Engineering energy-efficient Saccharomyces cerevisiae for methanol and CO₂ assimilation

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 - Fdh1_sc, Adh2_m, Aox_m, and Rgi2_m - 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.