Alcohol dehydrogenase-mediated methanol dissimilation increases carbon efficiency in synthetic autotrophic yeast

The efficient production of food and biochemicals using microorganisms that utilize single-carbon feedstocks presents a promising approach for advancing a circular bioeconomy. Komagataella phaffii (formerly Pichia pastoris) is a methylotrophic yeast already widely used in industry, making it an attractive host for such applications. Recently, K. phaffii was converted into an autotrophic strain capable of assimilating CO₂ into both biomass and secreted organic acids, using energy derived from dissimilation of methanol to CO₂. In these strains, methanol oxidation is catalysed by an alcohol oxidase (Aox2), which transfers electrons to oxygen without conserving reducing equivalents. To address this limitation, in this study we explored redirecting methanol dissimilation through the native alcohol dehydrogenase (Adh2), coupling methanol oxidation with NADH generation to improve carbon efficiency. By deleting AOX2 and overexpressing ADH2, we generated Adh2-based autotrophic strains that exhibited growth rates comparable to the parental strain (0.007 h^-1), while reducing specific CO₂ production by 53% and increasing biomass yield (Y_X/MeOH) by 59%. We further applied this strategy to convert previously developed autotrophic strains producing itaconic acid and lactic acid into Adh2-dependent strains. Optimizing ADH2 expression through multicopy integration resulted in strains with approximately two-fold higher molar carbon efficiency (Y_{(X + P)/CO2}) while achieving elevated product titers-2.2-fold for itaconic acid and 3.8-fold for lactic acid-relative to the parental strains. Our findings demonstrate that alcohol dehydrogenase-mediated methanol dissimilation can significantly improve yield and productivity of autotrophic K. phaffii strains, with broad implications for sustainable bioproduction from one-carbon substrates.