Alcohol oxidase-free methanol assimilation boosts carbon efficiency and product yield in industrial yeasts.

Methanol is an attractive single-carbon feedstock for scalable bioprocesses that avoids the consumption of agricultural products. Native yeast methylotrophy relies on alcohol oxidase (Aox), which dissipates reducing power and limits carbon conversion efficiency. Here, we replace Aox with the native, NAD+-dependent alcohol dehydrogenase 2 and eliminate the methanol dissimilatory branch by deleting formaldehyde dehydrogenase (Fld) in the yeasts Komagataella phaffii and Ogataea parapolymorpha. Engineered strains of both species sustain methylotrophic growth despite each modification being individually growth-defective. FLD deletion eliminates competition for cytosolically produced formaldehyde and enforces flux coupling between methanol utilization and NADH formation. Adaptive laboratory evolution over 30-50 generations yielded growth rates comparable to industrial yeast strains. In methanol-limited chemostats, evolved Adh-Δfld strains reduced specific CO₂ production 2-fold while increasing biomass yield 1.4-fold. The biomass-per-methanol yields of 0.48gg-1 for K. phaffii and 0.43gg-1 for O. parapolymorpha are the highest reported for each species. The Adh-based metabolic framework also enabled gram-per-liter itaconic acid production with higher product yield and lower CO₂ evolution than Aox-based strains, underscoring its utility for growth-coupled bioproduction. Adh-coupled methanol oxidation represents a generalizable strategy to increase carbon efficiency in other C1 utilization pathways and yeast species, providing a foundation for more sustainable biomanufacturing processes with yeasts.