酵母菌的生長與代謝所需的能量主要透過呼吸作用(respiration)以及發酵作用(fermentation)來產生。在酵母菌中，每一分子的葡萄糖透過呼吸作用產生的ATP，相較於發酵作用多了約16倍。在環境中含有可發酵性碳源時，不論是否存在氧氣，大部分的酵母菌都會選擇進行發酵作用，而非進行呼吸作用，這個現象稱之為Crabtree effect。有此現象的酵母菌稱為Crabtree-positive酵母菌，例如Saccharomyces cerevisiae；相反的，Kluyveromyces lactis是Crabtree-negative酵母菌，主要進行呼吸作用，不會因為環境中含有可發酵性碳源而轉為發酵作用。另外，S. cerevisiae具有多達約20種對應不同環境的六碳糖轉運蛋白質(Hexose Transport，HXT)，而對於葡萄糖有高親和力的六碳糖轉運蛋白質，會在低濃度葡萄糖的環境中表現，攝取環境中稀少的葡萄糖，但是攝取速率相較於低親和力六碳糖轉運蛋白質慢，相比之下K. lactis只具有4種六碳糖轉運蛋白質。而為了研究S. cerevisiae中對葡萄糖具有高親和力的六碳糖轉運蛋白質置入到K. lactis中是否會影響K. lactis的生長速率。首先，比較S. cerevisiae中屬於實驗室菌株的BY4741以及野生型菌株RM11-1a對於葡萄糖具有高親和力的六碳糖轉運蛋白質的序列差異，發現兩者近乎沒有差異後挑選BY4741進行片段放大後插入含持續表現型啟動子 (constitutive promoter) TEF的質體中，再透過電脈衝穿孔(electroporation)的方式將含目標基因Hxt2、Hxt5、Hxt6及Hxt10的質體置入K. lactis中，而透過呼吸作用抑制劑 (Antimycin A)，選出具有發酵能力的K. lactis (KB101)作為觀察的目標菌株。結果顯示S. cerevisiae中對於葡萄糖具有高親和力的六碳糖轉運蛋白質可以縮短K. lactis在呼吸作用時doubling time所需的時間，而發酵作用則沒有明顯的差異，或許是因為高親和力的六碳糖轉運蛋白質攝取葡萄糖的速率不足以提升發酵所需的碳源，未來可以考慮放入複合種類的高親和力葡萄糖轉運蛋白質，或許可以縮短其在發酵生長時doubling time所需的時間，或者使用可以快速攝取大量葡萄糖的低親和力的六碳糖轉運蛋白質來增加攝取葡萄的速率，以補足進行發酵作用時所需的碳源。
關鍵字：克魯維乳酸酵母菌、六碳糖轉運蛋白質、葡萄糖高親和力轉運蛋白質、TEF啟動子、生長曲線

The energy required for yeast growth is mainly produced by respiration and fermentation processes. In yeast, ATP produced by one glucose molecule through the respiration process approximates 16 times more than that of the fermentation process. When the fermentable carbon source is available, some yeasts prefer the fermentation process rather than the respiration process to metabolize sugars no matter the oxygen is present or not. This phenomenon is called the Crabtree effect. Yeasts with aerobic fermentation capability are called Crabtree-positive yeasts, such as Saccharomyces cerevisiae; on the other hand, Crabtree-negative yeasts such as Kluyveromyces lactis mainly perform respiration process rather than fermentation process when the oxygen is present. In addition, S. cerevisiae has about 20 different kinds of hexose transporter proteins (Hxtps) to accommodate various environmental conditions. Hexose transporters with high glucose binding affinity express and uptake scarce glucose. However, the glucose uptake of these transporters is less efficient than that of transporters with low glucose binding affinity. In contrast, K. lactis contains only four hexose transporter genes. In this study, four high glucose binding affinity hexose transporter genes of S. cerevisiae were overexpressed in K. lactis for examining the effects of glucose transport on the growth of K. lactis. In this study, Hxt2, Hxt5, Hxt6, and Hxt10 of S. cerevisiae were over-expressed in K. lactis KB101 strain to check if these genes affect the respiration or fermentation processes of K. lactis. Analyses results showed that the hexose transporters with high glucose binding affinity decreased the doubling time of K. lactis under respiration growth conditions, while posed no significant effect on the growth of K. lactis under fermentation growth conditions. It is possible that the slow glucose transport capacity of the high glucose binding affinity hexose transporters is not able to provide enough glucose for fermentation growth.

Key Words: Kluyveromyces lactis, Hexose Transporter (HXT), High Glucose binding Affinity Transporter, TEF Promoter, Growth Curve.

Bitzilekis, S., & Barnett, J. A. (1997). Exponential growth rates of species of the yeast genus Kluyveromyces. FEMS Microbiol Lett, 146(2), 189-190. doi:10.1111/j.1574-6968.1997.tb10191.x
Breunig, K. D., Bolotin-Fukuhara, M., Bianchi, M. M., Bourgarel, D., Falcone, C., Ferrero, I. I., . . . Zeeman, A. M. (2000). Regulation of primary carbon metabolism in Kluyveromyces lactis. Enzyme Microb Technol, 26(9-10), 771-780. doi:10.1016/s0141-0229(00)00170-8
Crabtree, H. G. (1929). Observations on the carbohydrate metabolism of tumours. Biochem J, 23(3), 536-545. doi:10.1042/bj0230536
de Boer, H. A., Comstock, L. J., & Vasser, M. (1983). The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A, 80(1), 21-25. doi:10.1073/pnas.80.1.21
Diaz-Ruiz, R., Uribe-Carvajal, S., Devin, A., & Rigoulet, M. (2009). Tumor cell energy metabolism and its common features with yeast metabolism. Biochim Biophys Acta, 1796(2), 252-265. doi:10.1016/j.bbcan.2009.07.003
Dominguez, A., Ferminan, E., Sanchez, M., Gonzalez, F. J., Perez-Campo, F. M., Garcia, S., . . . Lopez, M. C. (1998). Non-conventional yeasts as hosts for heterologous protein production. Int Microbiol, 1(2), 131-142. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/10943351
Gancedo, J. M. (1998). Yeast carbon catabolite repression. Microbiol Mol Biol Rev, 62(2), 334-361. doi:10.1128/MMBR.62.2.334-361.1998
Goffrini, P., Wesolowski-Louvel, M., Ferrero, I., & Fukuhara, H. (1990). RAG1 gene of the yeast Kluyveromyces lactis codes for a sugar transporter. Nucleic Acids Res, 18(17), 5294. doi:10.1093/nar/18.17.5294
Hahn-Hagerdal, B., Hallborn, J., Jeppsson, H., Meinander, N., Walfridsson, M., Ojamo, H., . . . Zimmermann, F. K. (1996). Redox balances in recombinant Saccharomyces cerevisiae. Ann N Y Acad Sci, 782, 286-296. doi:10.1111/j.1749-6632.1996.tb40569.x
Horak, J. (2013). Regulations of sugar transporters: insights from yeast. Curr Genet, 59(1-2), 1-31. doi:10.1007/s00294-013-0388-8
Karhumaa, K., Wu, B., & Kielland-Brandt, M. C. (2010). Conditions with high intracellular glucose inhibit sensing through glucose sensor Snf3 in Saccharomyces cerevisiae. J Cell Biochem, 110(4), 920-925. doi:10.1002/jcb.22605
Kotyk, A., & Kleinzeller, A. (1967). Affinity of the yeast membrane carrier for glucose and its role in the Pasteur effect. Biochim Biophys Acta, 135(1), 106-111. doi:10.1016/0005-2736(67)90012-0
Milkowski, C., Krampe, S., Weirich, J., Hasse, V., Boles, E., & Breunig, K. D. (2001). Feedback regulation of glucose transporter gene transcription in Kluyveromyces lactis by glucose uptake. J Bacteriol, 183(18), 5223-5229. doi:10.1128/JB.183.18.5223-5229.2001
Moriya, H., & Johnston, M. (2004). Glucose sensing and signaling in Saccharomyces cerevisiae through the Rgt2 glucose sensor and casein kinase I. Proc Natl Acad Sci U S A, 101(6), 1572-1577. doi:10.1073/pnas.0305901101
Mortimer, R. K., & Johnston, J. R. (1986). Genealogy of principal strains of the yeast genetic stock center. Genetics, 113(1), 35-43. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/3519363
Newcomb, L. L., Diderich, J. A., Slattery, M. G., & Heideman, W. (2003). Glucose regulation of Saccharomyces cerevisiae cell cycle genes. Eukaryot Cell, 2(1), 143-149. doi:10.1128/EC.2.1.143-149.2003
Ozcan, S., Dover, J., & Johnston, M. (1998). Glucose sensing and signaling by two glucose receptors in the yeast Saccharomyces cerevisiae. EMBO J, 17(9), 2566-2573. doi:10.1093/emboj/17.9.2566
Ozcan, S., & Johnston, M. (1999). Function and regulation of yeast hexose transporters. Microbiol Mol Biol Rev, 63(3), 554-569. doi:10.1128/MMBR.63.3.554-569.1999
Ozier-Kalogeropoulos, O., Malpertuy, A., Boyer, J., Tekaia, F., & Dujon, B. (1998). Random exploration of the Kluyveromyces lactis genome and comparison with that of Saccharomyces cerevisiae. Nucleic Acids Res, 26(23), 5511-5524. doi:10.1093/nar/26.23.5511
Partow, S., Siewers, V., Bjorn, S., Nielsen, J., & Maury, J. (2010). Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae. Yeast, 27(11), 955-964. doi:10.1002/yea.1806
Polish, J. A., Kim, J.-H., & Johnston, M. (2005). How the Rgt1 Transcription Factor of Saccharomyces cerevisiae Is Regulated by Glucose. Genetics, 169(2), 583-594. doi:10.1534/genetics.104.034512
Rajaei, N. (2015). Regulation and mechanism of mating-type switching in Kluyveromyces lactis. Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm …,
Reifenberger, E., Boles, E., & Ciriacy, M. (1997). Kinetic characterization of individual hexose transporters of Saccharomyces cerevisiae and their relation to the triggering mechanisms of glucose repression. Eur J Biochem, 245(2), 324-333. doi:10.1111/j.1432-1033.1997.00324.x
Reifenberger, E., Freidel, K., & Ciriacy, M. (1995). Identification of novel HXT genes in Saccharomyces cerevisiae reveals the impact of individual hexose transporters on glycolytic flux. Mol Microbiol, 16(1), 157-167. doi:10.1111/j.1365-2958.1995.tb02400.x
Sicard, D., & Legras, J. L. (2011). Bread, beer and wine: yeast domestication in the Saccharomyces sensu stricto complex. C R Biol, 334(3), 229-236. doi:10.1016/j.crvi.2010.12.016
Siso, M. G., Ramil, E., Cerdán, M., & Freire-Picos, M. (1996). Respirofermentative metabolism in Kluyveromyces lactis: ethanol production and the Crabtree effect. Enzyme and Microbial Technology, 18(8), 585-591.
Steensma, H. Y., de Jongh, F. C. M., & Linnekamp, M. (1988). The use of electrophoretic karyotypes in the classification of yeasts: Kluyveromyces marxianus and K. lactis. Current Genetics, 14(4), 311-317. doi:10.1007/BF00419987
Steiner, S., & Philippsen, P. (1994). Sequence and promoter analysis of the highly expressed TEF gene of the filamentous fungus Ashbya gossypii. Mol Gen Genet, 242(3), 263-271. doi:10.1007/BF00280415
Tiukova, I. A., Moller-Hansen, I., Belew, Z. M., Darbani, B., Boles, E., Nour-Eldin, H. H., . . . Borodina, I. (2019). Identification and characterisation of two high-affinity glucose transporters from the spoilage yeast Brettanomyces bruxellensis. FEMS Microbiol Lett, 366(17). doi:10.1093/femsle/fnz222
Trumbly, R. J. (1992). Glucose repression in the yeast Saccharomyces cerevisiae. Mol Microbiol, 6(1), 15-21. doi:10.1111/j.1365-2958.1992.tb00832.x
Turcotte, B., Liang, X. B., Robert, F., & Soontorngun, N. (2010). Transcriptional regulation of nonfermentable carbon utilization in budding yeast. FEMS Yeast Res, 10(1), 2-13. doi:10.1111/j.1567-1364.2009.00555.x
Van Urk, H., Voll, W. S., Scheffers, W. A., & Van Dijken, J. P. (1990). Transient-state analysis of metabolic fluxes in crabtree-positive and crabtree-negative yeasts. Appl Environ Microbiol, 56(1), 281-287. doi:10.1128/aem.56.1.281-287.1990
Wésolowski-Louvel, M., Breunig, K. D., & Fukuhara, H. (1996). Kluyveromyces lactis. In Nonconventional yeasts in biotechnology (pp. 139-201): Springer.
Weirich, J., Goffrini, P., Kuger, P., Ferrero, I., & Breunig, K. D. (1997). Influence of mutations in hexose-transporter genes on glucose repression in Kluyveromyces lactis. Eur J Biochem, 249(1), 248-257. doi:10.1111/j.1432-1033.1997.t01-1-00248.x
Wieczorke, R., Krampe, S., Weierstall, T., Freidel, K., Hollenberg, C. P., & Boles, E. (1999). Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. FEBS Lett, 464(3), 123-128. doi:10.1016/s0014-5793(99)01698-1
Wright, M. C., & Philippsen, P. (1991). Replicative transformation of the filamentous fungus Ashbya gossypii with plasmids containing Saccharomyces cerevisiae ARS elements. Gene, 109(1), 99-105. doi:10.1016/0378-1119(91)90593-z