Screening CYP450 genes from Gleditsia japonica Miq. and identifying CYP710A157 and CYP71D752 functions in the catalysis of echinocystic acid and betulin

The triterpenes and saponin compounds of Gleditsia japonica Miq. play a key role in the suppression of various human tumor cell lines. Cytochrome P450 monooxygenases (CYP450s) are critical for the triterpene skeleton diversification and functional modification. This study systematically analyzed 104 full-length GjCYP450 genes in G. japonica from northeast China, classifying them into nine clans using bioinformatics. Co-expression modules and response patterns of GjCYP450s with triterpene pathway genes were constructed. Four genes - CYP710A157, CYP714E97, CYP716A377, and CYP71D752 - were selected for functional studies based on their high expression in different tissues of G. japonica and their homology with triterpenoid-related CYP450s in Arabidopsis thaliana. Co-expression of the CYP710A157 gene with the BpY gene (encoding β-amyrin synthase), and CYP71D752 with the BpW gene (encoding lupeol synthase) in tobacco significantly enhanced the catalytic efficiency of echinocystic acid (EA) and betulin (BT) compared to the control, by achieving 10.22-fold and 3.73-fold increases, respectively. Overexpression of CYP710A157 and CYP71D752 in Saccharomyces cerevisiae JWy602 yielded EA and BT at 3.25 mg l⁻¹ and 13.84 mg l⁻¹, respectively, whereas no product accumulation was detected in the control. Additionally, CYP710A157 and CYP714E97 enhanced yeast alkaline tolerance (500 mmol l⁻¹ Na₂CO₃), while CYP716A377 and CYP71D752 improved their salt tolerance (10% NaCl). We reported the catalytic activity of CYP450 genes responsible for EA and BT synthesis within the CYP710A and CYP71D subfamilies in G. japonica for the first time here. These findings provide valuable genetic resources for plants' triterpene biosynthesis, including ginsenosides, and betulinic acid, and insights into regulating the triterpene metabolic network in G. japonica.