Metabolic engineering of Pichia pastoris as an industrial chassis enables biosynthesis of dopamine from methanol

Methanol, as a renewable and carbon-neutral single-carbon (C1) feedstock, has emerged as an ideal carbon source for green biomanufacturing due to its non-competition with food resources and scalability for industrial production. Here, we report the first efficient biosynthesis of dopamine from methanol through systematic metabolic engineering strategies in P. pastoris. Specifically, overexpressing high-activity tyrosine hydroxylase and enhancing shikimate pathway flux yielded 579 mg/L dopamine. To prevent degradation, key dopamine catabolic enzymes (PAS_chr1-4_0441) were knocked out. By reinforcing NADH regeneration and accelerating methanol assimilation, the titer increased to 1533 mg/L, an 84.2-fold increase from the first-generation strain. Finally, we optimized the fermentation process in a 15 L fermenter to minimize dopamine autoxidation, achieving a highest reported dopamine titer using methanol as the sole carbon source to date (12.2 g/L). This study not only validates methanol as a high-performance substrate for industrial microbiology, but also establishes a critical foundation for synthesizing dopamine and its derivatives from C1 feedstocks.