Adaptive laboratory evolution enhances methanol-driven CO₂ fixation in Sporomusa sphaeroides KIAC

Acetogens are microorganisms that reduce carbon dioxide (CO₂) into acetate via the Wood-Ljungdahl pathway, a process that necessitates an external electron donor. While hydrogen gas is the conventional electron donor, its low aqueous solubility and severe gas-liquid mass transfer limitations significantly restrict industrial scalability. In contrast, methanol has emerged as a promising alternative one-carbon liquid feedstock due to its complete miscibility and abundant availability. This study demonstrates the efficacy of utilizing methanol as an electron donor for CO₂ reduction in the novel acetogen Sporomusa sphaeroides KIAC. To enhance its innate methanol utilization capacity, adaptive laboratory evolution was performed under methanol-CO₂ selection pressure. The resulting evolved strain, mKIAC, exhibited a 3.2-fold increase in methanol consumption rate compared to the wild type (WT). Subsequent genomic and transcriptomic analyses elucidated the genetic basis linking the evolved genotype to its enhanced phenotype. A critical point mutation was identified in the mtaB gene of mKIAC, which encodes a methanol-binding corrinoid methyltransferase, with the mutation predicted to be located near the catalytic zinc-binding pocket. Consistently, a cell-free enzymatic assay using MtaB heterologously produced via an E. coli-based cell-free protein synthesis system demonstrated that the MtaB mutant exhibited a 1.76-fold increase in methanol conversion rate compared to the WT. Collectively, this study highlights the potential of methanol-driven CO₂ utilization as a robust platform for sustainable, carbon-neutral bioprocesses.