Design of a rhodopsin-based light-driven proton pump to improve acid tolerance of yeast

Abstract

The enhancement of acid stress tolerance in yeast is critical for advancing its industrial application in biomanufacturing, given yeast's capacity to synthesize a wide range of acidic chemicals. Intracellular acidity can be mitigated by endogenous proton pumps; however, this process consumes substantial ATP (Adenosine Triphosphate) and imposes a metabolic burden on cells. To address this problem, this study introduced a light-driven proton pump in yeast to regulate intracellular acidity. The rhodopsin dR from Natrinema thermotolerans was expressed heterologously in Saccharomyces cerevisiae. However, it was found that dR could not be correctly localized to the plasma membrane. To realize its proton pump function, dR was relocated to the plasma membrane by fusing the signal peptide MLS to the N-terminus of dR. The activation of dR-mediated proton translocation across the membrane was successfully achieved through the application of light and retinene. The ability of the system to pump protons is enhanced with light intensity. This system significantly enhanced the survival ability of yeast in acidic environments. An increase in cell biomass of 6.6% was observed at a pH of 2.3 in comparison to the control. This study has expanded the application of photosensitive proteins for acid tolerance and provides a new strategy for the optimization of light-driven biosystems, which can help to enhancepotential of yeast in the biomanufacturing.