Modifying redox potential and its impact on metabolic fluxes in Saccharomyces cerevisiae

[摘要] ENGLISH ABSTRACT: The production of glycerol by Saccharomyces cerevisiae under anaerobic conditions isessential for maintaining the intracellular redox balance thereby allowing continuousenergy generation through conversion of sugars into ethanol. In addition, glycerol canact as an osmolyte and is synthesized to maintain turgor pressure under hyperosmoticconditions. The production of ethanol from sugars can be a redox-neutral process,where the NAD+ (nicotinamide adenine dinucleotide) that is consumed during theglycolytic conversion of glyceraldehyde-3-phosphate to pyruvate is later regenerated bythe reduction of acetaldehyde to ethanol. However, in particular the redirection ofmetabolic flux of pyruvate to biomass formation leads to excess NADH formation. Theintracellular redox balance in these conditions is then primarily maintained throughformation of glycerol which is control by two main enzymes, namely Gpd1p and Gpd2p.Deletion of the genes coding for these two proteins leads to accumulation of NADH andrenders the cells incapable of maintaining their fermentative ability and growth underanaerobic conditions.The goal of this study was to investigate the growth, fermentative ability and metabolitesynthesis of various gpd1Δgpd2Δ double mutant (DM) strains in which the redoxbalancing potential was partially restored through expression of native or heterologousgenes. Strains were constructed by introducing alternative NADH oxidizing pathways ormanipulating existing pathways to favour the oxidation of excess NADH. Morespecifically, the modifications included (i) sorbitol formation; (ii) establishing a pathwayfor propane-1,2-diol formation; and (iii) increasing ethanol formation. Apart fromgenetically manipulating the gpd1Δgpd2Δ double mutant, the addition of pyruvateduring growth was also investigated. The experiments were carried out under oxygenlimited conditions in a high sugar medium and the fermented product was analyzed fortotal sugar consumed, biomass and primary and secondary metabolites formed by thedifferent strains. The relationships between sugar consumption, growth and metaboliteproduction by different strains were investigated by comparing the data generated fromthe different strains by using multivariate data analysis tools. Analysis of the pathwaysinvolved in the production of primary (acids, ethanol and other metabolites) andsecondary metabolites (aroma compounds) were also carried out in order to establishflux modification in comparison to the wild type (WT) strain. The results revealed that these manipulations improved the fermentative capacity of thegpd1Δgpd2Δ double mutant, suggesting a partial recovery of NAD+ regeneration ability,albeit not to the extent of the WT strain. As expected a significant correlation was foundbetween sugar consumption and ethanol and biomass formation. Ethanol yields but notfinal concentrations were increased by the genetic manipulations. Sorbitol by DM(srlD)and DM(SOR1) strains and propane-1,2-diol by DM(gldA, GRE3, mgsA) strain wereformed in significant amounts although at lower molar yields than glycerol.Furthermore, by genetic manipulation the yield of secondary metabolites (isobutanol,isoamyl alcohol, 2-phenyl ethanol and isobutyric acid) was increased whereas the ethylacetate concentration and yield decreased. The results indicate that aroma compoundproperties of wine yeasts could be favourably changed by manipulating the glycerolsynthesizing pathway. The addition of pyruvate during the growth of gpd1Δgpd2Δdouble mutant contributes to excess NADH re-oxidation through additional ethanolformation.

[发布机构] Stellenbosch University