熱休克蛋白104啟動子置換之酵母菌特性

Characterization of Saccharomyces cerevisiae with chromosomal promoter replacement of Hsp104

賴昱璇(Lai, Yushiuan)

指導教授 ： 黃光策

國立中正大學/工學院/化學工程學系/碩士(2012年)

摘要

熱休克蛋白104與熱休克蛋白70/40會將已聚集的蛋白分離並重新摺疊，當細胞遭受環境壓力時，細胞體內熱休克蛋白104的濃度會在短時間內急遽上升，使堆積在細胞質內已失活的聚集蛋白質恢復活性，而維持細胞的正常生理，達到保護細胞的作用。然而熱休克蛋白104無法長時間的表現，使得酵母菌無法在高溫下長期發酵，因此實驗室之前建構重組酵母菌Kyokai6-Hsp104R26-kan，將熱休克蛋白104的啟動子置換為熱休克蛋白26啟動子，以延長熱休克蛋白104的表現時間，並且由耐熱性實驗證實重組菌(Kyokai6-Hsp104R26-kan)較原生菌能忍受高溫環境，且可達46℃。本研究更進一步探討重組菌生長的其他特性及耐受性，結果發現於42℃高濃度的葡萄糖及酒精環境下，重組菌的耐受性皆較原生菌佳。當培養液中酒精濃度高於10%時，原生菌及重組菌的菌體濃度會下降，但重組菌株於三小時後即能適應高濃度的酒精生長環境，而使菌體量達到平穩。重組菌在含有240 g/L葡萄糖培養基中對於菌體生長的抑制作用並不明顯，但對於酸性的培養基則較敏感，顯示重組菌最佳生長環境為中性。

關鍵字

熱休克蛋白104

並列摘要

Heat-shock protein 104 (Hsp104) together with Hsp70, and Hsp40 are involved in disaggregating insoluble protein aggregates. In addition, Hsp104 regulates prion assembly and disassembly. Under extreme stress, Hsp104 can be induced and is essential for cell survival. However, the induction of Hsp104 is transient so that the cells would not survive after a long period of stress. To prolong the expression of Hsp104 during long-term stress, our laboratory previously constructed a Saccharomyces cerevisiae mutant, Kyokai 6-Hsp104R26-kan, in which the regulatory promoter region of gene Hsp104 was replaced with that of Hsp26. Up to 46℃, the recombinant strain (Kyokai 6-Hsp104R26-kan) shows much better thermotolerance than wild type from the spot assay. In addition, the tolerances for high concentration glucose and ethanol were also enhanced in the recombinant strain. When the concentration of ethanol in the medium was up to or higher than 10%, both strains showed a decrease of cell density. However, the recombinant strain, but not wild type, quickly adapted the high ethanol environment after 3 hours. The effect of glucose inhibition up to concentration 240 g/l was insignificant in the recombinant strain. The sensitivity for pH was increased in recombinant strain and its optimal pH for cell growth was shifted from acidic toward neutral medium.

並列關鍵字

Heat shock protein 104

參考文獻

Arnold, C. E. & Wittrup, K. D. (1994). The stress response to loss of signal recognition particle function in Saccharomyces cerevisiae. Journal of Biological Chemistry 269, 30412-30418.

Bai, F. W., Anderson, W. A. & Moo-Young, M. (2008). Ethanol fermentation technologies from sugar and starch feedstocks. Biotechnology Advances 26, 89-105.

Benjaphokee, S., Hasegawa, D., Yokota, D., Asvarak, T., Auesukaree, C., Sugiyama, M., Kaneko, Y., Boonchird, C. & Harashima, S. (2011). Highly efficient bioethanol production by a Saccharomyces cerevisiae strain with multiple stress tolerance to high temperature, acid and ethanol. New Biotechnology 29, 379-386.

Birch, R. M. & Walker, G. M. (2000). Influence of magnesium ions on heat shock and ethanol stress responses of Saccharomyces cerevisiae. Enzyme and Microbial Technology 26, 678-687.

Bosl, B., Grimminger, V. & Walter, S. (2006). The molecular chaperone Hsp104--A molecular machine for protein disaggregation. Journal of Structural Biology 156, 139-148.