葡萄糖是所有生物最有利生長的碳源，通常透過呼吸作用來代謝，但 Saccharomyces cerevisiae即便是在有氧環境下，更喜歡以發酵作用來代謝所有可發酵糖類，此現象稱為Crabtree effect，然而1分子葡萄糖經發酵作用途徑只能釋放出2個分子ATP，經呼吸作用途徑能釋放出約32個分子ATP，但屬於Crabtree-positive的酵母菌S. cerevisiae為何更喜歡以發酵作用以及如何以發酵作用來代謝可發酵醣類，而不是以呼吸作用代謝的這個問題至今仍未知。而與Crabtree-negative的酵母菌相比，Crabtree-positive的酵母菌必須具有葡萄糖轉運能力更強的六碳糖轉運蛋白質(hexose transporter)，以支持能量轉換效率較差的有氧發酵方式進行生長，因此在這項研究中，研究六碳糖轉運蛋白質對Crabtree-negative酵母菌Kluyveromyces lactis生長的影響。K. lactis是一種乳酸酵母菌，通常用於工業上的異源蛋白質表現和生產，我在K. lactis中大量表現S. cerevisiae的六碳糖轉運蛋白質基因Hxt1、Hxt3及Hxt9，觀察外源的六碳糖轉運蛋白質是否會影響K. lactis中的葡萄糖代謝過程。實驗結果顯示在會抑制呼吸作用的抗黴素A (antimycin A)存在下野生型KB101能夠生長，野生型GG799卻是緩慢生長，而除了ScHxt9，大量表現ScHxt1與ScHxt3基因後提高了野生型KB101在抗黴素A存在下以發酵作用生長的速度，而在GG799中大量表現ScHxt1、ScHxt3與ScHxt9的基因時，在以呼吸作用進行生長時的速度變快，但卻無法提高添加抗黴素A後以發酵作用生長的速度，綜合以上結果顯示，外源的六碳糖轉運蛋白質會影響K. lactis的生長狀況，未來更需透過更多的實驗驗證觀察六碳糖轉運蛋白質對呼吸作用與發酵作用的影響。

Glucose is the most favorable carbon source for all living cells and usually is metabolized by respiration process. However, S. cerevisiae prefers using fermentation process for metabolizing all the fermentable sugars even in the presence of oxygen, and this phenomenon is called Crabtree effect. However, one molecule of glucose can only generate 2 molecules of ATP through fermentation process, and one molecule of glucose generates about 32 molecules of ATP through respiration process. It is speculated that the Crabtree-positive yeasts must have more efficient glucose transporter(s) to comendate the low energy efficient aerobic fermentation life-style than the Crabtree-negative yeasts. In this study, I studied the effect of glucose transporters to the growth of a Crabtree-negative yeast Kluyveromyces lactis. K. lactis is a dairy yeast and is often use in industrials for heterologous protein expression and production. Here hexose transporter genes, Hxt1, Hxt3 and Hxt9, of S. cerevisiae were expressed in K. lactis to check whether the heterogous hexose transporters affect the glucose metabolism of K. lactis. Expression analysis showed that except for ScHxt9, ScHxt1 and ScHxt3 improve the growth rate of wild-type KB101 significantly in the presence of antimycin A which inhibits respiration. ScHxt1, ScHxt3 and ScHxt9 improved the growth rate of wild-type GG799 under respiration process, whereas they did not under fermentation process. In summary, the hexose transporters with high glucose transport ability will affect the growth of K. lactis.

Baumann, K., M. Carnicer, M. Dragosits, A. B. Graf, J. Stadlmann, P. Jouhten, H. Maaheimo, B. Gasser, J. Albiol, D. Mattanovich and P. Ferrer (2010). "A multi-level study of recombinant Pichia pastoris in different oxygen conditions." BMC systems biology 4: 141-141.
Billard, P., S. Menart, J. Blaisonneau, M. Bolotin-Fukuhara, H. Fukuhara and M. Wesolowski-Louvel (1996). "Glucose uptake in Kluyveromyces lactis: role of the HGT1 gene in glucose transport." J Bacteriol 178(20): 5860-5866.
Boles, E. and C. P. Hollenberg (1997). "The molecular genetics of hexose transport in yeasts." FEMS Microbiology Reviews 21(1): 85-111.
Carlson, M. (1987). "Regulation of sugar utilization in Saccharomyces species." J Bacteriol 169(11): 4873-4877.
Celenza, J. L. and M. Carlson (1986). "A yeast gene that is essential for release from glucose repression encodes a protein kinase." Science 233(4769): 1175-1180.
Conant, G. C. and K. H. Wolfe (2007). "Increased glycolytic flux as an outcome of whole-genome duplication in yeast." Molecular systems biology 3: 129-129.
Cotton, P., A. Soulard, M. Wésolowski-Louvel and M. Lemaire (2012). "The SWI/SNF KlSnf2 subunit controls the glucose signaling pathway to coordinate glycolysis and glucose transport in Kluyveromyces lactis." Eukaryotic cell 11(11): 1382-1390.
De Deken, R. H. (1966). "The Crabtree effect: a regulatory system in yeast." J Gen Microbiol 44(2): 149-156.
Dynesen, J., H. P. Smits, L. Olsson and J. Nielsen (1998). "Carbon catabolite repression of invertase during batch cultivations of Saccharomyces cerevisiae: the role of glucose, fructose, and mannose." Applied Microbiology and Biotechnology 50(5): 579-582.
Enquist-Newman, M., A. M. E. Faust, D. D. Bravo, C. N. S. Santos, R. M. Raisner, A. Hanel, P. Sarvabhowman, C. Le, D. D. Regitsky, S. R. Cooper, L. Peereboom, A. Clark, Y. Martinez, J. Goldsmith, M. Y. Cho, P. D. Donohoue, L. Luo, B. Lamberson, P. Tamrakar, E. J. Kim, J. L. Villari, A. Gill, S. A. Tripathi, P. Karamchedu, C. J. Paredes, V. Rajgarhia, H. K. Kotlar, R. B. Bailey, D. J. Miller, N. L. Ohler, C. Swimmer and Y. Yoshikuni (2014). "Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform." Nature 505(7482): 239-243.
Estruch, F., M. A. Treitel, X. Yang and M. Carlson (1992). "N-terminal mutations modulate yeast SNF1 protein kinase function." Genetics 132(3): 639-650.
Flores, C.-L., C. Rodríguez, T. Petit and C. Gancedo (2000). "Carbohydrate and energy-yielding metabolism in non-conventional yeasts1." FEMS Microbiology Reviews 24(4): 507-529.
Gancedo, J. M. (1998). "Yeast carbon catabolite repression." Microbiol Mol Biol Rev 62(2): 334-361.
Goffrini, P., M. Wesolowski-Louvel, I. Ferrero and H. Fukuhara (1990). "RAG1 gene of the yeast Kluyveromyces lactis codes for a sugar transporter." Nucleic acids research 18(17): 5294-5294.
Hagman, A. and J. Piškur (2015). "A study on the fundamental mechanism and the evolutionary driving forces behind aerobic fermentation in yeast." PloS one 10(1): e0116942-e0116942.
Hedbacker, K. and M. Carlson (2006). "Regulation of the nucleocytoplasmic distribution of Snf1-Gal83 protein kinase." Eukaryotic cell 5(12): 1950-1956.
Hu, Z., J. O. Nehlin, H. Ronne and C. A. Michels (1995). "MIG1-dependent and MIG1-independent glucose regulation of MAL gene expression in Saccharomyces cerevisiae." Current Genetics 28(3): 258-266.
Jacoby, J., C. P. Hollenberg and J. J. Heinisch (1993). "Transaldolase mutants in the yeast Kluyveromyces lactis provide evidence that glucose can be metabolized through the pentose phosphate pathway." Mol Microbiol 10(4): 867-876.
Johnston, M. (1999). "Feasting, fasting and fermenting: glucose sensing in yeast and other cells." Trends in Genetics 15(1): 29-33.
Jordan, P., J.-Y. Choe, E. Boles and M. Oreb (2016). "Hxt13, Hxt15, Hxt16 and Hxt17 from Saccharomyces cerevisiae represent a novel type of polyol transporters." Scientific Reports 6(1): 23502.
Kayikci, Ö. and J. Nielsen (2015). "Glucose repression in Saccharomyces cerevisiae." FEMS yeast research 15(6): fov068.
Kellis, M., B. W. Birren and E. S. Lander (2004). "Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae." Nature 428(6983): 617-624.
Kim, J.-H., J. Polish and M. Johnston (2003). "Specificity and regulation of DNA binding by the yeast glucose transporter gene repressor Rgt1." Molecular and cellular biology 23(15): 5208-5216.
Kishi, T., T. Seno and F. Yamao (1998). "Grr1 functions in the ubiquitin pathway in Saccharomyces cerevisiae through association with Skp1." Mol Gen Genet 257(2): 143-148.
Krieger, F., M. Spinka, R. Golbik, G. Hübner and S. König (2002). "Pyruvate decarboxylase from Kluyveromyces lactis." European Journal of Biochemistry 269(13): 3256-3263.
Kruckeberg, A. L. (1996). "The hexose transporter family of Saccharomyces cerevisiae." Archives of Microbiology 166(5): 283-292.
Li, F. N. and M. Johnston (1997). "Grr1 of Saccharomyces cerevisiae is connected to the ubiquitin proteolysis machinery through Skp1: coupling glucose sensing to gene expression and the cell cycle." Embo j 16(18): 5629-5638.
Liang, H. and R. F. Gaber (1996). "A novel signal transduction pathway in Saccharomyces cerevisiae defined by Snf3-regulated expression of HXT6." Molecular biology of the cell 7(12): 1953-1966.
Liti, G., F. Wardrop, G. Cardinali, A. Martini and G. Walker (2001). "Differential responses to antimycin A and expressions of the Crabtree effect in selected Klyuveromyces spp." Annals of Microbiology 51.
Lundin, M., J. O. Nehlin and H. Ronne (1994). "Importance of a flanking AT-rich region in target site recognition by the GC box-binding zinc finger protein MIG1." Molecular and cellular biology 14(3): 1979-1985.
Marger, M. D. and M. H. Saier (1993). "A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport." Trends in Biochemical Sciences 18(1): 13-20.
McCartney, R. R. and M. C. Schmidt (2001). "Regulation of Snf1 Kinase: ACTIVATION REQUIRES PHOSPHORYLATION OF THREONINE 210 BY AN UPSTREAM KINASE AS WELL AS A DISTINCT STEP MEDIATED BY THE Snf4 SUBUNIT*." Journal of Biological Chemistry 276(39): 36460-36466.
Merico, A., S. Galafassi, J. Piškur and C. Compagno (2009). "The oxygen level determines the fermentation pattern in Kluyveromyces lactis." FEMS Yeast Research 9(5): 749-756.
Milkowski, C., S. Krampe, J. Weirich, V. Hasse, E. Boles and K. D. Breunig (2001). "Feedback regulation of glucose transporter gene transcription in Kluyveromyces lactis by glucose uptake." J Bacteriol 183(18): 5223-5229.
Nourani, A., M. Wesolowski-Louvel, T. Delaveau, C. Jacq and A. Delahodde (1997). "Multiple-drug-resistance phenomenon in the yeast Saccharomyces cerevisiae: involvement of two hexose transporters." Mol Cell Biol 17(9): 5453-5460.
Otterstedt, K., C. Larsson, R. M. Bill, A. Ståhlberg, E. Boles, S. Hohmann and L. Gustafsson (2004). "Switching the mode of metabolism in the yeast Saccharomyces cerevisiae." EMBO reports 5(5): 532-537.
Ozcan, S., J. Dover, A. G. Rosenwald, S. Wölfl and M. Johnston (1996). "Two glucose transporters in Saccharomyces cerevisiae are glucose sensors that generate a signal for induction of gene expression." Proceedings of the National Academy of Sciences of the United States of America 93(22): 12428-12432.
Ozcan, S., J. Dover, A. G. Rosenwald, S. Wölfl and M. Johnston (1996). "Two glucose transporters in Saccharomyces cerevisiae are glucose sensors that generate a signal for induction of gene expression." Proc Natl Acad Sci U S A 93(22): 12428-12432.
Ozcan, S. and M. Johnston (1999). "Function and regulation of yeast hexose transporters." Microbiology and Molecular Biology Reviews 63(3): 554-+.
Pritchard, L. and D. B. Kell (2002). "Schemes of flux control in a model of Saccharomyces cerevisiae glycolysis." Eur J Biochem 269(16): 3894-3904.
Pronk, J. T., H. Yde Steensma and J. P. Van Dijken (1996). "Pyruvate metabolism in Saccharomyces cerevisiae." Yeast 12(16): 1607-1633.
Reifenberger, E., E. Boles and M. Ciriacy (1997). "Kinetic Characterization of Individual Hexose Transporters of Saccharomyces Cerevisiae and their Relation to the Triggering Mechanisms of Glucose Repression." European Journal of Biochemistry 245(2): 324-333.
Reifenberger, E., K. Freidel and M. Ciriacy (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.
Ring, J., C. Sommer, D. Carmona-Gutierrez, C. Ruckenstuhl, T. Eisenberg and F. Madeo (2012). "The metabolism beyond programmed cell death in yeast." Experimental cell research 318(11): 1193-1200.
Rolland, F., J. Winderickx and J. M. Thevelein (2002). "Glucose-sensing and -signalling mechanisms in yeast." FEMS Yeast Res 2(2): 183-201.
Santos, A. M., W. B. Silveira, L. G. Fietto, R. L. Brandão and I. M. Castro (2014). "Kinetics and regulation of lactose transport and metabolism in Kluyveromyces lactis JA6." World Journal of Microbiology and Biotechnology 30(7): 1977-1983.
Sauro, H. M. (2017). "Control and regulation of pathways via negative feedback." Journal of the Royal Society, Interface 14(127): 20160848.
Skowyra, D., K. L. Craig, M. Tyers, S. J. Elledge and J. W. Harper (1997). "F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex." Cell 91(2): 209-219.
Tarrío, N., M. Becerra, M. E. Cerdán and M. I. G. Siso (2006). "Reoxidation of cytosolic NADPH in Kluyveromyces lactis." FEMS Yeast Research 6(3): 371-380.
Thomson, J. M., E. A. Gaucher, M. F. Burgan, D. W. De Kee, T. Li, J. P. Aris and S. A. Benner (2005). "Resurrecting ancestral alcohol dehydrogenases from yeast." Nature genetics 37(6): 630-635.
Tu, J. and M. Carlson (1994). "The GLC7 type 1 protein phosphatase is required for glucose repression in Saccharomyces cerevisiae." Molecular and cellular biology 14(10): 6789-6796.
Vincent, O., R. Townley, S. Kuchin and M. Carlson (2001). "Subcellular localization of the Snf1 kinase is regulated by specific beta subunits and a novel glucose signaling mechanism." Genes & development 15(9): 1104-1114.
Wésolowski-Louvel, M., P. Goffrini, I. Ferrero and H. Fukuhara (1992). "Glucose transport in the yeast Kluyveromyces lactis. I. Properties of an inducible low-affinity glucose transporter gene." Mol Gen Genet 233(1-2): 89-96.
Wieczorke, R., S. Krampe, T. Weierstall, K. Freidel, C. P. Hollenberg and E. Boles (1999). "Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae." Febs Letters 464(3): 123-128.