Redox engineering by ectopic expression ofglutamate dehydrogenase genes links NADPH availability and NADH oxidation with coldgrowth in Saccharomyces cerevisiae

Microbial Cell Factories

Research

Lidia Ballester-Tomás¹  Francisca Randez-Gil¹  Jose Antonio Prieto¹  Roberto Pérez-Torrado¹ 

[1] Department of Biotechnology, Instituto de Agroquímica y Tecnología de los Alimentos (CSIC), Avda. Agustín Escardino 7, 46980, Paterna, Valencia, Spain;

关键词: Wine yeast;    Winemaking;    Low temperature;    Fermentation;    Tryptophan;    ROS accumulation;    GDH1;    GDH2;    GPD1;    GRE3;   

DOI  :  10.1186/s12934-015-0289-2

 received in 2015-05-05, accepted in 2015-06-23,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundCold stress reduces microbial growth and metabolism being relevantin industrial processes like wine making and brewing. Knowledge on the coldtranscriptional response of Saccharomycescerevisiae suggests the need of a proper redox balance.Nevertheless, there are no direct evidence of the links between NAD(P) levelsand cold growth and how engineering of enzymatic reactions requiring NAD(P) maybe used to modify the performance of industrial strains at lowtemperature.ResultsRecombinant strains of S.cerevisiae modified for increased NADPH- and NADH-dependent Gdh1and Gdh2 activity were tested for growth at low temperature. A high-copy numberof the GDH2-encoded glutamate dehydrogenasegene stimulated growth at 15°C, while overexpression of GDH1 had detrimental effects, a difference likely caused bycofactor preferences. Indeed, neither the Trp−character of the tested strains, which could affect the synthesis of NAD(P), norchanges in oxidative stress susceptibility by overexpression of GDH1 and GDH2account for the observed phenotypes. However, increased or reduced NADPHavailability by knock-out or overexpression of GRE3, the NADPH-dependent aldose reductase gene, eliminated orexacerbated the cold-growth defect observed in YEpGDH1 cells. We alsodemonstrated that decreased capacity of glycerol production impairs growth at 15but not at 30°C and that 15°C-grown baker’s yeast cells display higherfermentative capacity than those cultivated at 30°C. Thus, increasing NADHoxidation by overexpression of GDH2 wouldhelp to avoid perturbations in the redox metabolism induced by a higherfermentative/oxidative balance at low temperature. Finally, it is shown thatoverexpression of GDH2 increases notably thecold growth in the wine yeast strain QA23 in both standard growth medium andsynthetic grape must.ConclusionsRedox constraints limit the growth of S.cerevisiae at temperatures below the optimal. An adequate supplyof NAD(P) precursors as well as a proper level of reducing equivalents in theform of NADPH are required for cold growth. However, a major limitation is theincreased need of oxidation of NADH to NAD+ at lowtemperature. In this scenario, our results identify the ammonium assimilationpathway as a target for the genetic improvement of cold growth in industrialstrains.

【 授权许可】

CC BY   
© Ballester-Tomás et al. 2015