Abstract
Methanol is a promising one-carbon (C1) feedstock for microbial bioconversion; however, engineered Saccharomyces cerevisiae often faces energetic constrains during its assimilation. Here, we develop SC-AOX25, an energy-efficient methylotrophic S. cerevisiae, through engineering of heterologous methanol-formaldehyde-formate (MFF) oxidation pathways coupled with adaptive laboratory evolution. SC-AOX25 efficiently generates adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NADH) during methanol metabolism while co-assimilating methanol-derived intermediates (formaldehyde, formate, and CO₂) via native glyoxylate-serine cycle, pentose phosphate pathway, and reductive glycine pathway. Key energy modules - Fdh1sc, Adh2m, Aoxm, and Rgi2m - are characterized for their roles in ATP/NADH synthesis and methylotrophic growth. Formaldehyde-induced DNA-protein crosslinks (DPCs) and large repeated DNA fragments suggest strategies for methanol detoxification and phenotype enhancement. Utilizing SC-AOX25, we enable CO₂ assimilation through non-native Calvin cycle during methanol fermentation, establishing the engineered strain as a robust and energy-efficient methylotrophic platform for further C1 engineering.
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Data availability
The RNA-seq data and genome resequencing data generated in this study have been deposited in the NCBI SRA database under Bioproject PRJNA1238490 and PRJNA1238044, respectively. The proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE repository under accession PXD064258 and PXD064984. Source data are provided with this paper.
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Acknowledgements
This work was supported by the National Science Foundation of China (32401209), National Key R&D Program of China (2022YFA0912800), National Natural Science Foundation of China (22178233), Talents Program of Sichuan Province, Fundamental Research Funds for the Central Universities (SCU2025D014), Double First-Class University Plan of Sichuan University, State Key Laboratory of Polymer Materials Engineering (sklpme 2020-03-01), Suzhou Municipal High-Level Talent Entrepreneurship Fund, Tianfu Emei Program of Sichuan Province (2022-EC02-00073-CG) the Center of Synthetic Biology and Integrated Bioengineering (WU2022A006, WU2022A007, WU2023A009), Research Plan for Westlake University Funded Scientific Research Project (WU2022C032), Zhejiang Key Laboratory of Low-Carbon Intelligent Synthetic Biology (2024ZY01025), and Shenzhen Science and Technology Program (RCYX20231211090115013). We thank Wenwen Zhang and Xiaoyan Xu at Mass Spectrometry & Metabolomics Core Facility of Westlake University for MS analysis.
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W.Z. and Y.W. conceived and designed the study. Y.W. supervised the project. W.Z. and N.L. performed the experiments and data processing, and analyzed the data. B.C. completed the analysis of the structure of Aoxm and Adh2m, respectively. H.S. and X.F. were involved in constructing some of the engineered yeast strains needed for the study. W.Z. and Y.W. wrote and revised the manuscript with input of all authors. J.L., J.G., and B.W. contributed to the review, editing, and final approval of the manuscript. They provided valuable insights and suggestions to improve the quality of the research paper.
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Zhong, W., Liu, N., Chen, B. et al. Engineering energy-efficient Saccharomyces cerevisiae for methanol and CO2 assimilation. Nat Commun (2026). https://doi.org/10.1038/s41467-026-68516-y
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DOI: https://doi.org/10.1038/s41467-026-68516-y
