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標題: 太古生物耐鹽保濕相容質在食品與農業應用潛力研發
Application Potential of Archaeal Compatible Solutes in Food and Agriculture
作者: 賴美津
關鍵字: 食品科技;基礎研究
相容質與滲透壓的基礎與應用研究,從抗鹽相容質glycerol、trehalose和近年發展的高鹽細菌生產的ectoine在發酵工業、食品界、化妝美容界、製藥與醫療等的應用,可以想見微生物因應鹽與滲透壓力的小分子相容質在基礎研究與應用的重要性。 極端厭氧的甲烷太古生物 (Archaea, 又稱古菌、古生菌或古細菌),以甲烷的生合成方式提供細胞所需的能量,能生存於各式溫度(2- 117 ℃)、酸鹼度(中性至強鹼)及鹽度(淡水至飽合鹽度)等環境,是唯一能適應多變化環境的太古生物。 我們實驗室收集國內外能生長在各種不同鹽度的甲烷菌,長期探討甲烷菌對鹽逆境的反應與適應機制。我們的研究顯示嗜鹽甲烷菌具有一高親和性、高專一性,需能且受滲透壓調控的ABC型式的甜菜鹼運輸系統Bta,會自胞外環境優先攝取相容質甜菜鹼。而當胞外環境缺乏相容質的提供時,能自體生合成相容質β-glutamine、Nε-acetyl-β-lysine及甜菜鹼 (betaine)於細胞體內,以維持膨壓及保護胞內蛋白等大分子。某些相容質具有特殊風味,廣泛應用於食品添加工業上,在農漁業上的應用逐漸受到矚目,已有研究指出帶有相容質基因的轉殖株對於高鹽、低溫、乾旱等環境因子具有耐受性。另外在飼料中添加相容質對畜牧業也極有幫助。絕對厭氧的甲烷太古生物能生長在淡水至飽和鹽環境,對鹽逆境與滲透壓逆境的適應是生物界中最寬廣的,期由對甲烷太古生物在鹽逆境的反應與適應機制相關基因與調控的了解及應用潛力的分析,進而提高生物抗鹽抗旱的能力。我們已獲得嗜鹽甲烷菌Methanohalophilus portucalensis FDF1相容質betaine的生合成酵素GSMT與SDMT的基因。在甲烷菌的共通相容質Nε-acetyl-β-lysine生合成基因部分,我們已分別獲得海洋甲烷菌Methanosarcina mazei N2M9705、耐鹽甲烷菌Methanocalculus chunghsingensis K1F9705b與嗜鹽甲烷菌Methanohalophilus portucalensis FDF1的lysine 2,3-aminomutase (ablA)與β-lysine acetyltransferase (ablB)基因;以及淡水型甲烷菌M. mazei N2M9705的相容質β-glutamine 的生合成基因。在此三年期計畫期間,除了探討甲烷菌自體生合成相容質基因對於環境逆境的調控機制以及分析異源表現蛋白活性之外,並將生合成相容質β-glutamine、Nε-acetyl-β-lysine及betaine的基因組,分別轉殖到大腸桿菌、阿拉伯芥與斑馬魚上,分析其相容質生成累積量與探討是否能提高其對鹽度的耐受性,增加轉殖動、植物對於環境逆境的適應性。將更近一步測試並量產相容質,評估在食品工業上與農林畜牧與養殖業的應用。

Archaea as the strict anaerobic, low redox required methanogen, hyperthermophiles that can grow up to 121 ℃, microbes that enjoy the 1000 atm pressure and extreme halophiles that could grow at saturated NaCl. As we facing the extreme living environmental challenge now and in near future, the extreme adaptation genes, extremozymes and strategies from Archaea may help us to fight for the global warming.The osmotic strength of the environment is one of the parameters that determine the ability of organisms to proliferate in a given habitat. Changes in the extracellular osmolarity have the same physiochemical effects on cell from all living domains and the responses to osmotic shifts have considerable similarities in all organisms. Strict anaerobic methanogenic archaea can survive at broad range of salt stress (0-4.5 M NaCl) from freshwater, marine to hypersaline environments. The extreme and board range of salt adaptations made the Methanoarchaea the best model for studying salt-stress response of archaea and osmoadaptation. To encounter the changing osmotic stress in hypersaline environments, halophilic methanogen-Methanohalophilus portucalensis could transport compatible solute (osmolyte) betaine through an energy-requiring, high-affinity and highly specific betaine transport system which was regulated by betaine and osmotic stresses. Without the extracellular supply of betaine, cell could de novo synthesize betaine, Nε-acetyl-β-lysine, β-glutamate and β-glutamine as compatible solutes to increase the internal osmolarity and protect cellular macromolecules.In order to reveal how methanogen deal with salt stress with osmolytes, we have already obtained the (1).osmolyte betaine synthesizing genes for GSMT and SDMT from halophilic methanogen- M. portucalensis; (2). osmolyte Nε-acetyl-β-lysine synthesizing genes encoding for lysine-2,3-aminomutase (ablA) and β-lysine acetyltransferase (ablB) from marine Methanosarcina mazei strain N2M9705, halotolerant Methanocalculus chungshingensis and halophilic M. portucalensis and (3). osmolyte β-glutamine synthesizing genes encoding for glutamine synthetase (glnA1) from Methanosarcina mazei strain N2M9705.In this grant proposal, we plan to (1) transform osmolytes betaine, Nε-acetyl-β-lysine and β-glutamine synthesizing gene sets separately to osmolyte transport and synthesis defective mutant E. coli KH13(ΔbetT, ΔputPA, ΔproP, and ΔproU)to test for the accumulation of osmolytes and salt stress tolerance. (2). transform and over-express each osmolytes synthesizing gene in E. coli BL21 (DE3)-RIL and purified protein for enzyme activity assay and further structure analysis. (3). transform and over-express osmolytes betaine, Nε-acetyl-β-lysine and β-glutamine synthesizing gene sets separately into E. coli BL21 (DE3)-RIL and tested for intracellular osmolyte accumulation and salt/temperature stress tolerance. (4). transform and over-express osmolytes betaine, Nε-acetyl-β-lysine and β-glutamine synthesizing gene sets separately into Corynebacterium glutamicum for osmolytes production. These osmolytes can be used for salt, chilling, drought protectants and humectants for food and agriculture industries. (5). transformed and over-expressed osmolytes betaine, Nε-acetyl-β-lysine and β-glutamine synthesizing gene sets separately to Arabidopsis thaliana and Zebrafish and tested for intracellular osmolyte accumulation and salt/ temperature stress tolerance. The study in methanogenic archaea, the widest salt growth range of living organism, at salt stress-response should gain the basic knowledge in osmoregulation and application in anti-salt and drought organisms and macromolecules.
其他識別: NSC97-2313-B005-013-MY3
Appears in Collections:生命科學系所

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