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

Area of Science:

• Biotechnology
• Metabolic Engineering
• Yeast Physiology

Background:

• Saccharomyces cerevisiae utilizes facilitated diffusion for hexose uptake, impacting ATP yield in anaerobic cultures.
• Proton-coupled hexose transport can improve ethanol yield due to lower net ATP yield during sugar metabolism.

Purpose of the Study:

• To replace the native hexose transport system in S. cerevisiae with proton symporters.
• To enhance anaerobic ethanol yield by engineering hexose-proton symport mechanisms.

Main Methods:

• Introduction of heterologous glucose- and fructose-proton symporters into an hxt⁰ yeast strain.
• Employing evolutionary engineering for anaerobic growth adaptation.
• Cultivation in anaerobic, hexose-limited chemostats and construction of a sugar-negative platform strain for sucrose fermentation.

Main Results:

• Hexose-proton symporter expression restored aerobic growth and increased anaerobic ethanol yield by up to 17.2% (1.51 to 1.77 mol mol hexose^-1).
• Biomass yield decreased by 44.0-47.6% in engineered strains compared to controls.
• A platform strain engineered for sucrose fermentation showed a 16.6% increase in anaerobic ethanol yield and a 46.6% decrease in biomass yield.

Conclusions:

• Replacement of endogenous hexose transporters with hexose-proton symport provides a proof-of-concept for improving anaerobic ethanol yield in S. cerevisiae.
• Engineered strains exhibit reduced ATP yield and enhanced sugar-to-ethanol conversion efficiency.
• The developed sugar-negative platform strain is a valuable resource for future cell factory development and sugar transport studies.