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

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

de Valk, S.C. (TU Delft BT/Industriele Microbiologie)
Bouwmeester, Susan E. (Student TU Delft)
de Hulster, A.F. (TU Delft BT/Industriele Microbiologie)
Mans, R. (TU Delft BT/Industriele Microbiologie)

2022

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.

Bioethanol
Energy metabolism
Evolutionary engineering
Sugar transport
Yeast physiology

http://resolver.tudelft.nl/uuid:32ee8b57-291b-4e36-903e-b3ae762ceb76

https://doi.org/10.1186/s13068-022-02145-7

Biotechnology for Biofuels and Bioproducts, 15 (1)

Institutional Repository

journal article

© 2022 S.C. de Valk, Susan E. Bouwmeester, A.F. de Hulster, R. Mans