利用基因優化的解脂耶氏酵母生產高價值二羧酸｜國立臺灣科技大學博碩士論文系統

簡易檢索 / 詳目顯示

回結果列表

研究生：	趙君翰 Chun-Han Chao
論文名稱：	利用基因優化的解脂耶氏酵母生產高價值二羧酸 Massive Production of Fatty Acids and High Value Dicarboxylic Acids in Yarrowia lipolytica
指導教授：	蔡伸隆 Shen-Long Tsai
口試委員:	李振綱 Cheng-Kang Lee 王勝仕 Sheng-Shih Wang
學位類別：	碩士 Master
系所名稱：	工程學院 - 化學工程系 Department of Chemical Engineering
論文出版年：	2021
畢業學年度：	109
語文別：	中文
論文頁數：	94
中文關鍵詞：	解脂耶氏酵母、癸二酸、硫酯酶、代謝工程、β氧化、ω氧化
外文關鍵詞：	Yarrowia lipolytica, Sebacic acid, Thioesterase, Metabolic engineering, β-oxidation, ω-oxidation
相關次數：	點閱：240 下載：0
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

摘要    I
ABSTRACT    II
致謝    III
目錄    V
圖目錄    IX
表目錄    XI
壹、    緒論    1
1    前言    1
2    研究動機與目的    2
3    研究內容    3
貳、    文獻回顧    5
1    解脂耶氏酵母(Yarrowia lipolytica)的介紹    5
2    解脂耶氏酵母的代謝路徑    7
2. 1    解脂耶氏酵母的碳源代謝路徑    7
2. 2    葡萄糖的代謝路徑與硫酯酶對脂肪酸碳鏈長度之影響    9
2. 3    葡萄糖代謝路徑與β氧化 (β-Oxidation)    12
2. 4    ω氧化 (ω-Oxidation)    15
3    癸二酸的應用    18
參、    實驗材料與方法    19
1    實驗摘要    19
2 材料與儀器    21
2. 1    藥品    21
2. 2    實驗用之生物製劑及套組    25
2. 3    儀器    27
2. 4    菌株    29
2. 5    質體    30
2. 6    引子(Primer)    31
3    實驗方法    33
3. 1    大腸桿菌培養方法    33
3. 2    酵母菌之培養    33
3. 3    小量質體純化法(Miniprep)    34
3. 4    小量質體純化法(ZR Plasmid Miniprep – Classic Kit)    36
3. 5    小量質體純化法(Tools Plasmid Mini Kit)    37
3. 6    提取酵母菌基因組(YeaStar Genomic DNA Kit)    38
3. 7    聚合酶鏈鎖反應(Polymerase chain reaction, PCR)    39
3. 8    瓊脂糖凝膠電泳分析(Agarose gel electrophoresis)    41
3. 9    瓊脂糖凝膠回收DNA (DNA recovery)    43
3. 10    酶切反應(Digestion)    44
3. 11    核酸接合反應(DNA ligation)    46
3. 12    Gibson assembly    47
3. 13    大腸桿菌勝任細胞(Ultra competent cell)的製備    49
3. 14    大腸桿菌之轉型作用(Transformation)    51
3. 15    大腸桿菌電脈衝穿孔術之勝任細胞(Electrocompetent cell)的製備    52
3. 16    大腸桿菌之電脈衝穿孔術(Electroporation)    53
3. 17    線性化(Linearization)    54
3. 18    解脂耶氏酵母的轉型作用    55
3. 19    菌株保存    57
3. 20    解脂耶氏酵母的發酵測試    58
3. 21    發酵液的分析前處理    59
3. 22    氣相層析質譜儀(GC-MS)的操作    60
3. 23    繪製癸二酸二(三甲基矽基)酯的檢量線    64
肆、    結果與討論    65
1    菌株的發酵條件測試    65
2. 1 發酵條件測試-底物濃度(辛酸)對於癸二酸產量的影響    65
2. 2 發酵條件測試-pH值對於癸二酸產量的影響    68
2 改植菌株的發酵測試    69
2. 1 過量表達硫酯酶CpFatB1，且剔除GUT2或MFE1是否會影響癸二酸的產量    70
2. 2 過量表達ω氧化基因FAO1、ALK1及硫酯酶CpFatB1，且剔除GUT2或MFE1是否會影響癸二酸的產量    71
2. 3 過量表達ω氧化基因ALK1、CPR1及硫酯酶CpFatB1，且剔除GUT2或MFE1是否會影響癸二酸的產量    72
2. 4 癸二酸累積能力較高的改殖菌株與原始菌株Po1g的比較    74
伍、    結論    76
1 研究結果結論    76
2 未來工作    78
陸、    參考文獻    79
柒、    質體建構    85
1 欲剔除基因的質體建構    85
1. 1 pGUT2int質體建構    85
1. 2 pMFE1int質體建構    86
1. 3 pYAL1int質體建構    87
2 欲過表達基因的質體建構    88
2. 1 pGUT2int-Leu質體建構    88
2. 2 pMFE1int-Leu質體建構    89
2. 3 pCpFatB1質體建構    90
2. 4 pYAL1int-CpFatB1質體建構    91
2. 5 pGUT2int-CpFatB1-Leu質體建構    92
2. 6 pMFE1int-CpFatB1-Leu質體建構    93

                                

[1] H. Liu, T. Cheng, M. Xian, Y. Cao, F. Fang, H.J.B.a. Zou, Fatty acid from the renewable sources: a promising feedstock for the production of biofuels and biobased chemicals, 32(2) (2014) 382-389.
[2] W. Runguphan, J.D.J.M.e. Keasling, Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals, 21 (2014) 103-113.
[3] J.M. Ageitos, J.A. Vallejo, P. Veiga-Crespo, T.G.J.A.m. Villa, biotechnology, Oily yeasts as oleaginous cell factories, 90(4) (2011) 1219-1227.
[4] L. Liu, A. Pan, C. Spofford, N. Zhou, H.S.J.M.e. Alper, An evolutionary metabolic engineering approach for enhancing lipogenesis in Yarrowia lipolytica, 29 (2015) 36-45.
[5] G. Dwivedi, M.J.R. Sharma, S.E. Reviews, Impact of cold flow properties of biodiesel on engine performance, 31 (2014) 650-656.
[6] K. Ahuja, S. Singh, Sebacic Acid Market Size By Application (Plasticizers, Lubricants, Solvents, Adhesives, Chemical Intermediates), Industry Analysis Report, Regional Outlook, Application Potential, Price Trends, Competitive Market Share & Forecast, 2019 – 2026, 2019. https://www.gminsights.com/industry-analysis/sebacic-acid-market. (Accessed February 2020 2020).
[7] W.H. Organization, Prioritization of pathogens to guide discovery, research and development of new antibiotics for drug-resistant bacterial infections, including tuberculosis, World Health Organization, 2017.
[8] M. Beed, R. Sherman, S.J.C.E.i.A. Holden, Critical Care, Pain, Fungal infections and critically ill adults, 14(6) (2014) 262-267.
[9] A. Rywińska, P. Juszczyk, M. Wojtatowicz, M. Robak, Z. Lazar, L. Tomaszewska, W.J.B. Rymowicz, Bioenergy, Glycerol as a promising substrate for Yarrowia lipolytica biotechnological applications, 48 (2013) 148-166.
[10] K. Qiao, S.H.I. Abidi, H. Liu, H. Zhang, S. Chakraborty, N. Watson, P.K. Ajikumar, G.J.M.e. Stephanopoulos, Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica, 29 (2015) 56-65.
[11] M. Tai, G.J.M.e. Stephanopoulos, Engineering the push and pull of lipid biosynthesis in oleaginous yeast Yarrowia lipolytica for biofuel production, 15 (2013) 1-9.
[12] J.-M. Nicaud, C. Madzak, P. van den Broek, C. Gysler, P. Duboc, P. Niederberger, C.J.F.y.r. Gaillardin, Protein expression and secretion in the yeast Yarrowia lipolytica, 2(3) (2002) 371-379.
[13] G. Barth, C.J.F.m.r. Gaillardin, Physiology and genetics of the dimorphic fungus Yarrowia lipolytica, 19(4) (1997) 219-237.
[14] R. Ledesma-Amaro, J.-M.J.T.i.B. Nicaud, Metabolic engineering for expanding the substrate range of Yarrowia lipolytica, 34(10) (2016) 798-809.
[15] P. Fickers, P.-H. Benetti, Y. Waché, A. Marty, S. Mauersberger, M. Smit, J.-M.J.F.y.r. Nicaud, Hydrophobic substrate utilisation by the yeast Yarrowia lipolytica, and its potential applications, 5(6-7) (2005) 527-543.
[16] N.D.J.C.j.o.b. Lindley, Bioconversion and biodegradation of aliphatic hydrocarbons, 73(S1) (1995) 1034-1042.
[17] R. Tsugawa, T. Nakase, T. Kobayashi, K. Yamashita, S.J.A. Okumura, B. Chemistry, Fermentation of n-Paraffins by Yeast: Part III. α-Ketoglutarate Productivity of Various Yeast, 33(6) (1969) 929-938.
[18] J.-T. Kim, S.G. Kang, J.-H. Woo, J.-H. Lee, B.C. Jeong, S.-J.J.A.M. Kim, Biotechnology, Screening and its potential application of lipolytic activity from a marine environment: characterization of a novel esterase from Yarrowia lipolytica CL180, 74(4) (2007) 820-828.
[19] R. Margesin, F.J.A. Schinner, E. Microbiology, Efficiency of indigenous and inoculated cold-adapted soil microorganisms for biodegradation of diesel oil in alpine soils, 63(7) (1997) 2660-2664.
[20] A.V. Bankar, A.R. Kumar, S.S.J.A.m. Zinjarde, biotechnology, Environmental and industrial applications of Yarrowia lipolytica, 84(5) (2009) 847-865.
[21] D.-C. Chen, J.-M. Beckerich, C.J.A.m. Gaillardin, biotechnology, One-step transformation of the dimorphic yeast Yarrowia lipolytica, 48(2) (1997) 232-235.
[22] P. Fickers, M. Le Dall, C. Gaillardin, P. Thonart, J.J.J.o.m.m. Nicaud, New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica, 55(3) (2003) 727-737.
[23] C.M. Schwartz, M.S. Hussain, M. Blenner, I.J.A.s.b. Wheeldon, Synthetic RNA polymerase III promoters facilitate high-efficiency CRISPR–Cas9-mediated genome editing in Yarrowia lipolytica, 5(4) (2016) 356-359.
[24] C. Madzak, Yarrowia lipolytica strains and their biotechnological applications: how natural biodiversity and metabolic engineering could contribute to cell factories improvement, (2021).
[25] Z. Lazar, T. Rossignol, J. Verbeke, A.-M. Crutz-Le Coq, J.-M. Nicaud, M.J.J.o.I.M. Robak, Biotechnology, Optimized invertase expression and secretion cassette for improving Yarrowia lipolytica growth on sucrose for industrial applications, 40(11) (2013) 1273-1283.
[26] J. Blazeck, A. Hill, L. Liu, R. Knight, J. Miller, A. Pan, P. Otoupal, H.S.J.N.c. Alper, Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production, 5(1) (2014) 1-10.
[27] A. Beopoulos, T. Chardot, J.-M.J.B. Nicaud, Yarrowia lipolytica: A model and a tool to understand the mechanisms implicated in lipid accumulation, 91(6) (2009) 692-696.
[28] A. Beopoulos, Z. Mrozova, F. Thevenieau, M.-T. Le Dall, I. Hapala, S. Papanikolaou, T. Chardot, J.-M.J.A. Nicaud, e. microbiology, Control of lipid accumulation in the yeast Yarrowia lipolytica, 74(24) (2008) 7779-7789.
[29] K. Mlícková, E. Roux, K. Athenstaedt, S. d'Andrea, G.n. Daum, T. Chardot, J.-M.J.A. Nicaud, e. microbiology, Lipid accumulation, lipid body formation, and acyl coenzyme A oxidases of the yeast Yarrowia lipolytica, 70(7) (2004) 3918-3924.
[30] J.L.J.B. Adrio, bioengineering, Oleaginous yeasts: promising platforms for the production of oleochemicals and biofuels, 114(9) (2017) 1915-1920.
[31] C. Landriscina, G.V. Gnoni, E. Quagliariello, Fatty-Acid Biosynthesis, European Journal of Biochemistry 29(1) (1972) 188-196.
[32] P. von Wettstein-Knowles, J.G. Olsen, K.A. McGuire, A. Henriksen, Fatty acid synthesis, The FEBS Journal 273(4) (2006) 695-710.
[33] O. Tehlivets, K. Scheuringer, S.D.J.B.e.B.A.-M. Kohlwein, C.B.o. Lipids, Fatty acid synthesis and elongation in yeast, 1771(3) (2007) 255-270.
[34] I.B. Lomakin, Y. Xiong, T.A.J.C. Steitz, The crystal structure of yeast fatty acid synthase, a cellular machine with eight active sites working together, 129(2) (2007) 319-332.
[35] C.D. Rutter, S. Zhang, C.V.J.A.m. Rao, biotechnology, Engineering Yarrowia lipolytica for production of medium-chain fatty acids, 99(17) (2015) 7359-7368.
[36] T. Czabany, K. Athenstaedt, G.J.B.e.B.A.-M. Daum, C.B.o. Lipids, Synthesis, storage and degradation of neutral lipids in yeast, 1771(3) (2007) 299-309.
[37] C.F. Kurat, K. Natter, J. Petschnigg, H. Wolinski, K. Scheuringer, H. Scholz, R. Zimmermann, R. Leber, R. Zechner, S.D.J.J.o.B.C. Kohlwein, Obese yeast: triglyceride lipolysis is functionally conserved from mammals to yeast, 281(1) (2006) 491-500.
[38] H. Wang, M.-T. Le Dall, Y. Waché, C. Laroche, J.-M. Belin, J.-M.J.C.b. Nicaud, biophysics, Cloning, sequencing, and characterization of five genes coding for acyl-CoA oxidase isozymes in the yeast Yarrowia lipolytica, 31(2) (1999) 165-174.
[39] R. Wang, C.J.A.o.b. Thorpe, biophysics, The reductive half-reaction in acyl-CoA oxidase from Candida tropicalis: Interaction with acyl-CoA analogues and an unusual thioesterase activity, 286(2) (1991) 504-510.
[40] M. Gatter, A. Forster, K. Bar, M. Winter, C. Otto, P. Petzsch, M. Jezkova, K. Bahr, M. Pfeiffer, F. Matthaus, G. Barth, A newly identified fatty alcohol oxidase gene is mainly responsible for the oxidation of long-chain omega-hydroxy fatty acids in Yarrowia lipolytica, FEMS Yeast Res 14(6) (2014) 858-72.
[41] B. Dujon, D. Sherman, G. Fischer, P. Durrens, S. Casaregola, I. Lafontaine, J. de Montigny, C. Marck, C. Neuvéglise, E. Talla, N. Goffard, L. Frangeul, M. Aigle, V. Anthouard, A. Babour, V. Barbe, S. Barnay, S. Blanchin, J.-M. Beckerich, E. Beyne, C. Bleykasten, A. Boisramé, J. Boyer, L. Cattolico, F. Confanioleri, A. de Daruvar, L. Despons, E. Fabre, C. Fairhead, H. Ferry-Dumazet, A. Groppi, F. Hantraye, C. Hennequin, N. Jauniaux, P. Joyet, R. Kachouri, A. Kerrest, R. Koszul, M. Lemaire, I. Lesur, L. Ma, H. Muller, J.-M. Nicaud, M. Nikolski, S. Oztas, O. Ozier-Kalogeropoulos, S. Pellenz, S. Potier, G.-F. Richard, M.-L. Straub, A. Suleau, D. Swennen, F. Tekaia, M. Wésolowski-Louvel, E. Westhof, B. Wirth, M. Zeniou-Meyer, I. Zivanovic, M. Bolotin-Fukuhara, A. Thierry, C. Bouchier, B. Caudron, C. Scarpelli, C. Gaillardin, J. Weissenbach, P. Wincker, J.-L. Souciet, Genome evolution in yeasts, Nature 430(6995) (2004) 35-44.
[42] J. Wang, B. Zhang, S. Chen, Oleaginous yeast Yarrowia lipolytica mutants with a disrupted fatty acyl-CoA synthetase gene accumulate saturated fatty acid, Process Biochemistry 46(7) (2011) 1436-1441.
[43] K. Mlickova, E. Roux, K. Athenstaedt, S. d'Andrea, G. Daum, T. Chardot, J.M. Nicaud, Lipid accumulation, lipid body formation, and acyl coenzyme A oxidases of the yeast Yarrowia lipolytica, Appl Environ Microbiol 70(7) (2004) 3918-24.
[44] S. Picataggio, T. Rohrer, K. Deanda, D. Lanning, R. Reynolds, J. Mielenz, L.D. Eirich, Metabolic Engineering of Candida Tropicalis for the Production of Long–Chain Dicarboxylic Acids, Bio/Technology 10(8) (1992) 894-898.
[45] M.S. Smit, M.M. Mokgoro, E. Setati, J.M. Nicaud, alpha,omega-Dicarboxylic acid accumulation by acyl-CoA oxidase deficient mutants of Yarrowia lipolytica, Biotechnol Lett 27(12) (2005) 859-64.
[46] H.J. Wang, M.-T. Le Dall, Y. Waché, C. Laroche, J.-M. Belin, C. Gaillardin, J.-M.J.J.o.b. Nicaud, Evaluation of acyl coenzyme A oxidase (Aox) isozyme function in the n-alkane-assimilating yeast Yarrowia lipolytica, 181(17) (1999) 5140-5148.
[47] M. Gatter, A. Förster, K. Bär, M. Winter, C. Otto, P. Petzsch, M. Ježková, K. Bahr, M. Pfeiffer, F.J.F.y.r. Matthäus, A newly identified fatty alcohol oxidase gene is mainly responsible for the oxidation of long-chain ω-hydroxy fatty acids in Yarrowia lipolytica, 14(6) (2014) 858-872.
[48] U. Scheller, T. Zimmer, D.r. Becher, F. Schauer, W.-H.J.J.o.B.C. Schunck, Oxygenation cascade in conversion of n-alkanes to α, ω-dioic acids catalyzed by cytochrome P450 52A3, 273(49) (1998) 32528-32534.
[49] G.J. Garssen, J.G. Vliegenthart, J.J.B.J. Boldingh, An anaerobic reaction between lipoxygenase, linoleic acid and its hydroperoxides, 122(3) (1971) 327-332.
[50] J.J. Chen, B.P.J.F.R.B. Yu, Medicine, Alterations in mitochondrial membrane fluidity by lipid peroxidation products, 17(5) (1994) 411-418.
[51] K. Hirakawa, S. Kobayashi, T. Inoue, S. Endoh-Yamagami, R. Fukuda, A.J.J.o.B.C. Ohta, Yas3p, an Opi1 family transcription factor, regulates cytochrome P450 expression in response to n-alkanes in Yarrowia lipolytica, 284(11) (2009) 7126-7137.
[52] H. Takai, R. Iwama, S. Kobayashi, H. Horiuchi, R. Fukuda, A.J.F.g. Ohta, biology, Construction and characterization of a Yarrowia lipolytica mutant lacking genes encoding cytochromes P450 subfamily 52, 49(1) (2012) 58-64.
[53] T. Iida, T. Sumita, A. Ohta, M.J.Y. Takagi, The cytochrome P450ALK multigene family of an n‐alkane‐assimilating yeast, Yarrowia lipolytica: cloning and characterization of genes coding for new CYP52 family members, 16(12) (2000) 1077-1087.
[54] B. Cornils, P.J.U.s.E.o.I.C. Lappe, Dicarboxylic acids, aliphatic, (2000).
[55] S. Wang, P. Qin, X. Fang, Z. Zhang, S. Wang, X.J.S.E. Liu, A novel sebacic acid/expanded graphite composite phase change material for solar thermal medium-temperature applications, 99 (2014) 283-290.
[56] J. Kim, K.-W. Lee, T.E. Hefferan, B.L. Currier, M.J. Yaszemski, L.J.B. Lu, Synthesis and evaluation of novel biodegradable hydrogels based on poly (ethylene glycol) and sebacic acid as tissue engineering scaffolds, 9(1) (2008) 149-157.
[57] D.G. Gibson, L. Young, R.Y. Chuang, J.C. Venter, C.A. Hutchison, 3rd, H.O. Smith, Enzymatic assembly of DNA molecules up to several hundred kilobases, Nat Methods 6(5) (2009) 343-5.
[58] W.J. Dower, J.F. Miller, C.W. Ragsdale, High efficiency transformation of E. coli by high voltage electroporation, Nucleic Acids Res 16(13) (1988) 6127-6145.
[59] N.M. Calvin, P.C. Hanawalt, High-efficiency transformation of bacterial cells by electroporation, Journal of Bacteriology 170(6) (1988) 2796-2801.
[60] A. Kretzschmar, C. Otto, M. Holz, S. Werner, L. Hubner, G. Barth, Increased homologous integration frequency in Yarrowia lipolytica strains defective in non-homologous end-joining, Curr Genet 59(1-2) (2013) 63-72.
[61] J.W. Szostak, T.L. Orr-Weaver, R.J. Rothstein, F.W. Stahl, The double-strand-break repair model for recombination, Cell 33(1) (1983) 25-35.
[62] T.L. Orr-Weaver, J.W. Szostak, Yeast recombination: the association between double-strand gap repair and crossing-over, Proc Natl Acad Sci U S A 80(14) (1983) 4417-21.
[63] D.C. Chen, J.M. Beckerich, C. Gaillardin, One-step transformation of the dimorphic yeast Yarrowia lipolytica, Applied Microbiology and Biotechnology 48(2) (1997) 232-235.
[64] L. Han, Y. Peng, Y. Zhang, W. Chen, Y. Lin, Q. Wang, Designing and Creating a Synthetic Omega Oxidation Pathway in Saccharomyces cerevisiae Enables Production of Medium-Chain alpha, omega-Dicarboxylic Acids, Front Microbiol 8 (2017) 2184.
[65] H.T. Thanh, L. Beney, H. Simonin, T.X.S. Nguyen, P. Gervais, J.-M. Belin, F.J.B.e.B.A.-B. Husson, Toxicity of fatty acid hydroperoxides towards Yarrowia lipolytica: implication of their membrane fluidizing action, 1768(9) (2007) 2256-2262.

全文公開日期 2031/10/12 (校內網路)
全文公開日期本全文未授權公開 (校外網路)
全文公開日期本全文未授權公開 (國家圖書館：臺灣博碩士論文系統)

簡易檢索 / 詳目顯示

相關論文