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 <i>Yarrowia lipolytica</i> through integrated metabolic pathway reconstruction and adaptive laboratory evolution. First, overexpression of <i>DGA1</i> and <i>LRO1</i> 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 <i>Y. lipolytica</i>, establishing the reference for synthesizing squalene and its derived compounds in engineered <i>Y. lipolytica</i>.