臺灣博碩士論文加值系統

聚羥基烷酸(polyhydroxyalkanates, PHAs)是微生物中在營養不平衡的條件下，將多餘的碳源轉換成脂質堆積於胞內之聚合物。巨大芽孢桿菌(Bacillus megaterium)具有堆積polyhydroxybutyrate(PHB)的能力，其合成PHAs之主要基因包括phaPQRBC，其中phaC在其合成機制中扮演聚合反應的角色。本研究目的在於選殖B. megaterium合成PHAs的這些相關基因，並將其於大腸桿菌DH5α內表現。實驗並以添加4% 葡萄糖、4% 琥珀酸鹽以及1% 甘油作為碳源，以探討其生成之產物的產率及物理性質。研究結果發現，B. megaterium於LB培養液中添加1% 甘油生長情況較佳，產物則為PHB。而在重組大腸桿菌方面，菌株EYT09(DH5α / pBluescript II SK (+)::phaRBC)可成功的表現經起始密碼子改變phaC之PhaC蛋白質(39.82 KDa)。另外在LB培養液添加1% 甘油培養下，重組大腸桿菌EYT09及EYT11 (DH5α / pBluescript II SK (+)::phaC)在氣相層析儀分析結果中，可發現於6.3分鐘中有一吸收鋒面的存在，產率分別為3.67%及11.50%；然而進一步分析發現此產物並非與PHAs相關之產物。雖然如此，本實驗仍然成功的在大腸桿菌內表現B. megaterium之PhaB與PhaC蛋白質。

Polyhydroxyalkanoates (PHA) are polyesters accumulated by various bacteria under unbalanced growth conditions when the carbon substrate is in excess to other nutrients such as nitrogen, sulfur, phosphorus or oxygen. B. megaterium, the first microorganism that has been discovered for the ability to accumulate intracellular polyhydroxybutyrate (PHB), was demonstrated to be able to accumulate polyhydroxyvalerate (PHV) when supply the bacterium with certain carbon source (e.g. sodium succinate). Biosynthesis of PHB in B. megaterium involves genes including phaPQRBC. The ability and mechanisms (and genes involved) in PHV synthesis in this bacterium have not been described before.
In this study, we have examined the production of PHB and PHV by B. megaterium using different carbon sources. Production of PHB in recombinant Escherichia coli harboring the B. megaterium PHB synthesis genes (phaP, phaQ, phaR, phaB and phaC) was also evaluated. Here we attempt to clone these genes into suitable E. coli host in order to (1) construct a recombinant strain of E. coli using PHB synthesis genes of B. megaterium to produce PHB; (2) examine if PHV (or other PHA) can be
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produced in the recombinant Escherichia coli when supplemented with different carbon sources.
The results of thesis shown that B. megaterium was able to produced PHB when grown with a variety of carbon sources including glycerol. Expression of PhaB and PhaC protein can be detected in the recombinant Escherichia coli ETY09 (DH5α / pBluescript II SK (+)::phaRBC) and EYT11 (DH5α / pBluescript II SK (+)::phaC). However, there was no detectable polyhydroxyalkanoates in these recombinant strains by Gas chromatography and FTIR. Some other gene(s) of B. megaterium might be required in order to produce PHB in the recombinant E. coli and this remained to be investigated.

中文摘要 ..................................................................................................... I
Abstract ...................................................................................................... II
致謝 .......................................................................................................... IV
目錄 ............................................................................................................ V
圖索引 ...................................................................................................... IX
表索引 ...................................................................................................... XI
第一章 前言............................................................................................. 1
1.1 簡介.............................................................................................. 1
1.2 研究動機 ..................................................................................... 2
1.3 實驗菌株介紹 ............................................................................. 3
1.3.1 簡介巨大芽孢桿菌 .......................................................... 3
1.3.2 簡介羅爾斯頓氏菌 .......................................................... 3
1.4 PHAs的性質 ................................................................................ 4
1.5 PHAs的構造 ................................................................................ 4
1.6 PHAs的代謝機制 ........................................................................ 5
1.6.1 PHAs的合成路徑 ............................................................. 5
1.6.2 代謝途徑I:醣類降解作用 ............................................... 5
1.6.3 代謝途徑II:脂肪酸氧化作用 ......................................... 6
1.6.4代謝途徑III:脂肪酸合成作用 ......................................... 7
1.7 PHAs的主要生合成基因 ............................................................ 8
1.8 PHAs的應用與生產 .................................................................. 11
VI
1.9本研究架構及流程 .................................................................... 12
第二章 材料與方法 ................................................................................ 13
2.1 實驗材料 ................................................................................... 13
2.1.1 菌株及表現質體 ............................................................ 13
2.1.2 引子 ................................................................................ 15
2.1.3 培養基 ............................................................................ 16
2.1.4 藥品及酵素 .................................................................... 16
2.2 實驗方法 ................................................................................... 16
2.2.1 菌種保存與前培養 ........................................................ 16
2.2.2 菌體生長情況測定 ........................................................ 17
2.2.3 染色體DNA之純化 ...................................................... 17
2.2.4 聚合酶鏈鎖反應 ............................................................ 18
2.2.5 重組質體之篩選 ............................................................ 19
2.2.6 質體DNA之純化 .......................................................... 19
2.2.7 洋菜膠體DNA萃取 ...................................................... 20
2.2.8 限制酶切割 .................................................................... 21
2.2.9 DNA接合作用 ................................................................ 21
2.2.10 大腸桿菌勝任細胞製備 .............................................. 22
2.2.11 轉型作用 ....................................................................... 22
2.2.12 菌株鑑定 ...................................................................... 23
2.2.13 蛋白質電泳分析 .......................................................... 23
2.2.14 重組大腸桿菌培養 ...................................................... 25
2.2.15 PHAs之萃取純化 ......................................................... 26
2.2.16 PHAs性質鑑定方法 ..................................................... 27
VII
2.2.17 穿透式電子顯微鏡 ...................................................... 30
2.2.18 PHAs含量計算及檢量線 ............................................. 32
第三章 結果與討論 ................................................................................ 33
3.1 探討B.megaterium不同培養條件之生長測試 ....................... 33
3.2 PHAs組成分析 .......................................................................... 34
3.2.1 PHAs標準品定性分析 ................................................... 34
3.2.2 標準品定量分析 ............................................................ 35
3.3 B. megaterium體內所累積的PHAs定性定量分析 ................ 37
3.4 B.megaterium之PHAs合成基因phaPQRBC選殖 ................ 38
3.4.1 B. megaterium之染色體純化 ......................................... 38
3.4.2 聚合酶鏈鎖反應引子設計 ............................................ 39
3.4.3 重組大腸桿菌之質體確認 ............................................ 44
3.4.4 探討添加誘導劑(IPTG)重組大腸桿菌之生長曲線 .... 45
3.4.5 重組大腸桿菌之蛋白質表現 ........................................ 46
3.4.6 重組大腸桿菌之定性定量分析 .................................... 48
3.4.7 基因組分析 .................................................................... 48
3.5 聚合酶鏈鎖反應定點突變引子設計 ....................................... 50
3.5.1 選殖B. megaterium PHAs合成基因起始密碼子定點突變片段於pBluescript SK (+)載體 ........................................... 54
3.5.2 重組大腸桿菌EYT09之質體確認 .............................. 56
3.5.3重組大腸桿菌EYT09之蛋白質表現 ........................... 56
3.5.4 重組大腸桿菌EYT09之PHAs組成分析 ................... 58
3.5.5 重組大腸桿菌EYT11之物理性質分析 ...................... 62
3.6 選殖R. eutropha BCRC13036 之PHAs合成基因片段phaAB
VIII
........................................................................................................... 65
3.6.1 選殖phaCBM基因片段與phaABRE基因片段於pBluescript SK (+)載體 ............................................................ 66
3.6.2 重組大腸桿菌EYT12之質體確認 .............................. 67
3.6.3 重組大腸桿菌EYT12之蛋白質表現 .......................... 68
3.6.4 重組大腸桿菌EYT12之定性定量分析 ...................... 68
第四章 結論及未來展望 ........................................................................ 70
第五章 參考文獻 .................................................................................... 73
第六章 附錄............................................................................................. 78
6.1 B. megaterium 合成基因序列 ................................................... 78
6.2 實驗藥品 ................................................................................... 80
6.3實驗儀器 .................................................................................... 82
6.4 尼羅紅染色試劑 ....................................................................... 82
6.5 酵素及抗生素 ........................................................................... 83
6.6 DNA 電泳試劑 .......................................................................... 83
6.7 蛋白質電泳分析之試劑 ........................................................... 84
6.8 染色體DNA及質體DNA純化試劑 ...................................... 85
6.9 重組質體篩選之試劑 ............................................................... 85
6.10 TEM前處理使用試劑 ............................................................. 85
6.11 EYT09之基因序列鑑定結果 .................................................. 86
6.12 EYT12之基因序列鑑定結果.................................................. 87
6.13 phaCRE及phaCBM之比對 ........................................................ 88

Brandl, H., Gross, R. A., W, L. R. & C, F. R. (1998). Pseudomonas oleovorans as a source of poly(_-hydroxyalkanoates) for potential applications as biodegradable polyesters. Appl Environ Microbiol 54, 1977-1982.
Chinwetkitvanich, S., Randall, C. W. & Panswad, T. (2004). Effects of phosphorus limitation and temperature on PHA production in activated sludge. Water Sci Technol 50, 135-143.
Elbanna, K., Lutke-Eversloh, T., Jendrossek, D., Luftmann, H. & Steinbuchel, A. (2004). Studies on the biodegradability of polythioester copolymers and homopolymers by polyhydroxyalkanoate (PHA)-degrading bacteria and PHA depolymerases. Arch Microbiol 182, 212-225.
Fukui, T., Shiomi, N. & Doi, Y. (1998). Expression and characterization of (R)-specific enoyl coenzyme A hydratase involved in polyhydroxyalkanoate biosynthesis by Aeromonas caviae. J Bacteriol 180, 667-673.
Green, P. R., Kemper, J., Schechtman, L., Guo, L., Satkowski, M., Fiedler, S., Steinbuchel, A. & Rehm, B. H. (2002). Formation of short chain length/medium chain length polyhydroxyalkanoate copolymers by fatty acid beta-oxidation inhibited Ralstonia eutropha. Biomacromolecules 3, 208-213.
Hoffmann, N., Steinbuchel, A. & Rehm, B. H. (2000). Homologous functional expression of cryptic phaG from Pseudomonas oleovorans establishes the transacylase-mediated polyhydroxyalkanoate biosynthetic pathway. Appl Microbiol Biotechnol 54, 665-670.
Jan, S., Roblot, C., Goethals, G., Courtois, J., Courtois, B., Saucedo,
74
J.E., et al. (1995). Study of parameters affecting poly(3-hydroxybutyrate) quantification by gas chromatography. Anal Biochem 225, 258-263.
Kichise, T., Taguchi, S. & Doi, Y. (2002). Enhanced accumulation and changed monomer composition in polyhydroxyalkanoate (PHA) copolyester by in vitro evolution of Aeromonas caviae PHA synthase. Appl Environ Microbiol 68, 2411-2419.
Lee, T. R., Lin, J. S., Wang, S. S. & Shaw, G. C. (2004). PhaQ, a new class of poly-beta-hydroxybutyrate (phb)-responsive repressor, regulates phaQ and phaP (phasin) expression in Bacillus megaterium through interaction with PHB. J Bacteriol 186, 3015-3021.
Lemos, P. C., Serafim, L. S., Santos, M. M., Reis, M. A. M. & Santos, H. (2003). Metabolic pathway for propionate utilization by phosphorus-accumulating organisms in activated sludge: C-13 labeling and in vivo nuclear magnetic resonance. APPLIED AND ENVIRONMENTAL MICROBIOLOGY 69, 241-251.
Lu, Q., Han, J., Zhou, L., Zhou, J. & Xiang, H. (2008). Genetic and biochemical characterization of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthase in Haloferax mediterranei. J Bacteriol 190, 4173-4180.
Lu, X., Zhang, W., Jian, J., Wu, Q. & Chen, G. Q. (2005). Molecular cloning and functional analysis of two polyhydroxyalkanoate synthases from two strains of Aeromonas hydrophila spp. FEMS Microbiol Lett 243, 149-155.
Luengo, J. M., Garcia, B., Sandoval, A., Naharro, G. & Olivera, E. R. (2003). Bioplastics from microorganisms. Curr Opin Microbiol 6, 251-260.
Lutke-Eversloh, T., Bergander, K., Luftmann, H. & Steinbuchel, A.
75
(2001). Identification of a new class of biopolymer: bacterial synthesis of a sulfur-containing polymer with thioester linkages. Microbiology 147, 11-19.
Madison, L. L. & Huisman, G. W. (1999). Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol Mol Biol Rev 63, 21-53.
Maehara, A., Taguchi, S., Nishiyama, T., Yamane, T. & Doi, Y. (2002). A repressor protein, PhaR, regulates polyhydroxyalkanoate (PHA) synthesis via its direct interaction with PHA. J Bacteriol 184, 3992-4002.
McCool, G. J. & Cannon, M. C. (1999). Polyhydroxyalkanoate inclusion body-associated proteins and coding region in Bacillus megaterium. J Bacteriol 181, 585-592.
McCool, G. J. & Cannon, M. C. (2001). PhaC and PhaR are required for polyhydroxyalkanoic acid synthase activity in Bacillus megaterium. J Bacteriol 183, 4235-4243.
Ojumu, T. V., AttahDaniel, B. E., Betiku, E. & Solomon, B. O. (2003). Auto-hydrolysis of lignocellulosics under extremely low sulphuric acid and high temperature conditions in batch reactor. Biotechnology and Bioprocess Engineering 8, 291-293.
Park, S. J. & Lee, S. Y. (2004). New fadB homologous enzymes and their use in enhanced biosynthesis of MediumChain-Length polyhydroxyalkanoates in fadB mutant Escherichia coli. Biotechnology and Bioengineering 86, 681-686.
Rehm, B. H. A. & Steinbuchel, A. (1999). Biochemical and genetic analysis of PHA synthases and other proteins required for PHA synthesis. International Journal of Biological Macromolecules 25, 3-19.
76
Rehm, B. H. A. (2003). Polyester synthases: natural catalysts for plastics. Biochemical Journal 376, 15-33.
Sabirova, J. S., Ferrer, M., Lunsdorf, H., Wray, V., Kalscheuer, R., Steinbuchel, A., Timmis, K. N. & Golyshin, P. N. (2006). Mutation in a "tesB-like" hydroxyacyl-coenzyme A-specific thioesterase gene causes hyperproduction of extracellular polyhydroxyalkanoates by Alcanivorax borkumensis SK2. J Bacteriol 188, 8452-8459.
Sang, Y. L. (1996b). Plastic bacteria ? Progress and prospects for polyhydroxyalkanoate production in bacteria. . Trends Biotechnol 14, 431-438.
Sato, S., Nomura, C. T., Abe, H., Doi, Y. & Tsuge, T. (2007). Poly [(R)-3-hydroxybutyrate] formation in Escherichia coli from glucose through an enoyl-CoA hydratase-mediated pathway. Journal of Bioscience and Bioengineering 103, 38-44.
Sudesh, K., Abe, H., and Doi, Y. (2000). Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. Prog Polym Sci 25, 1503-1555.
Takase, K., Matsumoto, K., Taguchi, S. & Doi, Y. (2004). Alteration of substrate chain-length specificity of type II synthase for polyhydroxyalkanoate biosynthesis by in vitro evolution: in vivo and in vitro enzyme assays. Biomacromolecules 5, 480-485.
Takeda, M., Kitashima, K., Adachi, K., Hanaoka, Y., Suzuki, I. & Koizumi, J. (2000). Cloning and expression of the gene encoding thermostable poly(3-hydroxybutyrate) depolymerase. J Biosci Bioeng 90, 416-421.
Tsuge, T., Fukui, T., Matsusaki, H., Taguchi, S., Kobayashi, G., Ishizaki, A. & Doi, Y. (2000). Molecular cloning of two (R)-specific enoyl-CoA hydratase genes from Pseudomonas
77
aeruginosa and their use for polyhydroxyalkanoate synthesis. FEMS Microbiol Lett 184, 193-198.
Tsuge, T., Yano, K., Imazu, S., Numata, K., Kikkawa, Y., Abe, H., Taguchi, S. & Doi, Y. (2004). Biosynthesis of polyhydroxyalkanoate (PHA) copolymer from fructose using wild-type and laboratory-evolved PHA synthases. Macromolecular Bioscience 5, 112-117.
Valappil, S. P., Boccaccini, A. R., Bucke, C. & Roy, I. (2007). Polyhydroxyalkanoates in Gram-positive bacteria: insights from the genera Bacillus and Streptomyces. Antonie Van Leeuwenhoek 91, 1-17.
Wang, C., Sheng, X., Equi, R. C., Trainer, M. A., Charles, T. C. & Sobral, B. W. (2007). Influence of the poly-3-hydroxybutyrate (PHB) granule-associated proteins (PhaP1 and PhaP2) on PHB accumulation and symbiotic nitrogen fixation in Sinorhizobium meliloti Rm1021. J Bacteriol 189, 9050-9056.
Weiner, R. M. (1997). Biopolymers from marine prokaryotes. Trends in Biotechnology 15, 390-394.
唐世珍 (2007). 利用微生物生產不同種類聚羥基烷酸. 元智大學.
張先明 (2008). 以本土根瘤菌降解酚生產聚羥基烷酸與其動力學探討. 元智大學.
莊育泉 (2005). 聚羥基烷酸生產菌株的快速檢測、分離與鑑定. 元智大學.