代谢工程改造酿酒酵母高效合成熊果酸

Metabolic engineering of Saccharomyces cerevisiae for efficient synthesis of ursolic acid

熊果酸是一种五环三萜类化合物,具有抗炎、抗氧化等作用,广泛应用于医药、化妆品等领域。为提高酿酒酵母中熊果酸的产量,该研究采用了代谢工程的“推-拉”策略。首先,通过异源表达和优化CADDS、CaCYP716A83和HtCPR,实现了熊果酸的从头合成,其产量为5.4 mg/L。随后,通过扩大内质网面积,为膜蛋白酶提供更多的附着面积,熊果酸产量达到40.3 mg/L。由于熊果酸合成路径中涉及多个氧化还原反应,NADPH是必需的辅因子,强化NADPH供给后,熊果酸产量提升至48.2 mg/L。此外,通过优化电子传递链,利用细胞色素b5参与第二个电子向P450酶转移,增强反应中的电子供给,熊果酸产量达到74.6 mg/L。最后,将扩大内质网、增强NADPH及电子传递、调节CaCYP716A83和HtCPR拷贝数等策略进行迭代整合,熊果酸产量达到84.3 mg/L。该研究为构建高效合成萜类化合物的微生物细胞工厂提供了参考。

Ursolic acid,a pentacyclic triterpene compound with anti-inflammatory and antioxidant effects,has been widely applied in medicine,cosmetics,and various other fields.In this study,a“push-pull”metabolic engineering strategy was employed to increase the production of ursolic acid in Saccharomyces cerevisiae.Firstly,de novo biosynthesis of ursolic acid was achieved by heterologous expression and optimization of CADDS,CaCYP716A83,and HtCPR genes,resulting in 5.4 mg/L of ursolic acid.Subsequently,an expansion of the endoplasmic reticulum area was conducted to provide more adherent surface for membrane protease activity,obtaining 40.3 mg/L of ursolic acid.Due to multiple REDOX reactions was involved in the ursolic acid synthesis pathway,in which the NADPH was an essential cofactor.By enhancing the supply of NADPH,the ursolic acid titer was increased to 48.2 mg/L.Additionally,optimization of the electron transport chain and utilization of cytochrome b5 for efficient electron transfer to P450 enzyme further enhanced the electron supply,resulting in 74.6 mg/L of ursolic acid.Finally,by iteratively integrating these strategies including expanding endoplasmic reticulum area,enhancing NADPH availability,and electron transport efficiency and regulating the copy number of CaCYP716A83 and HtCPR genes,the ursoilc acid production was further enhanced to 84.3 mg/L.This study offers valuable insights for the development of efficient microbial cell factories for terpenoid synthesis,serving as a reference in the field.