Abstract: To improve the level of squalene production by engineered strain YLM-1 (Po1f, Δku70, ΔMHY1) ofYarrowia lipolytica, theGUT2gene was first knocked out and the endogenousDGA1gene was overexpressed. This modification successfully yielded the engineered strain YLM-3. This strain exhibited enhanced lipid droplet formation and synthesized squalene at a titer of 64.86 mg/L (8.43 mg/g). Subsequently, a polycistronic expression system was constructed using theIGG6sequence to overexpress key genes of the squalene biosynthesis pathway (ScHMG1,IDI, andSQS). This strategy generated the engineered strain YLM-4, which demonstrated significantly enhanced squalene synthesis capacity, achieving a titer of 491.28 mg/L (63.08 mg/g). Finally, fermentation medium optimization revealed that strain YLM-4 could produce squalene at a titer of up to 1225.34 mg/L (62.96 mg/g) after 72 h of fermentation in YPD-70 fermentation medium. This study achieved efficient squalene synthesis inY. lipolytica, laying a solid foundation for the subsequent synthesis of downstream squalene derivatives.

Key words: Yarrowia lipolytica, squalene, metabolic engineering, lipid droplets, polycistronic expression system