Metabolic engineering of Pichia pastoris for sustainable production of 1,8-cineole from methanol

Methanol is an ideal feedstock for biomanufacturing and can be produced in large quantities from carbon dioxide via photocatalysis or electrolytic reduction, contributing to global carbon neutrality. In this study, we described an engineered Pichia pastoris strain capable of efficiently producing the monoterpene 1,8-cineole from methanol. First, a remarkable high-level synthesis of 1,8-cineole was achieved in Pichia pastoris by introducing an exogenous 1,8-cineole synthase, strengthening the MVA pathway, and suppressing the competing pathway. Subsequently, we significantly improved the yield of 1,8-cineole by reconstructing the methanol assimilation pathway and regulating the non-oxidative branch of the pentose phosphate pathway. To mitigate formaldehyde toxicity and redirect carbon flux toward central carbon metabolism for enhanced cellular vitality, the conversion of formic acid to serine was strengthened by overexpressing the ADE3 and SHM2 genes, resulting in an 82.2% increase in 1,8-cineole yield. Finally, we obtained 386.3 mg/L of 1,8-cineole from methanol in a 5 L bioreactor. This study established a metabolic engineering platform for the efficient synthesis of 1,8-cineole in Pichia pastoris based on methanol assimilation, providing a new strategy for the green biomanufacturing of terpenoids and offering a reference for the high-value utilization of methanol in synthetic biology.