Role of Hexose Transport in Control of Glycolytic Flux in Saccharomyces cerevisiae

ABSTRACT The yeast Saccharomyces cerevisiae predominantly ferments glucose to ethanol at high external glucose concentrations, irrespective of the presence of oxygen. In contrast, at low external glucose concentrations and in the presence of oxygen, as in a glucose-limited chemostat, no ethanol is produced. The importance of the external glucose concentration suggests a central role for the affinity and maximal transport rates of yeast's glucose transporters in the control of ethanol production. Here we present a series of strains producing functional chimeras between the hexose transporters Hxt1 and Hxt7, each of which has distinct glucose transport characteristics. The strains display a range of decreasing glycolytic rates resulting in a proportional decrease in ethanol production. Using these strains, we show for the first time that at high glucose levels, the glucose uptake capacity of wild-type S. cerevisiae does not control glycolytic flux during exponential batch growth. In contrast, our chimeric Hxt transporters control the rate of glycolysis to a high degree. Strains whose glucose uptake is mediated by these chimeric transporters will undoubtedly provide a powerful tool with which to examine in detail the mechanism underlying the switch between fermentation and respiration in S. cerevisiae and will provide new tools for the control of industrial fermentations.

[1]  Jens Nielsen,et al.  Increasing galactose consumption by Saccharomyces cerevisiae through metabolic engineering of the GAL gene regulatory network , 2000, Nature Biotechnology.

[2]  J. Pronk,et al.  Regulation of carbon metabolism in chemostat cultures of Saccharomyces cerevisiae grown on mixtures of glucose and ethanol , 1995, Yeast.

[3]  E. Boles,et al.  Kinetic characterization of individual hexose transporters of Saccharomyces cerevisiae and their relation to the triggering mechanisms of glucose repression. , 1997, European journal of biochemistry.

[4]  Carlos Gancedo,et al.  Trehalose‐6‐phosphate, a new regulator of yeast glycolysis that inhibits hexokinases , 1993, FEBS letters.

[5]  K. Brindle,et al.  Effects of overexpression of phosphofructokinase on glycolysis in the yeast Saccharomyces cerevisiae. , 1992, Biochemistry.

[6]  J. Ramos,et al.  Relationship between low- and high-affinity glucose transport systems of Saccharomyces cerevisiae , 1988, Journal of bacteriology.

[7]  J. Diderich,et al.  Functional analysis of the hexose transporter homologue HXT5 in Saccharomyces cerevisiae , 2001, Yeast.

[8]  J. Pronk,et al.  Glucose Uptake Kinetics and Transcription of HXTGenes in Chemostat Cultures of Saccharomyces cerevisiae * , 1999, The Journal of Biological Chemistry.

[9]  J. Berden,et al.  Growth and Glucose Repression Are Controlled by Glucose Transport in Saccharomyces cerevisiae Cells Containing Only One Glucose Transporter , 1999, Journal of bacteriology.

[10]  K. Mauch,et al.  Determination of in vivo kinetics of the starvation-induced Hxt5 glucose transporter of Saccharomyces cerevisiae. , 2002, FEMS yeast research.

[11]  D. Fell Metabolic control analysis: a survey of its theoretical and experimental development. , 1992, The Biochemical journal.

[12]  P. Goffrini,et al.  Respiration-Dependent Utilization of Sugars in Yeasts: a Determinant Role for Sugar Transporters , 2002, Journal of bacteriology.

[13]  J. Hauf,et al.  Simultaneous genomic overexpression of seven glycolytic enzymes in the yeast Saccharomyces cerevisiae. , 2000, Enzyme and microbial technology.

[14]  G. Fink,et al.  A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance , 1984, Molecular and General Genetics MGG.

[15]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[16]  H. Kacser,et al.  The control of flux. , 1995, Biochemical Society transactions.

[17]  F. Zimmermann,et al.  Overproduction of glycolytic enzymes in yeast , 1989, Yeast.

[18]  I. Paulsen,et al.  Major Facilitator Superfamily , 1998, Microbiology and Molecular Biology Reviews.

[19]  M. Johnston,et al.  Three different regulatory mechanisms enable yeast hexose transporter (HXT) genes to be induced by different levels of glucose , 1995, Molecular and cellular biology.

[20]  E. Postma,et al.  Kinetics of growth and glucose transport in glucose‐limited chemostat cultures of Saccharomyces cerevisiae CBS 8066 , 1989, Yeast.

[21]  A. H. Rose Energy-Yielding Metabolism , 1968 .

[22]  M. Saier,et al.  A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport. , 1993, Trends in biochemical sciences.

[23]  Stefan Hohmann,et al.  Switching the mode of metabolism in the yeast Saccharomyces cerevisiae , 2004, EMBO reports.

[24]  D A Fell,et al.  Physiological control of metabolic flux: the requirement for multisite modulation. , 1995, The Biochemical journal.

[25]  J. Nielsen,et al.  Flux distributions in anaerobic, glucose-limited continuous cultures of Saccharomyces cerevisiae. , 1997, Microbiology.

[26]  Pronk,et al.  Regulation of fermentative capacity and levels of glycolytic enzymes in chemostat cultures of Saccharomyces cerevisiae. , 2000, Enzyme and microbial technology.

[27]  Duboc,et al.  An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. , 2000, Enzyme and microbial technology.

[28]  C. Hollenberg,et al.  Concurrent knock‐out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae , 1999, FEBS letters.

[29]  W. A. Scheffers,et al.  Effect of benzoic acid on metabolic fluxes in yeasts: A continuous‐culture study on the regulation of respiration and alcoholic fermentation , 1992, Yeast.

[30]  W. A. Scheffers,et al.  Continuous measurement of ethanol production by aerobic yeast suspensions with an enzyme electrode , 1984, Applied Microbiology and Biotechnology.

[31]  J. D. de Winde,et al.  During the initiation of fermentation overexpression of hexokinase PII in yeast transiently causes a similar deregulation of glycolysis as deletion of Tps1 , 1998, Yeast.

[32]  M. Walsh,et al.  Affinity of glucose transport in Saccharomyces cerevisiae is modulated during growth on glucose , 1994, Journal of bacteriology.

[33]  J. Heinisch Isolation and characterization of the two structural genes coding for phosphofructokinase in yeast , 2004, Molecular and General Genetics MGG.

[34]  L. Oehlen,et al.  Decrease in glycolytic flux in Saccharomyces cerevisiae cdc35-1 cells at restrictive temperature correlates with a decrease in glucose transport. , 1994, Microbiology.

[35]  A. Blomberg,et al.  Use of microcalorimetric monitoring in establishing continuous energy balances and in continuous determinations of substrate and product concentrations of batch‐grown Saccharomyces cerevisiae , 1991, Biotechnology and bioengineering.

[36]  H. Westerhoff,et al.  Control of glycolytic dynamics by hexose transport in Saccharomyces cerevisiae. , 2001, Biophysical journal.

[37]  A. Kruckeberg,et al.  Yeast sugar transporters. , 1993, Critical reviews in biochemistry and molecular biology.

[38]  R. F. Rosenzweig Regulation of fitness in yeast overexpressing glycolytic enzymes: parameters of growth and viability. , 1992, Genetical research.

[39]  B. Teusink,et al.  Strategies to determine the extent of control exerted by glucose transport on glycolytic flux in the yeast Saccharomyces bayanus. , 1999, Microbiology.

[40]  L. Gustafsson,et al.  Glycolytic flux is conditionally correlated with ATP concentration in Saccharomyces cerevisiae: a chemostat study under carbon- or nitrogen-limiting conditions , 1997, Journal of bacteriology.

[41]  M. Ciriacy,et al.  Identification of novel HXT genes in Saccharomyces cerevisiae reveals the impact of individual hexose transporters on qlycolytic flux , 1995, Molecular microbiology.

[42]  C. Hollenberg,et al.  The molecular genetics of hexose transport in yeasts. , 1997, FEMS microbiology reviews.

[43]  Hans V. Westerhoff,et al.  Intracellular Glucose Concentration in Derepressed Yeast Cells Consuming Glucose Is High Enough To Reduce the Glucose Transport Rate by 50% , 1998, Journal of bacteriology.

[44]  F. Gamo,et al.  The mutation DGT1-1 decreases glucose transport and alleviates carbon catabolite repression in Saccharomyces cerevisiae , 1994, Journal of bacteriology.

[45]  O. Käppeli,et al.  Regulation of glucose metabolism in growing yeast cells. , 1981, Advances in microbial physiology.

[46]  S. Ho,et al.  Site-directed mutagenesis by overlap extension using the polymerase chain reaction. , 1989, Gene.

[47]  Mark Johnston,et al.  Function and Regulation of Yeast Hexose Transporters , 1999, Microbiology and Molecular Biology Reviews.

[48]  A. Kruckeberg,et al.  The hexose transporter family of Saccharomyces cerevisiae , 1996, Archives of Microbiology.

[49]  A. Brown,et al.  Pyruvate kinase (Pyk1) levels influence both the rate and direction of carbon flux in yeast under fermentative conditions. , 2001, Microbiology.

[50]  L. Bisson,et al.  Involvement of kinases in glucose and fructose uptake by Saccharomyces cerevisiae. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[51]  M. Ciriacy,et al.  Glucose uptake and catabolite repression in dominant HTR1 mutants of Saccharomyces cerevisiae , 1993, Journal of bacteriology.