De Novo Biosynthesis of Sabinene from Methanol by Multiple Engineered Pichia pastoris

Sabinene, a high-value monoterpene with broad industrial applications, is facing growing demand. To enable sustainable and cost-efficient production, we engineered the yeast Pichia pastoris for sabinene production from methanol. Through systematic metabolic engineering, we implemented a multipronged strategy to optimize sabinene biosynthesis: (1) enhancing precursor supply and synthase activity via fusion proteins (ERG20^WW-tNPPS1 and ERG20^WW-t37SabS1 (H561F)), (2) redirecting carbon flux through the phosphoketolase-phosphotransacetylase pathway, and (3) upregulating the mevalonate pathway via overexpression of a truncated HMGR. The engineered strain achieved record-high sabinene titers of 3.24 g/L in shake flasks and 6.38 g/L in a 3-L bioreactor. Notably, this yield surpasses those achieved with conventional sugar-based feedstocks in other microbial hosts, underscoring the potential of methanol as a superior carbon source for terpenoid biosynthesis. This work not only establishes P. pastoris as a promising platform for sabinene production, but also paves the way for methanol-based biomanufacturing of diverse natural products.