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

Abstract

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.