Transcriptomic insights into methanol utilization in Pichia pastoris lacking AOX genes under co-feeding conditions.

The methylotrophic yeast Pichia pastoris (P. pastoris) exhibits remarkable capability for methanol-driven protein biosynthesis, positioning it as an attractive platform for carbon-neutral biomanufacturing utilizing methanol as a renewable feedstock. However, challenges arising from methanol metabolism, particularly the accumulation of toxic formaldehyde intermediates, significantly hinder efficient methanol biotransformation. To address this limitation, we implemented a metabolic engineering strategy involving dual knockout of alcohol oxidase genes (aox1 and aox2) combined with glycerol co-substrate supplementation. Using enhanced green fluorescent protein (EGFP) as a model heterologous product, we demonstrated that the ΔAOX1/2 strain achieved superior protein productivity in glycerol-methanol co-feeding cultures. Under optimized conditions (0.5% methanol + 0.4% glycerol), the engineered strain attained a biomass density of 38.5 (OD600) and EGFP fluorescence intensity of 494,723 units, representing improvements of 32.8% and 53.6%, respectively, compared to the wild-type (WT) strain cultivated with 1% methanol alone. Transcriptome profiling revealed that the observed enhancement in protein synthesis originated from optimized methanol utilization through coordinated upregulation of both assimilatory and dissimilatory metabolic modules. This study demonstrates that alcohol oxidase suppression coupled with glycerol co-metabolism constitutes an effective strategy to alleviate methanol-derived metabolic stress while enhancing heterologous protein yields in P. pastoris.