Systematic metabolic engineering of Komagataella phaffii for methanol-to-lactate bioconversion.

Methanol is an attractive C1 feedstock for sustainable biomanufacturing. However, efficient conversion of methanol into value-added products remains challenging due to metabolic compartmentalization, accumulation of toxic intermediates, and carbon loss in methylotrophic yeasts. In this study, we established a methanol-to-lactate bioconversion platform in Komagataella phaffii through a systematic metabolic engineering strategy that integrates methanol assimilation, detoxification, and downstream carbon flux redirection. Initial expression of a heterologous lactate dehydrogenase enabled efficient lactic acid production from glycerol but failed under methanol conditions, revealing a methanol-specific metabolic bottleneck. Reinforcement of methanol oxidation and oxidative stress mitigation improved methanol utilization and initiated lactic acid production, yet substantial carbon loss persisted through formaldehyde dissimilation. Blocking this dissimilatory pathway led to severe growth defects, suggesting its indispensable role in redox homeostasis and formaldehyde tolerance. In contrast, optimization of the downstream pyruvate node by eliminating competing ethanol formation and enhancing lactate export significantly improved both productivity and yield. The final engineered strain achieved a lactic acid titer of 0.53 g/L with a methanol-based yield of 63.8 mg lactic acid/g methanol. These results reveal that efficient methanol-based lactic acid production requires a holistic strategy that couples robust C1 assimilation with targeted redirection of cytosolic carbon flux and effective product secretion. This work provides a foundational framework for developing scalable C1-based biorefineries for organic acid production.