Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23834
標題: 選殖與同源表現小球藻Chlorella sp. DT產氫酶
Cloning and homologous expression of hydrogenase in Chlorella sp. DT
作者: 郭婷婷
Kuo, Ting-Ting
關鍵字: Chlorella
產氫
hydrogenase
hydrogen
產氫酶
出版社: 生命科學系所
引用: Abendroth, G.V., Stripp, S., Silakov, A., Croux, C., Soucaille, P., Girbal, L., and Happe, T. (2008). Optimized over-expression of [FeFe] hydrogenase with high specific activity in Clostridium acetobutylicum. Int. J. Hydrogen Energy 33: 6076-6081. Antal, T.K., Krendeleva, T.E., Laurinavichene, T.V., Makarova, V.V., Ghirardi, M.L., Rubin, A.B., Tsygankov, A.A., and Seibert, M. (2003). The dependence of algal H2 production on photosystem II and O2 consumption activities in sulfur-deprived Chlamydomonas reinhardtii cells. Biochim. Biophys. Acta 1607: 153-160. Asada, Y., Koik, Y., Schnackenberg, J., Miyake, M., Uemur, I., and Miyak, J. (2000). Heterologous expression of clostridial hydrogenase in the cyanobacterium Synechococcus PCC7942. Biochim. Biophys. Acta 1490: 269-278. Balk, J., Pierik, A.J., Netz, D.J.A., Muhlenhoff, U., and Lill, R. (2004). The hydrogenase-like Nar1P is essential for maturation of cytosolic and nuclear iron-sulphur proteins. EMBO J. 23: 2105-2115. Balk, J., Pierik, A.J., Netz, D.J.A., Muhlenhoff, U., and Lill, R. (2005). Nar1P, a conserved eukaryotic protein with similarity to Fe-only hydrogenases, functions in cytosolic iron-sulphur protein biogenesis. Biochem. Society 33: 86-89. Bricker, T.M., and Frankel, L.K. (2008). The psbo1 Mutant of Arabidopsis Cannot Efficiently Use Calcium in Support of Oxygen Evolution by Photosystem II. J. Biol. Chem. 283: 29022-29027. Cammack, R. (1999). Hydrogenase sophistication. Nature 397: 214-215. Chader, S., Hacene, H., and Agathos, S.N. (2009). Study of hydrogen production by three strains of Chlorella isolated from the soil in the Algerian Sahara. Int. J. Hydrogen Energy 34: 4941-4946. Chen, H.P. (2007). Comparative analysis of energy-conserving and energy-dissipating system in bamboo mitochondria from Bambusa oldhamii and Phyllostachys edulis. Master thesis of the Department of Life Science, National Chung-Hsing University, Taiwan. Chen, M.W. (2005). Functional expression of mercuric reductase in microalga Chlorella sp. DT. Master thesis of the Department of Life Science, National Chung-Hsing University, Taiwan. Chen, P.C., and Lai, C.L. (1996). Physiological adaptation during cell dehydration and rewetting of a newly-isolated Chlorella species. Physiol. Plant 96: 453-457. De La Rivas, J., and Roman, A. (2005). Structure and evolution of the extrinsic proteins that stabilize the oxygen-evolving engine. Photochem. Photobiol. Sci. 4: 1003-1010. Enami, I., Okumura, A., Nagao, R., Suzuki, T., Iwai, M., and Shen, J.R. (2008). Structures and functions of the extrinsic proteins of photosystem II from different species. Photosynth. Res. 98: 349-363. Florin, L., Tsokoglou, A., and Happe, T. (2001). A novel type of iron hydrogenase in the green alga Scenedesmus obliquus is linked to the photosynthetic electron transport chain. J. Biol. Chem. 276: 6125-6132. Florin, L., Tsokoglou, A., and Happe, T. (2001). A Novel type of iron hydrogenase in the green alga Scenedesmus obliquus is linked to the photosynthetic electron transport chain. J. Biol. Chem. 276: 6125-6132. Forestier, M., King, P., Zhang, L.P., Posewitz, M., Schwarzer, S., Happe, T., Ghirardi, M.L., and Seibert, M. (2003). Expression of two [Fe]-hydrogenases in Chlamydomonas reinhardtii under anaerobic conditions. Eur. J. Biochem. 270: 2750-2758. Gaffron, H. (1940). The oxyhydrogen reaction in green algae and the reduction of carbon dioxide in the dark. Science 91: 529-530. Gaffron, H. (1940). The oxyhydrogen reaction in green algae and the reduction of carbon dioxide in the dark. Science 91: 529-530 Gaffron, H., and Rubin, J. (1942). Fermentative and photochemical production of hydrogen in algae. J. Gen. Physiol. 20: 219-240 Ghirardi, M. L., Posewitz, M.C., Maness, P.C., Dubini, A., Yu, J., and Seibert, M. (2007). Hydrogenases and hydrogen photoproduction in oxygenic photosynthetic organisms. Annu. Rev. Plant Biol. 58: 71-91. Girbal, L., Abendroth, G.V., Winkler, M., Benton, P.M., Meynial-Salles, I., Croux, C., Peters, J.W., Happe, T., and Soucaille, P. (2005). Homologous and Heterologous Overexpression in Clostridium acetobutylicum and Characterization of Pureified Clostridial and Algal Fe-Only Hydrogenase with High Specific Activities. Appl. Environ. Microbiol. 71: 2777-2781. Godman, J.E., Molnar, A., Baulcombe, D.C., and Balk, J. (2010). RNA silencing of hydrogenase(-like) genes and investigation of their physiological roles in the green alga Chlamydomonas reinhardtii. Biochem. J. 431: 345-351. Happe, T., and Kaminsk, A. (2002). Differential regulation of the Fe-hydrogenase during anaerobic adaptation in the green alga Chlamydomonas reinhardtii. Eur. J. Biochem. 269: 1022-1032. Happe, T., and Kaminski, A. (2002). Differential regulation of the Fe-hydrogenase during anaerobic adaptation in the green alga Chlamydomonas reinhardtii. Eur. J. Biochem. 269: 1022-1032. Happe, T., and Naber, J.D. (1993). Isolation, characterization and N-terminal amino acid sequence of hydrogenase from the green alga Chlamydomonas reinhardtii. Eur. J. Biochem. 214: 475-481. Happe, T., Hemschemeier, A., Winkler, M., and Kaminski, A. (2002). Hydrogenases in green algae: do they save the algae's life and solve our energy problems? Trends Plant Science 7: 246-250. Happe, T., Mosler, B., and Naber, J.D. (1994). Induction, localization and metal content of hydrogenase in the green alga Chlamydomonas reinhardtii. Eur. J. Biochem. 222: 769-774. Hemschemeier, A., Fouchard, S., Cournac, L., Peltier, G., and Happe, T. (2008). Hydrogen production by Chlamydomonas reinhardtii: elaborate interplay of electron source and sink. Planta 229: 397-407. Huang, C.C., Chen, M.W., Hsieh, J.L., Lin, W.H., Chen, P.C., and Chien, L.F. (2006). Expression of mercuric reductase from Bacillus megaterium MB1 in eukaryotic microalga Chlorella sp. DT: an approach for mercury phytoremediation. Appl. Microbiol. Biotechnol. 72: 197-205. Janneke, B., Antonio, J.P., Daili, J.A.N., Ulrich, M., and Roland, L. (2004). The hydrogenase-like Nar1p is essential for maturation of cytosolic and nuclear iron-sulphur proteins. EMBO J. 23: 2105-2115. Kim, J.P., Kang, C.D., Park, T.H., Kim, M.S., and Sim, S.J. (2006). Enhanced hydrogen production by controlling light intensity in sulfur-deprived Chlamydomonas reinhardtii culture. Int. J. Hydrogen Energy 31: 1585-1590. Kosourov, S., Seibert, M., and Ghirardi, M.L. (2003). Effects of Extracellular pH on the Metabolic Pathways in Sulfur-Deprived, H2-Producing Chlamydomonas reinhardtii Cultures. Plant Cell Physiol. 44: 146-155. Laurinavichene, T., Tolstygina, I., and Tsygankov, A. (2004). The effect of light intensity on hydrogen production by sulfur-deprived Chlamydomonas reinhardtii. J. Biotech. 114: 143-151. Li, C.X., Parker, A., Menocal, E., Xiang, S.L., Borodyansky, L., and Fruehauf, J.H. (2006). Delivery of RNA Interference. Cell Cycle 5: 2103-2109. Lill, R., and Muhlenhoff, U. (2008). Maturation of Iron-Sulfur Proteins in Eukaryotes: Mechanisms, Connected Processes, and Diseases. Annu. Rev. Biochem. 77: 669-700. Lin, H.T. (2010). PsbA or PsbO suppression leads to hydrogenase induction in Chlorella sp. DT. Master thesis of the Department of Life Science, National Chung-Hsing University, Taiwan. Lin, L.P. (2005). Purchasing and collection of algal culture. In CHLORELLA- It's Ecology, Structure, Cultivation, Bioprocess and Application, 1st ed, L.P. Lin, ed (Yi Hsien Publisher Co, Ltd, Taipei, Taiwan), pp. 248-254. Lin, W.H. (2004). The change in Chlorella photosynthesis and superoxide dismutase activity under low temperature/relavively high irradiation stress. Master thesis of the Department of Life Science, National Chung-Hsing University, Taiwan. Ma, W., Chen, M., Wang, L., Wei, L., and Wang, Q. (2011). Treatment with NaHSO3 greatly enhances photobiological H2 production in the green alga Chlamydomonas reinhardtii. Bioresour. Technol. DOI: 10.1016/j.biortech.2011.03.052. Melis, A. (2007). Photosynthetic H2 metabolism in Chlamydomonas reinhardtii (unicellurlar green algae). Planta 226: 1075-1086. Melis, A., and Happe, T. (2001). Hydrogen Production. Green Algae as a Source of Energy. Plant Physiol. 127: 740-748. Melis, A., Zhang, L.P., Forestier, M., Ghirardi, M.L., and Seibert, M. (2000). Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii. Plant Physiol. 122: 127-135. Meuser, J.E., Boyd, E.S., Ananyev, G., Karns, D., Radakovits, R., Murthy, U.M.N., Ghirardi, M.L., Dismukes, G.C., Peters, J.W., and Posewitz, M.C. (2011). Evolutionary significance of an algal gene encoding an [FeFe]-hydrogenase with F-domain homology and hydrogenase activity in Chlorella variabilis NC64A. Planta DOI: 10.1007/s00425-011-1431-y. Murakami, R., Ifuku, K., Takabayashi, A., Shikanai, T., Endo, T., and Sato, F. (2002). Characterization of an Arabidopsis thaliana mutant with impaired psbO, one of two genes encoding extrinsic 33-kDa proteins in photosystem II. FEBS Lett. 523: 138-142. Murakami, R., Ifuku, K., Takabayashi, A., Shikanai, T., Endo, T., and Sato, F. (2005). Functional dissection of two Arabidopsis PsbO proteins PsbO1 and PsbO2. FEBS J. 272: 2165-2175. Posewitz, M.C., King, P.W., Smolinski, S.L., Zhang, L., Seibert, M., and Ghirardi, M.L. (2004). Discovery of Two Novel Radical S-Adenosylmethionine Proteins Required for the Assembly of an Active [Fe] Hydrogenase. J. Bio. Chem. 279: 25711-25720. Posewitz, M.C., King, P.W., Smolinski, S.L., Smith, R.D., Ginley, A.R., Ghirardi, M.L., and Seibert, M. (2005). Identification of genes required for hydrogenase activity in Chlamydomonas reinhardtii. Biochem. Soc. Trans. 33: 102-104. Rohr, J., Sarkar, N., Balenger, S., Jeong, B.r., and Cerutti, H. (2004). Tandem inverted repeat system for selection of effective transgenic RNAi strains in Chlamydomonas. Plant J. 40: 611-621. Skjanes, K., Knutsen, G., Kӓllqvist, T., and Lindblad, P. (2008). H2 production from marine and freshwater species of green algae during sulfur deprivation and considerations for bioreactor design. Int. J. Hydrogen Energy 33: 511-521. Small, I. (2007). RNAi for revealing and engineering plant gene functions. Curr. Opin. Biol. 18: 148-153. Stephenson, M., and Stickland, L.H. (1931). Hydrogenase: a bacterial enzyme activating molecular hydrogen. Biochem. J. 25: 205-214. Stirnberg, M., and HAppe, T. (2004). Identification of a cis-acting element controlling anaerobic expression of the HYDA gene from Chlamydomonas reinhardtii. In Biohydrogen III, J. Miyake, Y. Igarashi and M. Rӧgner (Elsevier, UK), pp. 117-127. Stripp, S.T., Goldet, G., Brandmayr, C., Sanganas, O., Vincent, K.A., Haumann, M., Armstrong, F.A., and Happe, T. (2009). How oxygen attacks [FeFe] hydrogenase from photosynthetic organisms. PNAS 106: 17331-17336. Taiz, L., and Zeiger, E. (2006). Photosynthesis: the light reactions. In Plant Physiology, 4th ed, L. Taiz and E. Zeiger, ed (Sinauer Associates Inc., Massachusetts, USA), pp. 125-158. Torzillo, G., Scoma, A., Faraloni, C., Ena, A., and Johanningmeier, U. (2009). Increased hydrogen photoproduction by means of a sulfur-deprived Chlamydomonas reinhardtii D1 protein mutant. Int. J. Hydrogen Energy 34: 4529-4536. Tsai, H.C. (2009). Cloning and expression of mitochondrial-encoded subunits Nad1, Nad4 and Nad5 of complex I (NADH:ubiquinone oxidoreductase) from Bambusa oldhamii and Phyllostachys edulis. Master thesis of the Department of Life Science, National Chung-Hsing University, Taiwan. Tsygankova, A.A., Kosourova, S.N., Tolstyginaa, I.V., Ghirardi, M.L., and Seibert, M. (2006). Hydrogen production by sulfur-deprived Chlamydomonas reinhardtii under photoautotrophic conditions. Int. J. Hydrogen Energy 31: 1547-1584. Vignais, P.M., Billoud, B. and Meyer, J. (2001). Classification and phylogeny of hydrogenase. FEMS Microbiol. Rev. 54: 455-501. Wang, H., Fan, X., Zhang, Y., Yang, D., and Guo, R. (2011). Sustained photo-hydrogen production by Chlorella pyrenoidosa without sulfur depletion. Biotechnol Lett. DOI: 10.1007/s10529-011-0584-x. Winkler, M., Heil, B., Heli, B., and Happe, T. (2002). Isolation and molecular characterization of the [Fe]-hydrogenase from the unicellular green alga Chlorella fusca. Biochim. Biophys. Acta 1576: 330-334. Winkler, M., Kuhlgert, S., Hippler, M., and Happe, T. (2009). Characterization of the Key Step for Light-driven Hydrogen Evolution in Green Algae. J. Biol. Chem. 284: 36620-36627. Wu, S.X., Yan, G.Y., Xu, L.L., Wang, Q.X., and Liu, X.L. (2010). Improvement of hydrogen production with expression of lba gene in chloroplast of Chlamydomonas reinhardtii. Int. J. Hydrogen Energy 35: 13419-13426. Wykoff, D.D., Davies, J.P., Melis, A., and Grossman, A.R. (1998). The regulation of photosynthetic electron transport during nutrient deprivation in Chlamydomonas reinhardtii. Plant Physol. 117: 129-139. Yi, X.P., Hargett, S.R., Frank, L.K., and Bricke, T.M. (2008). The effects of simultaneous RNAi suppression of PsbO and PsbP protein expression in photosystem II of Arabidopsis. Photosynth. Res. 98: 439-448. Yi, X.P., McChargue, M., Laborde, S., Frankel, L.K., and Bricke, T.M. (2005). The manganese-stabilizing protein is required for photosystem II assembly/stability and photoautotrophy in higher plants. J. Biol. Chem. 280: 16170-16174.
摘要: 綠藻具有與植物相似的光合作用系統能將太陽光轉換成電子。在無氧情況下,產氫酶(HydA)會接收由硫鐵蛋白(ferredoxin)而來的電子產生氫氣。產氫酶的基因(hydA)位於綠藻的核染色體,基因的轉錄以及酵素活性都會被氧氣所抑制。 小球藻Chlorella sp. DT(DT),在無氧或是缺硫情況下能夠產生氫氣。在這個研究中,我們希望在小球藻DT中同源過度表現產氫酶,使其能在一般情況下產生氫氣。 我們將小球藻DT hydA基因的genomic DNA和cDNA的核甘酸序列定序之後,經比對後發現,hydA基因在小球藻DT和其他綠藻之間具有高度保留性。接著將DT hydA基因編碼區域(coding region)構築至載體pHm3A-hydA、pHyg3-hydA和pRpsbOa-hydA中,利用已廣泛使用於植物的啟動子來驅動hydA 基因,並將這些載體轉殖於DT。利用聚合酶連鎖反應(PCR),在轉殖株中偵測到homologous DT hydA 片段,得知載體已成功地被轉殖於DT。再者,在這些轉殖株可以偵測到hydA基因mRNA的轉錄。以西方墨點法偵測,也觀察到HydA蛋白的表現。這些證據顯示,在有氧且含硫的情況下,hydA 基因在這些轉殖株中能夠被表現。若在無氧且含硫的情況下,轉殖株的氫氣產量是野生株的7到10倍左右。綜合以上結果,產氫酶已成功地被同源表現在DT轉殖株中,並且還增加了氫氣的產量。
Green algae have a photosynthetic system similar to plants but can produce hydrogen by using sun light under anaerobic conditions. The hydrogenase (HydA) of green algae accepts electrons directly from ferredoxin and generate hydrogen. HydA is nuclear-encoded by hydA gene, and its transcription and activity are strictly inhibited by oxygen. Chlorella sp.DT (DT), a Taiwanese isolate, was able to produce hydrogen under anaerobic or sulfur-deprived (-S) conditions. In this study, we attempted to homologously overexpressed hydA to enhance hydrogen production in DT under normal condition. The nucleotide sequences of genomic DNA and cDNA of DT hydA were sequenced, and found to be highly conserved as compared with other green algae. The coding region of DT hydA was constructed into plasmids and driven by the promoters which are commonly used in plants. The DT cells were transformed with plasmids pHm3A-hydA, pHyg3-hydA or pRpsbOa-hydA. The observation of homologous DT hydA fragment in DT transformants by PCR indicated that the plasmids were successfully transformed into DT. The observations of hydA transcript by RT-PCR and HydA protein by western blotting in certain DT transformants suggested that hydA could be expressed in these transformants under aerobic and S-supplied (+S) condition. The hydrogen contents in DT transformants are 7 to 10-fold higher than that in DT wild type under anaerobic and +S condition. These results suggested that the HydA protein was successfully homologously expressed to enhance hydrogen production in DT.
URI: http://hdl.handle.net/11455/23834
其他識別: U0005-2907201115424800
Appears in Collections:生命科學系所

文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.