Methanol-based biosynthesis of p-coumaric acid by engineered Pichia pastoris.

p-Coumaric acid (p-CA) is a key aromatic precursor for the biosynthesis of flavonoids, stilbenoids, and other high-value phenylpropanoids. While microbial production of p-CA typically relies on sugar-based substrates, methanol offers a sustainable and cost-effective alternative, though its use for aromatic biosynthesis remains unexplored. Here, we report the first de novo production of p-CA from methanol using engineered methylotrophic yeast Pichia pastoris. Through heterologous expression of a tyrosine ammonia-lyase and implementing a balanced push-pull strategy in the shikimate pathway using feedback-resistant variants of DAHP synthase (ARO4) and chorismate mutase (ARO7), carbon flux from methanol-derived C3 and C4 precursors was effectively redirected toward aromatic biosynthesis. Shake-flask studies revealed strong gene-dosage-dependent p-CA production, but strains with high-copy numbers suffered metabolic burden under high-density fermentation. Fed-batch bioreactor cultivation demonstrated that a moderate-copy strain achieved the highest titer of 704 ± 6 mg/L, outperforming high-copy variants in robustness and scalability. This study establishes P. pastoris as a promising chassis for methanol-based aromatic production and highlights the critical trade-off between pathway amplification and cellular fitness in C1 biomanufacturing.