De Novo Biosynthesis of Sabinene from Methanol by Multiple Engineered <i>Pichia pastoris</i>

Sabinene, a high-value monoterpene with broad industrial applications, is facing growing demand. To enable sustainable and cost-efficient production, we engineered the yeast <i>Pichia pastoris</i> 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 (<i>ERG20</i>^<i>WW</i>-<i>tNPPS1</i> and <i>ERG20</i>^<i>WW</i>-<i>t37SabS1</i> (<i>H561F</i>)), (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 <i>P. pastoris</i> as a promising platform for sabinene production, but also paves the way for methanol-based biomanufacturing of diverse natural products.