Integrated genetic and process engineering of Pichia pastoris for high-yield ergothioneine production via methanol-free cultivation.

Ergothioneine (EGT) is a histidine-derived thiol antioxidant with broad applications in food, pharmaceuticals and cosmetics, but its industrial production is limited by scarce natural sources and low heterologous host titers. Here, a high-yield, methanol-free EGT production platform was established in Pichia pastoris X33 via integrated genetic and process engineering: de novo pathway assembly first generated strain P4 with a shake-flask EGT titer of 99.6 ± 3.2 mg/L via screening of the strong constitutive promoter PGCW14, site-directed engineering of Schizophyllum commune methyltransferase ScEgt1a yielded the V17A mutant, which improved the titer to 188.0 ± 8.6 mg/L in strain P11 alongside accumulated hercynine intermediate, introduction of egtB from Methylobacterium pseudosasicola (encoding the hercynine-to-cys-hercynine conversion enzyme) to drive efficient conversion of the accumulated intermediate produced strain P12 with a titer of 328.2 ± 8.3 mg/L, and further systematic optimization of culture medium and process parameters elevated the final shake-flask EGT titer to 1023.3 ± 17.9 mg/L. This work delivers the first constitutive, methanol-free production strategy for EGT, providing a scalable chassis and transferable integrated genetic-process engineering framework that reaches an advanced level among reported yeast-based EGT production systems, offering a versatile blueprint for sustainable biomanufacture of EGT and other high-value sulfur-containing natural products.