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

doi:10.1016/j.cjche.2025.05.037

Chinese Journal of Chemical Engineering ›› 2025, Vol. 86 ›› Issue (10): 193-199.DOI: 10.1016/j.cjche.2025.05.037

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

Jingting He^1,2, Xiaosong Yu^3,4,5,6,7, Xin Liu^3,4,5, Lei Qin^3,4,5,6,7, Peng Cao^1,2, Chun Li^{1,2,3,4,5,6,7}

1. School of Chemistry and Chemical Engineering, Shihezi University, Shihezi 832003, China;
2. State Key Laboratory Incubation Base for Green Processing of Chemical Engineering, Shihezi 832003, China;
3. Department of Chemical Engineering, Tsinghua University, Beijing 100084, China;
4. Key Lab for Industrial Biocatalysis, Ministry of Education, Tsinghua University, Beijing 100084, China;
5. Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, China;
6. Beijing Key Laboratory of Recombinant Protein Synthetic Biomanufacturing, Beijing 100176, China;
7. State Key Laboratory of Green Biomanufacturing, Beijing 100029, China

Received:2025-03-30 Revised:2025-04-29 Accepted:2025-05-22 Online:2025-07-23 Published:2025-10-28
Contact: Peng Cao,E-mail:caop@shzu.edu.cn;Chun Li,E-mail:lichun@tsinghua.edu.cn
Supported by:
This work was supported by the National Key Research and Development Program of China (2023YFA0913600), the National Natural Science Foundation of China (22138006, 22278240).

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

Jingting He^1,2, Xiaosong Yu^3,4,5,6,7, Xin Liu^3,4,5, Lei Qin^3,4,5,6,7, Peng Cao^1,2, Chun Li^{1,2,3,4,5,6,7}

1. School of Chemistry and Chemical Engineering, Shihezi University, Shihezi 832003, China;
2. State Key Laboratory Incubation Base for Green Processing of Chemical Engineering, Shihezi 832003, China;
3. Department of Chemical Engineering, Tsinghua University, Beijing 100084, China;
4. Key Lab for Industrial Biocatalysis, Ministry of Education, Tsinghua University, Beijing 100084, China;
5. Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, China;
6. Beijing Key Laboratory of Recombinant Protein Synthetic Biomanufacturing, Beijing 100176, China;
7. State Key Laboratory of Green Biomanufacturing, Beijing 100029, China

通讯作者: Peng Cao,E-mail:caop@shzu.edu.cn;Chun Li,E-mail:lichun@tsinghua.edu.cn
基金资助:
This work was supported by the National Key Research and Development Program of China (2023YFA0913600), the National Natural Science Foundation of China (22138006, 22278240).

Abstract

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.

Key words: Synthetic biology, Rhodopsin, Saccharomyces cerevisiae, Light-driven proton pump, Acid stress tolerance

摘要： 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.

关键词: Synthetic biology, Rhodopsin, Saccharomyces cerevisiae, Light-driven proton pump, Acid stress tolerance

Jingting He, Xiaosong Yu, Xin Liu, Lei Qin, Peng Cao, Chun Li. Design of a rhodopsin-based light-driven proton pump to improve acid tolerance of yeast[J]. Chinese Journal of Chemical Engineering, 2025, 86(10): 193-199.

Jingting He, Xiaosong Yu, Xin Liu, Lei Qin, Peng Cao, Chun Li. Design of a rhodopsin-based light-driven proton pump to improve acid tolerance of yeast[J]. 中国化学工程学报, 2025, 86(10): 193-199.

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

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

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