Evolutionary Adaptation and Targeted Metabolic Engineering Synergistically Improve Methanol‐Derived Single‐Cell Protein Production in Pichia pastoris

Methanol is a promising renewable C1 feedstock for sustainable single-cell protein (SCP) production. However, its inherent cytotoxicity and metabolic trade-offs between cell growth and protein synthesis remain significant bottlenecks. Here, we established an "evolutionary-rational" dual-driven paradigm to construct a high-yield Pichia pastoris chassis. Through UV mutagenesis and adaptive laboratory evolution, we developed a highly tolerant strain A40, capable of growing in 70 g/L methanol. Notably, at 30 g/L methanol, A40 achieved a 3.4-fold higher maximum biomass than the wild-type. Whole-genome resequencing and reverse genetics revealed that this superior performance stems from a multi-gene synergistic network rather than a single dominant mutation. To further optimize SCP production, we rationally co-overexpressed nitrogen assimilation genes (GLN1, GDH1) and a translation elongation factor (PpeEF3) in the A40 background. This targeted metabolic engineering effectively redirected carbon flux toward protein biosynthesis. The engineered strain A40-2Ge3 achieved a peak intracellular crude protein content of 67.9% and a 51.3 g/L total titer in a 5-L bioreactor, representing a 23.1% increase over the wild-type strain. Collectively, this study provides deep insights into the synergistic mechanisms of methanol adaptation and establishes an efficient, scalable strategy for sustainable SCP production from C1 feedstocks.