Evolutionary engineered Saccharomyces cerevisiae wine yeast strains with increased in vivo flux through the pentose phosphate pathway.

Amplification of the flux toward the pentose phosphate (PP) pathway might be of interest for various S. cerevisiae based industrial applications. We report an evolutionary engineering strategy based on a long-term batch culture on gluconate, a substrate that is poorly assimilated by S. cerevisiae cells and is metabolized by the PP pathway. After adaptation for various periods of time, we selected strains that had evolved a greater consumption capacity for gluconate. (13)C metabolic flux analysis on glucose revealed a redirection of carbon flux from glycolysis towards the PP pathway and a greater synthesis of lipids. The relative flux into the PP pathway was 17% for the evolved strain (ECA5) versus 11% for the parental strain (EC1118). During wine fermentation, the evolved strains displayed major metabolic changes, such as lower levels of acetate production, higher fermentation rates and enhanced production of aroma compounds. These represent a combination of novel traits, which are of great interest in the context of modern winemaking.

[1]  A. Maujean,et al.  Formation of flavor components by the reaction of amino acid and carbonyl compounds in mild conditions. , 2000, Journal of agricultural and food chemistry.

[2]  G. Fink,et al.  Elements of a single MAP kinase cascade in Saccharomyces cerevisiae mediate two developmental programs in the same cell type: mating and invasive growth. , 1994, Genes & development.

[3]  Uwe Sauer,et al.  Metabolic Engineering of a Phosphoketolase Pathway for Pentose Catabolism in Saccharomyces cerevisiae , 2004, Applied and Environmental Microbiology.

[4]  P. Ribereau-gayon,et al.  Handbook of Enology , 2001 .

[5]  C. Grant,et al.  Protein S-thiolation targets glycolysis and protein synthesis in response to oxidative stress in the yeast Saccharomyces cerevisiae. , 2003, The Biochemical journal.

[6]  J. Dickinson,et al.  A genetic and biochemical analysis of the role of gluconeogenesis in sporulation of Saccharomyces cerevisiae. , 1986, Journal of general microbiology.

[7]  J. Mano,et al.  Importance of glucose-6-phosphate dehydrogenase in the adaptive response to hydrogen peroxide in Saccharomyces cerevisiae. , 1998, The Biochemical journal.

[8]  E. Boles,et al.  A Modified Saccharomyces cerevisiae Strain That Consumes l-Arabinose and Produces Ethanol , 2003, Applied and Environmental Microbiology.

[9]  P. Maitra,et al.  Induction of specific enzymes of the oxidative pentose phosphate pathway by glucono-delta-lactone in Saccharomyces cerevisiae. , 1992, Journal of general microbiology.

[10]  S. Sawayama,et al.  Ethanol production from xylose in engineered Saccharomyces cerevisiae strains: current state and perspectives , 2009, Applied Microbiology and Biotechnology.

[11]  Donald J. Nevins,et al.  A method for the analysis of sugars in plant cell-wall polysaccharides by gas-liquid chromatography , 1967 .

[12]  G. Braus,et al.  Differential Flo8p-dependent regulation of FLO1 and FLO11 for cell–cell and cell–substrate adherence of S. cerevisiae S288c , 2007, Molecular microbiology.

[13]  J. Nielsen,et al.  Impact of 'ome' analyses on inverse metabolic engineering. , 2004, Metabolic engineering.

[14]  Jean-Marie Sablayrolles,et al.  Design of a laboratory automatic system for studying alcoholic fermentations in anisothermal enological conditions. , 1987 .

[15]  Anne-Béatrice Dufour,et al.  The ade4 Package: Implementing the Duality Diagram for Ecologists , 2007 .

[16]  M. Casal,et al.  The use of genetically modified Saccharomyces cerevisiae strains in the wine industry , 2005, Applied Microbiology and Biotechnology.

[17]  P. Barré,et al.  Modulation of Glycerol and Ethanol Yields During Alcoholic Fermentation in Saccharomyces cerevisiae Strains Overexpressed or Disrupted for GPD1 Encoding Glycerol 3‐Phosphate Dehydrogenase , 1997, Yeast.

[18]  T. W. Jeffries,et al.  Metabolic engineering for improved fermentation of pentoses by yeasts , 2004, Applied Microbiology and Biotechnology.

[19]  D. Colavizza,et al.  Metabolic engineering of malolactic wine yeast. , 2006, Metabolic engineering.

[20]  S. Dequin,et al.  Glycerol export and glycerol-3-phosphate dehydrogenase, but not glycerol phosphatase, are rate limiting for glycerol production in Saccharomyces cerevisiae. , 2001, Metabolic engineering.

[21]  S. Dequin,et al.  Engineering of 2,3-Butanediol Dehydrogenase To Reduce Acetoin Formation by Glycerol-Overproducing, Low-Alcohol Saccharomyces cerevisiae , 2009, Applied and Environmental Microbiology.

[22]  Brigitte Cambon,et al.  Eukaryote-to-eukaryote gene transfer events revealed by the genome sequence of the wine yeast Saccharomyces cerevisiae EC1118 , 2009, Proceedings of the National Academy of Sciences.

[23]  S. Dequin,et al.  Glucose utilization of strains lacking PGI1 and expressing a transhydrogenase suggests differences in the pentose phosphate capacity among Saccharomyces cerevisiae strains. , 2008, FEMS yeast research.

[24]  Sylvie Dequin,et al.  Engineering of the Pyruvate Dehydrogenase Bypass inSaccharomyces cerevisiae: Role of the Cytosolic Mg2+ and Mitochondrial K+ Acetaldehyde Dehydrogenases Ald6p and Ald4p in Acetate Formation during Alcoholic Fermentation , 2000, Applied and Environmental Microbiology.

[25]  Uwe Sauer,et al.  Evolutionary Engineering of Saccharomyces cerevisiae for Anaerobic Growth on Xylose , 2003, Applied and Environmental Microbiology.

[26]  J. Pronk,et al.  Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle. , 2004, FEMS yeast research.

[27]  Merja Penttilä,et al.  Oxygen dependence of metabolic fluxes and energy generation of Saccharomyces cerevisiae CEN.PK113-1A , 2008, BMC Systems Biology.

[28]  R. Henry,et al.  An improved procedure for the methylation analysis of oligosaccharides and polysaccharides. , 1984, Carbohydrate research.

[29]  G. Fink,et al.  Feedback control of morphogenesis in fungi by aromatic alcohols. , 2006, Genes & development.

[30]  J. Jiménez,et al.  Adaptive evolution by mutations in the FLO11 gene. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[31]  J. Dickinson,et al.  'Fusel' alcohols induce hyphal-like extensions and pseudohyphal formation in yeasts. , 1996, Microbiology.

[32]  J. Nielsen,et al.  Network Identification and Flux Quantification in the Central Metabolism of Saccharomyces cerevisiae under Different Conditions of Glucose Repression , 2001, Journal of bacteriology.

[33]  M. Delgado,et al.  Isolation and characterization of a mutant from Saccharomyces cerevisiae lacking fructose 1,6-bisphosphatase. , 1984, European journal of biochemistry.

[34]  D. Kosman,et al.  The Yeast Copper/Zinc Superoxide Dismutase and the Pentose Phosphate Pathway Play Overlapping Roles in Oxidative Stress Protection* , 1996, The Journal of Biological Chemistry.

[35]  I. S. Pretorius,et al.  Expression of the Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae and its potential applications in wine production , 2003, Applied Microbiology and Biotechnology.

[36]  J. Pronk,et al.  Novel Evolutionary Engineering Approach for Accelerated Utilization of Glucose, Xylose, and Arabinose Mixtures by Engineered Saccharomyces cerevisiae Strains , 2008, Applied and Environmental Microbiology.

[37]  Candan Tamerler,et al.  Isolation of cobalt hyper-resistant mutants of Saccharomyces cerevisiae by in vivo evolutionary engineering approach. , 2009, Journal of biotechnology.

[38]  S. Dequin,et al.  Effects of GPD1 Overexpression in Saccharomyces cerevisiae Commercial Wine Yeast Strains Lacking ALD6 Genes , 2006, Applied and Environmental Microbiology.

[39]  J. Jiménez,et al.  Coding repeat instability in the FLO11 gene of Saccharomyces yeasts , 2008, Yeast.

[40]  P. Barré,et al.  Glycerol Overproduction by Engineered Saccharomyces cerevisiae Wine Yeast Strains Leads to Substantial Changes in By-Product Formation and to a Stimulation of Fermentation Rate in Stationary Phase , 1999, Applied and Environmental Microbiology.

[41]  P. Langridge,et al.  Decreasing acetic acid accumulation by a glycerol overproducing strain of Saccharomyces cerevisiae by deleting the ALD6 aldehyde dehydrogenase gene , 2002, Yeast.

[42]  I. S. Pretorius,et al.  Yeast modulation of wine flavor. , 2005, Advances in applied microbiology.

[43]  W. J. Dyer,et al.  A rapid method of total lipid extraction and purification. , 1959, Canadian journal of biochemistry and physiology.

[44]  S. Dequin,et al.  Functional analysis of the ALD gene family of Saccharomyces cerevisiae during anaerobic growth on glucose: the NADP+-dependent Ald6p and Ald5p isoforms play a major role in acetate formation. , 2004, Microbiology.

[45]  Jean-Marie Sablayrolles,et al.  Automatic detection of assimilable nitrogen deficiencies during alcoholic fermentation in oenological conditions , 1990 .

[46]  C. Wittmann,et al.  Characterization of the metabolic shift between oxidative and fermentative growth in Saccharomyces cerevisiae by comparative 13C flux analysis , 2005, Microbial cell factories.

[47]  G. Fink,et al.  Bakers' yeast, a model for fungal biofilm formation. , 2001, Science.

[48]  D. Ramón,et al.  Engineering the Saccharomyces cerevisiae isoprenoid pathway for de novo production of aromatic monoterpenes in wine. , 2008, Metabolic engineering.

[49]  Jack T. Pronk,et al.  Engineering of Saccharomyces cerevisiae for Efficient Anaerobic Alcoholic Fermentation of l-Arabinose , 2007, Applied and Environmental Microbiology.

[50]  Dorota Grabowska,et al.  The ALD6 Gene Product Is Indispensable for Providing NADPH in Yeast Cells Lacking Glucose-6-phosphate Dehydrogenase Activity* , 2003, The Journal of Biological Chemistry.

[51]  Pascal Ribéreau-Gayon,et al.  Handbook of Enology: The Microbiology of Wine and Vinifications , 2006 .

[52]  Dominique Schneider,et al.  Tests of parallel molecular evolution in a long-term experiment with Escherichia coli. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[53]  Candan Tamerler,et al.  Evolutionary engineering of multiple-stress resistant Saccharomyces cerevisiae. , 2005, FEMS yeast research.

[54]  S. Dequin,et al.  The potential of genetic engineering for improving brewing, wine-making and baking yeasts , 2001, Applied Microbiology and Biotechnology.

[55]  E. Nevoigt,et al.  Improvement of Saccharomyces yeast strains used in brewing, wine making and baking. , 2008, Advances in biochemical engineering/biotechnology.

[56]  B. Christensen,et al.  Isotopomer analysis using GC-MS. , 1999, Metabolic engineering.

[57]  E. Nevoigt,et al.  Reduced pyruvate decarboxylase and increased glycerol‐3‐phosphate dehydrogenase [NAD+] levels enhance glycerol production in Saccharomyces cerevisiae , 1996, Yeast.