Engineering proton-coupled hexose uptake in Saccharomyces cerevisiae for improved ethanol yield

Abstract

Background In the yeast Saccharomyces cerevisiae, which is widely applied for industrial bioethanol production, uptake of hexoses is mediated by transporters with a facilitated diffusion mechanism. In anaerobic cultures, a higher ethanol yield can be achieved when transport of hexoses is proton-coupled, because of the lower net ATP yield of sugar dissimilation. In this study, the facilitated diffusion transport system for hexose sugars of S. cerevisiae was replaced by hexose–proton symport. Results Introduction of heterologous glucose– or fructose–proton symporters in an hxt0 yeast background strain (derived from CEN.PK2-1C) restored growth on the corresponding sugar under aerobic conditions. After applying an evolutionary engineering strategy to enable anaerobic growth, the hexose–proton symporter-expressing strains were grown in anaerobic, hexose-limited chemostats on synthetic defined medium, which showed that the biomass yield of the resulting strains was decreased by 44.0-47.6%, whereas the ethanol yield had increased by up to 17.2% (from 1.51 to 1.77 mol mol hexose−1) compared to an isogenic strain expressing the hexose uniporter HXT5. To apply this strategy to increase the ethanol yield on sucrose, we constructed a platform strain in which all genes encoding hexose transporters, disaccharide transporters and disaccharide hydrolases were deleted, after which a combination of a glucose–proton symporter, fructose–proton symporter and extracellular invertase (SUC2) were introduced. After evolution, the resulting strain exhibited a 16.6% increased anaerobic ethanol yield (from 1.51 to 1.76 mol mol hexose equivalent−1) and 46.6% decreased biomass yield on sucrose. Conclusions This study provides a proof-of-concept for the replacement of the endogenous hexose transporters of S. cerevisiae by hexose-proton symport, and the concomitant decrease in ATP yield, to greatly improve the anaerobic yield of ethanol on sugar. Moreover, the sugar-negative platform strain constructed in this study acts as a valuable starting point for future studies on sugar transport or development of cell factories requiring specific sugar transport mechanisms.

Histidine-modified Fe 3 O 4 nanoparticles improving the ethanol yield and tolerance of Saccharomyces cerevisiae

“…However, the ethanol increase achieved in these studies was lower than that observed in the current study. Furthermore, certain approaches have resulted in a trade-off with biomass (de Valk et al, 2022), potentially compromising the maintenance of consistently high ethanol yield in successive batch fermentations, while others have shown limited reusability (Li et al, 2020), incurring potential costs. In this study, His-Fe 3 O 4 , characterized by its biocompatibility and cost-effectiveness, was introduced as an additive for co-culturing with S. cerevisiae .…”

From Saccharomyces cerevisiae to Ethanol: Unlocking the Power of Evolutionary Engineering in Metabolic Engineering Applications

“…The resulting metabolically engineered strain was then further adapted to anaerobic growth by an evolutionary engineering strategy, based on gradually decreasing oxygen levels from 100% air to 100% N 2 in a sequential batch reactor. The final evolved strains had a 17.2% increased ethanol yield, along with a 44-47.6% decrease in biomass formation [101].…”

Omics Sequencing of Saccharomyces cerevisiae Strain with Improved Capacity for Ethanol Production

“…In the absence of extracellular invertase activity of S. cerevisia, sucrose was internalized by proton symporters using ATP, which led to improving the anaerobic fermentation and ethanol yield from sugar [5]. In addition, the replacement of all endogenous hexose transporters with hexose-proton symport and extracellular invertase (SUC2) can increase ethanol yield and anaerobic growth of S. cerevisiae [41].…”