title: Redox engineering by ectopic expression of glutamate dehydrogenase genes links NADPH availability and NADH oxidation with cold growth in Saccharomyces cerevisiae.
abstract: BACKGROUND
  Cold stress reduces microbial growth and metabolism being relevant in industrial processes like wine making and brewing. Knowledge on the cold transcriptional response of Saccharomyces cerevisiae suggests the need of a proper redox balance. Nevertheless, there are no direct evidence of the links between NAD(P) levels and cold growth and how engineering of enzymatic reactions requiring NAD(P) may be used to modify the performance of industrial strains at low temperature.
  
  
  RESULTS
  Recombinant strains of S. cerevisiae modified for increased NADPH- and NADH-dependent Gdh1 and Gdh2 activity were tested for growth at low temperature. A high-copy number of the GDH2-encoded glutamate dehydrogenase gene stimulated growth at 15°C, while overexpression of GDH1 had detrimental effects, a difference likely caused by cofactor preferences. Indeed, neither the Trp(-) character of the tested strains, which could affect the synthesis of NAD(P), nor changes in oxidative stress susceptibility by overexpression of GDH1 and GDH2 account for the observed phenotypes. However, increased or reduced NADPH availability by knock-out or overexpression of GRE3, the NADPH-dependent aldose reductase gene, eliminated or exacerbated the cold-growth defect observed in YEpGDH1 cells. We also demonstrated that decreased capacity of glycerol production impairs growth at 15 but not at 30°C and that 15°C-grown baker's yeast cells display higher fermentative capacity than those cultivated at 30°C. Thus, increasing NADH oxidation by overexpression of GDH2 would help to avoid perturbations in the redox metabolism induced by a higher fermentative/oxidative balance at low temperature. Finally, it is shown that overexpression of GDH2 increases notably the cold growth in the wine yeast strain QA23 in both standard growth medium and synthetic grape must.
  
  
  CONCLUSIONS
  Redox constraints limit the growth of S. cerevisiae at temperatures below the optimal. An adequate supply of NAD(P) precursors as well as a proper level of reducing equivalents in the form of NADPH are required for cold growth. However, a major limitation is the increased need of oxidation of NADH to NAD(+) at low temperature. In this scenario, our results identify the ammonium assimilation pathway as a target for the genetic improvement of cold growth in industrial strains.
authors.affiliation: Department of Biotechnology, Instituto de Agroquímica y Tecnología de los Alimentos (CSIC), Avda. Agustín Escardino 7, 46980, Paterna, Valencia, Spain. lballester@iata.csic.es.
authors.forename: Lidia
authors.initials: L
authors.lastname: Ballester-Tomás
authors.affiliation: Department of Biotechnology, Instituto de Agroquímica y Tecnología de los Alimentos (CSIC), Avda. Agustín Escardino 7, 46980, Paterna, Valencia, Spain. randez@iata.csic.es.
authors.forename: Francisca
authors.initials: F
authors.lastname: Randez-Gil
authors.affiliation: Department of Biotechnology, Instituto de Agroquímica y Tecnología de los Alimentos (CSIC), Avda. Agustín Escardino 7, 46980, Paterna, Valencia, Spain. rober@iata.csic.es.
authors.forename: Roberto
authors.initials: R
authors.lastname: Pérez-Torrado
authors.affiliation: Department of Biotechnology, Instituto de Agroquímica y Tecnología de los Alimentos (CSIC), Avda. Agustín Escardino 7, 46980, Paterna, Valencia, Spain. prieto@iata.csic.es.
authors.forename: Jose Antonio
authors.initials: JA
authors.lastname: Prieto
mesh_terms: D005969:Glutamate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D060847:Metabolic Engineering; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D009249:NADP; D005969:Glutamate Dehydrogenase