Methanol-induced metabolic disturbances in protein expression in Pichia pastoris: A review of the interaction between amino acid synthesis and higher alcohol pathways.

Pichia pastoris is a prominent host for recombinant protein production, utilizing the methanol-inducible Alcohol Oxidase 1 (AOX1) promoter to express thousands of heterologous proteins. However, methanol induction, while driving high-level protein expression, simultaneously imposes a pronounced metabolic burden. This review highlights that a balanced and ample supply of amino acids is indispensable for efficient protein synthesis. The induction process leads to the accumulation of by-products, notably higher alcohols, which can negatively impact protein quality. The dynamic interplay between these two pathways is not fully understood. In addition, the lack of quantitative models for their competition over shared precursors hinders rational metabolic engineering and impedes the industrial exploitation of P. pastoris. Therefore, this review systematically dissects the link between methanol induction, amino acid metabolism, and higher alcohol synthesis. It specifically examines how methanol-triggered perturbations in amino acid metabolism propagate to modulate higher alcohol formation. Special attention is given to key metabolic nodes, such as pyruvate and α-ketoisovalerate, where carbon flux diverges between biosynthesis and by-product formation. This review provides significant implications for a deeper understanding of the metabolic processes in P. pastoris, offering comprehensive mechanistic insights and guidance for optimizing yeast fermentation and enhancing recombinant protein production.