Reengineering the Substrate Tunnel to Enhance the\nCatalytic Efficiency of Squalene Epoxidase

Squalene\nepoxidase plays a pivotal role in the biosynthesis of\nergosterol, its derivatives, and other triterpenoid compounds by catalyzing\nthe transformation of squalene into 2,3-oxidosqualene. However, its\nlow catalytic efficiency remains a primary bottleneck for the microbial\nsynthesis of triterpenoids. In this study, the catalytic activity\nof the squalene epoxidase from Saccharomyces cerevisiae was significantly improved by reshaping its substrate tunnel, resulting\nin a marked increase in the yield of the final product, ergosterol.\nFirst, the amino acid in the catalytic pocket of squalene epoxidase\nwas replaced with alanine (Ala), effectively reducing the steric hindrance,\nand thus, enhancing the affinity of the enzyme with its substrate.\nThen, the V249H/L343A mutant was obtained by redesigning the substrate\ntunnel of dominant mutant L343A, thus, increasing the titer of ergosterol.\nThe study also elucidated the mechanism behind the increased catalytic\nactivity of the V249H/L343A mutant through substrate tunnel parameter\nanalysis and molecular dynamics simulations. Finally, a titer of 3345\nmg/L of ergosterol was achieved by strains containing V249H/L343A\nin a 5 L bioreactor, with a specific yield of 84 mg/g dry cell weight\n(DCW), marking a 64% increase compared with the titer achieved by\nwild type strains. This study established a strong foundation for\nimproving the synthetic efficiency of ergosterol and other triterpenoid\ncompounds.