Engineering of a High-Performance Pichia pastoris Cell Factory for Methanol-Driven Biosynthesis of p -Coumaric Acid

The transition from sugar-based biorefining to C1-based biomanufacturing represents a pivotal step toward sustainable chemical production. In this study, we report the design and construction of a novel Pichia pastoris cell factory for the de novo biosynthesis of p-coumaric acid (p-CA), a valuable pharmaceutical precursor, using methanol as the carbon source. To overcome the inherent challenges of methanol and product toxicity, an engineered P. pastoris S12 strain with enhanced phospholipid biosynthesis was employed as the chassis, conferring superior tolerance to both methanol and p-CA. By integration of dual biosynthetic pathways originating from tyrosine and phenylalanine, a titer of 1.83 g/L was achieved. Systematic metabolic engineering, which includes the deregulation of feedback inhibition in the shikimate pathway, the enhancement of aromatic amino acid precursor supply, and improved methanol utilization, collectively increased p-CA production to 2.80 g/L in flask cultures. Remarkably, scale-up in a 7 L bioreactor yielded a final titer of 15.58 g/L, which is the highest level reported to date for p-CA production from any C1 feedstock. This work not only establishes a scalable and sustainable route for p-CA synthesis but also provides a strategic blueprint for advancing the C1-based biomanufacturing of complex plant secondary metabolites.