Channelling metabolic flux away from ethanol production by modification of gene expression under wine fermentation conditions

[摘要] ENGLISH ABSTRACT: There is a global demand for technologies to reduce ethanol levels in wine withoutcompromising wine quality. While several chemical and physical methods have been developedto reduce ethanol in finished wine, the target of an industrially applicable biological solution hasthus far not been met. Most attempted biological strategies have focused on developing newstrains of the main fermentative organism, the yeast Saccharomyces cerevisiae. Genemodification approaches have primarily focused on partially redirecting yeast carbonmetabolism away from ethanol production towards glycerol production. These techniques havemet with some moderate success, thus the focus of the current study was to re-direct carbonflux towards trehalose production by moderate over-expression of the TPS1 gene. This geneencodes trehalose-6-phosphate synthase, which converts glucose 6-phosphate and UDPglucoseto α,α-trehalose 6-phosphate. Previous data have shown that the overproduction oftrehalose restricts hexokinase activity reducing the amount of glucose that enters glycolysis.Nevertheless, preliminary TPS1 over-expression studies using multiple copy plasmids haveshown some promise, but also indicated significant negative impact on the general fermentationbehaviour of strains. In order to reduce such negative impacts of excessive trehaloseproduction, a new strategy consisting in increasing the expression of TPS1 only during specificgrowth phases and by a relatively minor degree was investigated. Our study employed a lowcopynumber episomal vector to drive moderate over-expression of the TPS1 gene in the widelyused industrial strain VIN13 at different stages during fermentation. The fermentations wereperformed in synthetic must with sugar levels representative of those found in real grape must.This, as well as the use of an industrial yeast strain, makes it easier to relate our results to realwinemaking conditions. A reduction in fermentation capacity was observed for all transformedstrains and controls. Expression profiles suggest that the DUT1 promoter certainly results inincreased TPS1 expression (up to 40%) during early exponential growth phase compared to thewild type strain (VIN13). TPS1 expression under the control of the GIP2 promoter regionshowed increased expression levels during early stationary phase (up to 60%). Chemicalanalysis of the yeast and the must at the end after fermentation showed an increase intrehalose production =in line with the expression data of TPS1. Importantly, glycerol productionwas also slightly increased, but without affecting acetic acid levels for the transformed strains.Although ethanol yield is not significantly lower in the DUT1-TPDS1 strain, s statisticallysignificantly lower ethanol yield is observed for over-expression under the GIP2 promotor.Increasing trehalose production during stationary phase appears therefore to be a morepromising approach at lowering ethanol yield and redirecting flux away from ethanol production.This controlled, growth phase specific over expression suggests a unique approach of loweringethanol yield while not impacting on the redox balance.

[发布机构] Stellenbosch University