Reference: Yin X, et al. (2024) Reengineering the Substrate Tunnel to Enhance the Catalytic Efficiency of Squalene Epoxidase. J Agric Food Chem 72(44):24599-24608

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Abstract


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

Reference Type
Journal Article
Authors
Yin X, Wei W, Chen Q, Zhang Y, Liu S, Gao S, Luo Z, Zhou J
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