Metabolic engineering and adaptive laboratory evolution enhance squalene production in Yarrowia lipolytica

Squalene, a multifunctional natural compound with diverse bioactivities, has significant potential in the nutraceutical and health industries. Microbial synthesis using engineered cell factories represents a sustainable alternative to conventional methods of extracting from plants or animals. This study systematically optimized squalene production in Yarrowia lipolytica through integrated metabolic pathway reconstruction and adaptive laboratory evolution. First, overexpression of DGA1 and LRO1 induced lipid droplet expansion and proliferation to achieve efficient steady-state accumulation of intracellular squalene. Second, the catalytic efficiency between ERG20 and SQS was enhanced by fusing ERG20-SQS through an enzyme fusion strategy for increasing the squalene synthesis flux. Then, the expression of ScHMG1, the rate-limiting enzyme of the MVA pathway, was further enhanced to optimize precursor supply. Finally, adaptive laboratory evolution induced by hydrogen peroxide generated the evolved strain SY8-H3, which produced 801.34 mg/L of squalene in shake-flask fermentation and 4.53 g/L via fed-batch fermentation in a 2.4 L bioreactor. This research firstly applies the oxidative stress-driven adaptive evolution to enhance squalene biosynthesis in Y. lipolytica, establishing the reference for synthesizing squalene and its derived compounds in engineered Y. lipolytica.