UHPLC-MS/MS分析酵母菌代謝物 58
三、化合物二次離子碎片分析 60
第三節、在酵母菌中提升特定基因tHMG1表現以增加下游三萜皂苷含量 66
一、建構tHMG1質體 66
二、在酵母菌過表達tHMG1基因 67
第六章、討論與未來展望 74
第七章、參考文獻 77
第八章、附錄 83

Ai, L.;Guo, W.;Chen, W.;Teng, Y.;Bai, L., The gal80 Deletion by CRISPR-Cas9 in Engineered Saccharomyces cerevisiae Produces Artemisinic Acid Without Galactose Induction. Curr. Microbiol. 2019, 76 (11), 1313-1319.
Alagu Lakshmi, S.;Shafreen, R. M. B.;Priya, A.;Shunmugiah, K. P., Ethnomedicines of Indian origin for combating COVID-19 infection by hampering the viral replication: using structure-based drug discovery approach. J. Biomol. Struct. Dyn. 2021, 39 (13), 4594-4609.
Basson, M. E.; Thorsness, M,; Rine, J., Saccharomyces cerevisiae contains two functional genes encoging 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Proc. Natl. Acad. Sci. 1986, 83, 5563-5567.
Behura, S. K.;Severson, D. W., Codon usage bias: causative factors, quantification methods and genome-wide patterns: with emphasis on insect genomes. Biol. rev. biol. proc. Camb. Philos. Soc. 2013, 88 (1), 49-61.
Bentley, F. K.; Zurbriggen, A.; Melis, A. Heterologous expression of the mevalonic acid pathway in cyanobacteria enhances endogenous carbon partitioning to isoprene. Mol Plant 2014, 7 (1), 71-86.
Chen, J. C.; Chiu, M. H.; Nie, R. L.; Cordell, G. A.; Qiu, S. X. Cucurbitacins and cucurbitane glycosides: structures and biological activities. Nat. Prod. Rep. 2005, 22 (3), 386-99.
Dai, L.; Liu, C.; Zhu, Y.; Zhang, J.; Men, Y.; Zeng, Y.; Sun, Y. Functional Characterization of Cucurbitadienol Synthase and Triterpene Glycosyltransferase Involved in Biosynthesis of Mogrosides from Siraitia grosvenorii. Plant Cell Physiol. 2015, 56 (6), 1172-82.
Dai, S.;Wang, C.;Zhao, X.;Ma, C.;Fu, K.;Liu, Y.;Peng, C.;Li, Y., Cucurbitacin B: A review of its pharmacology, toxicity, and pharmacokinetics. Pharmacol. Res. 2023, 187, 106587.
Dai, Z.;Wang, B.;Liu, Y.;Shi, M.;Wang, D.;Zhang, X.;Liu, T.;Huang, L.;Zhang, X., Producing aglycons of ginsenosides in bakers' yeast. Sci. Rep. 2014, 4, 3698.
Daly, R.;Hearn, M. T., Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J. Mol. Recognit. 2005, 18 (2), 119-38.
DeBose-Boyd, R. A., Feedback regulation of cholesterol synthesis: sterol-accelerated ubiquitination and degradation of HMG CoA reductase. Cell Res. 2008, 18 (6), 609-21.
Devendra, N. K.;Attard, E. G.;Raghunandan, D.;Seetharam, Y. N., Study on Seasonal Variation on the Content of Cucurbitacin of Various Vegetative Parts of Trichosanthes cucumerina L. var. cucumerina. Int. J. Plant Res. 2012, 1 (1), 25-28.
Dong, L.;Almeida, A.;Pollier, J.;Khakimov, B.;Bassard, J. E.;Miettinen, K.;Staerk, D.;Mehran, R.;Olsen, C. E.;Motawia, M. S.;Goossens, A.;Bak, S., An Independent Evolutionary Origin for Insect Deterrent Cucurbitacins in Iberis amara. Mol. Biol. Evol. 2021, 38 (11), 4659-4673.
Fu, H.;Liang, Y.;Zhong, X.;Pan, Z.;Huang, L.;Zhang, H.;Xu, Y.;Zhou, W.;Liu, Z., Codon optimization with deep learning to enhance protein expression. Sci. Rep. 2020, 10 (1), 17617.
Fukunaga, T.;Cha-Aim, K.;Hirakawa, Y.;Sakai, R.;Kitagawa, T.;Nakamura, M.;Nonklang, S.;Hoshida, H.;Akada, R., Designed construction of recombinant DNA at the ura3Delta0 locus in the yeast Saccharomyces cerevisiae. Yeast 2013, 30 (6), 243-53.
Garg, S.;Kaul, S. C.;Wadhwa, R., Cucurbitacin B and cancer intervention: Chemistry, biology and mechanisms (Review). Int. J. Oncol. 2018, 52 (1), 19-37.
Ge, W.;Chen, X.;Han, F.;Liu, Z.;Wang, T.;Wang, M.;Chen, Y.;Ding, Y.;Zhang, Q., Synthesis of Cucurbitacin B Derivatives as Potential Anti-Hepatocellular Carcinoma Agents. Molecules 2018, 23 (12).
Gibson, D. G.; Young, L.; Chuang, R. Y.; Venter, J. C.; Hutchison, C. A., 3rd; Smith, H. O. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 2009, 6 (5), 343-5.
Guo, H.; Wang, H.; Huo, Y.-x. Engineering of critical enzymes and pathways for improved triterpenoid biosynthesis in yeast. ACS Synth. Biol. 2020, 9 (9), 2214-2227.
Hassan, S. T. S.;Berchová-Bímová, K.;Petráš, J.;Hassan, K. T. S., Cucurbitacin B interacts synergistically with antibiotics against Staphylococcus aureus clinical isolates and exhibits antiviral activity against HSV-1. S. Afr. J. Bot. 2017, 108, 90-94.
Hua, D.;Fu, J.;Liu, L.;Yang, X.;Zhang, Q.;Xie, M., Change in Bitterness, Accumulation of Cucurbitacin B and Expression Patterns of CuB Biosynthesis-related Genes in Melon During Fruit Development. Hort. J. 2019, 88 (2), 253-262.
Jorda, T.;Puig, S., Regulation of Ergosterol Biosynthesis in Saccharomyces cerevisiae. Genes (Basel) 2020, 11 (7).
Kaushik, U.; Aeri, V.; Mir, S. R., Cucurbitacins - An insight into medicinal leads from nature. Pharmacogn Rev. 2015, 9 (17), 12-8.
Kim, Y. C.;Choi, D.;Cha, A.;Lee, Y. G.;Baek, N. I.;Rimal, S.;Sang, J.;Lee, Y.;Lee, S., Critical enzymes for biosynthesis of cucurbitacin derivatives in watermelon and their biological significance. Commun. Biol. 2020, 3 (1), 444.
Kirby, J.;Romanini, D. W.;Paradise, E. M.;Keasling, J. D., Engineering triterpene production in Saccharomyces cerevisiae-beta-amyrin synthase from Artemisia annua. FEBS J. 2008, 275 (8), 1852-9.
Klingenberg, M., Pigments of Rat Liver Microsomes. Arch. Biochem. Biophys. 1958, 75, 376-386.
Kurman, Y.;Kiliccioglu, I.;Dikmen, A. U.;Esendagli, G.;Bilen, C. Y.;Sozen, S.;Konac, E., Cucurbitacin B and cisplatin induce the cell death pathways in MB49 mouse bladder cancer model. Exp. Biol. Med. (Maywood) 2020, 245 (9), 805-814.
Lee, Y. S.; Park, H. S.; Lee, D. K.; Jayakodi, M.; Kim, N. H.; Koo, H. J.; Lee, S. C.; Kim, Y. J.; Kwon, S. W.; Yang, T. J. Integrated Transcriptomic and Metabolomic Analysis of Five Panax ginseng Cultivars Reveals the Dynamics of Ginsenoside Biosynthesis. Front. Plant Sci. 2017, 8, 1048.
Lundemo, M. T.;Woodley, J. M., Guidelines for development and implementation of biocatalytic P450 processes. Appl. Microbiol. Biotechnol. 2015, 99 (6), 2465-83.
Luo, F.; Li, Q.; Yu, L.; Wang, C.; Qi, H. High concentrations of CPPU promotes cucurbitacin B accumulation in melon (Cucumis melo var. makuwa Makino) fruit by inducing transcription factor CmBt. Plant Physiol. Biochem. 2020, 154, 770-781.
Ma, T.; Shi, B.; Ye, Z.; Li, X.; Liu, M.; Chen, Y.; Xia, J.; Nielsen, J.; Deng, Z.; Liu, T. Lipid engineering combined with systematic metabolic engineering of Saccharomyces cerevisiae for high-yield production of lycopene. Metab. Eng. 2019, 52, 134-142.
Malhotra, K.;Franke, J., Cytochrome P450 monooxygenase-mediated tailoring of triterpenoids and steroids in plants. Beilstein J. Org. Chem. 2022, 18, 1289-1310.
Marostica, L. L.;de Barros, A. L. B.;Oliveira, J.;Salgado, B. S.;Cassali, G. D.;Leite, E. A.;Cardoso, V. N.;Lang, K. L.;Caro, M. S. B.;Duran, F. J.;Schenkel, E. P.;de Oliveira, M. C.;Simoes, C. M. O., Antitumor effectiveness of a combined therapy with a new cucurbitacin B derivative and paclitaxel on a human lung cancer xenograft model. Toxicol. Appl. Pharmacol. 2017, 329, 272-281.
Morant, M.;Bak, S.;Møller, B. L.;Werck-Reichhart, D., Plant cytochromes P450: tools for pharmacology, plant protection and phytoremediation. Curr. Opin. Biotechnol. 2003, 14 (2), 151-162.
Nicolaou, K.; Yang, Z.; Liu, J.; Ueno, H.; Nantermet, P.; Guy, R.; Claiborne, C.; Renaud, J.; Couladorous, E.; Paulvannan, K., Total synthesis of taxol. Nature 1994, 367, 630.
Niu, Y.;Luo, H.;Sun, C.;Yang, T. J.;Dong, L.;Huang, L.;Chen, S., Expression profiling of the triterpene saponin biosynthesis genes FPS, SS, SE, and DS in the medicinal plant Panax notoginseng. Gene 2014, 533 (1), 295-303.
Paddon, C. J.; Westfall, P. J.; Pitera, D. J.; Benjamin, K.; Fisher, K.; McPhee, D.; Leavell, M. D.; Tai, A.; Main, A.; Eng, D.; Polichuk, D. R.; Teoh, K. H.; Reed, D. W.; Treynor, T.; Lenihan, J.; Fleck, M.; Bajad, S.; Dang, G.; Dengrove, D.; Diola, D.; Dorin, G.; Ellens, K. W.; Fickes, S.; Galazzo, J.; Gaucher, S. P.; Geistlinger, T.; Henry, R.; Hepp, M.; Horning, T.; Iqbal, T.; Jiang, H.; Kizer, L.; Lieu, B.; Melis, D.; Moss, N.; Regentin, R.; Secrest, S.; Tsuruta, H.; Vazquez, R.; Westblade, L. F.; Xu, L.; Yu, M.; Zhang, Y.; Zhao, L.; Lievense, J.; Covello, P. S.; Keasling, J. D.; Reiling, K. K.; Renninger, N. S.; Newman, J. D. High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 2013, 496 (7446), 528-32.
Pandian, B. A.; Sathishraj, R.; Djanaguiraman, M.; Prasad, P. V. V.; Jugulam, M. Role of Cytochrome P450 Enzymes in Plant Stress Response. Antioxidants 2020, 9 (5).
Peng, B.;Wood, R. J.;Nielsen, L. K.;Vickers, C. E., An Expanded Heterologous GAL Promoter Collection for Diauxie-Inducible Expression in Saccharomyces cerevisiae. ACS Synth. Biol. 2018, 7 (2), 748-751.
Polakowski T. ;Stahl U. ;C., L., Overexpression of a cytosolic hydroxymethylglutaryl-CoA reductase leads to squalene accumulation in yeast. Appl. Microbiol. Biotechnol. 1998, 49, 66-71.
Sang, J.;Dhakal, S.;Lee, Y., Cucurbitacin B Suppresses Hyperglycemia Associated with a High Sugar Diet and Promotes Sleep in Drosophila melanogaster. Mol. Cells 2021, 44 (2), 68-78.
Sawai, S.; Saito, K. Triterpenoid biosynthesis and engineering in plants. Front. Plant Sci. 2011, 2, 25.
Seo, C. R.; Yang, D. K.; Song, N. J.; Yun, U. J.; Gwon, A. R.; Jo, D. G.; Cho, J. Y.; Yoon, K.; Ahn, J. Y.; Nho, C. W.; Park, W. J.; Yang, S. Y.; Park, K. W. Cucurbitacin B and cucurbitacin I suppress adipocyte differentiation through inhibition of STAT3 signaling. Food Chem. Toxicol. 2014, 64, 217-24.
Shang, Y.; Ma, Y.; Zhou, Y.; Zhang, H.; Duan, L.; Chen, H.; Zeng, J.; Zhou, O.; Wang, S.; Gu, W., Biosynthesis, regulation, and domestication of bitterness in cucumber. Science 2014, 346, 1084-1088.
Sikorski, R. S.; Hieter, P., A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 1989, 122, 19-27.
Souza-Moreira, T. M.;Navarrete, C.;Chen, X.;Zanelli, C. F.;Valentini, S. R.;Furlan, M.;Nielsen, J.;Krivoruchko, A., Screening of 2A peptides for polycistronic gene expression in yeast. FEMS Yeast Res. 2018, 18 (5).
Sun, J.;Shao, Z.;Zhao, H.;Nair, N.;Wen, F.;Xu, J. H.;Zhao, H., Cloning and characterization of a panel of constitutive promoters for applications in pathway engineering in Saccharomyces cerevisiae. Biotechnol. Bioeng. 2012, 109 (8), 2082-92.
Varela, C.;Melim, C.;Neves, B. G.;Sharifi-Rad, J.;Calina, D.;Mamurova, A.;Cabral, C., Cucurbitacins as potential anticancer agents: new insights on molecular mechanisms. J. Transl. Med. 2022, 20 (1), 630.
Wong, G.;Lim, L. R.;Tan, Y. Q.;Go, M. K.;Bell, D. J.;Freemont, P. S.;Yew, W. S., Reconstituting the complete biosynthesis of D-lysergic acid in yeast. Nat. Commun. 2022, 13 (1), 712.
Xu, J.; Chen, Y.; Yang, R.; Zhou, T.; Ke, W.; Si, Y.; Yang, S.; Zhang, T.; Liu, X.; Zhang, L.; Xiang, K.; Guo, Y.; Liu, Y. Cucurbitacin B inhibits gastric cancer progression by suppressing STAT3 activity. Arch. Biochem. Biophys. 2020, 684, 108314.
Zhang, T.;Lei, J.;Yang, H.;Xu, K.;Wang, R.;Zhang, Z., An improved method for whole protein extraction from yeast Saccharomyces cerevisiae. Yeast 2011, 28 (11), 795-8.
Zhou, Y.; Ma, Y.; Zeng, J.; Duan, L.; Xue, X.; Wang, H.; Lin, T.; Liu, Z.; Zeng, K.; Zhong, Y.; Zhang, S.; Hu, Q.; Liu, M.; Zhang, H.; Reed, J.; Moses, T.; Liu, X.; Huang, P.; Qing, Z.; Liu, X.; Tu, P.; Kuang, H.; Zhang, Z.; Osbourn, A.; Ro, D. K.; Shang, Y.; Huang, S. Convergence and divergence of bitterness biosynthesis and regulation in Cucurbitaceae. Nat. Plants 2016, 2, 16183.