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.