Expanding the versatility of the protein master Pichia pastoris towards efficient biosynthesis of diterpenoid...

Non-conventional yeasts offer unique advantages as tailored cell factories. Pichia pastoris is a master of protein expression and secretion. However, engineering P. pastoris for the overproduction of secondary metabolites is challenging due to its tightly regulated metabolism. How can we overcome this metabolic rigidity for efficient biosynthesis of energy-demanding molecules such as sclareol, a valuable diterpenoid? In this research article, we developed a host-specific framework integrating systematic pathway-level rewiring of the sclareol biosynthetic pathway and central metabolism to alleviate precursor acetyl-coenzyme A and cofactor NADPH limitations. Regulatory-level remodeling identified metabolic factors that enhance cellular robustness and protein homeostasis, reprogrammed global transcriptional regulators for carbon and nitrogen flux balance, and tailored process-level fermentation to eliminate byproducts. These efforts enabled a sclareol titer of 27.8 g l^-1 in minimal medium, representing the highest diterpenoid production reported. This work redefines P. pastoris as a powerful chassis for natural product biosynthesis, providing a generalizable roadmap for engineering non-conventional hosts.