Functional identification of five CYP450 genes from birch responding to MeJA and SA in the synthesis of betulinic acid from lupitol

Abstract

Birch (Betula platyphylla Suk.) bark contains important pentacyclic triterpenes such as betulin and betulinic acid, which possess a range of biological effects, including anti-tumor and anti-HIV activity. In order to meet the worldwide demand for anti-tumor drugs, improvements in triterpenes' production and quality are of crucial importance. Cytochrome P450 monooxygenase (CYP450) is essential for the diversification and functional modification of the triterpene skeleton. In this study, five new CYP450 genes responding to methyl jasmonate (MeJA) and salicylic acid (SA) were cloned from birch. Phylogenetic tree analysis showed that five BpCYP450 genes were located in five subfamilies, named CYP94B89, CYP89S1, CYP97B62, CYP86B54, and CYP86A182. The five CYP450 genes in different tissues and their responses to different stresses were analyzed by quantitative RT-PCR. CYP89S1, CYP97B62, squalene epoxidase (BpSE), and dammarenediol synthase (BpDS) were highly expressed in leaves. The results of quantitative gene expression and correlation analysis suggested that CYP89S1, CYP97B62 with BpSS, BpW, BpDS, and BpY might be co-expressed, which may affect the synthesis of triterpenoids in birch as gene clusters or co-expression module. Ectopic expression showed that CYP89S1, CYP97B62, CYP86A182 had C-28 oxidation function and catalyzed the conversion of lupeol to betulinic acid. CYP97B62 gene has the highest catalytic efficiency, increasing the content of betulinic acid by 1136 %. In addition, co-expression of BpMYB21 and CYP86A182 can significantly enhance the conversion and synthesis efficiency of betulinic acid in tobacco (Nicotiana tabacum L.); CYP89S1 can enhance salt and alkali resistance in yeast (Saccharomyces cerevisiae). In this study, the discovery of five CYP450 provided new and important gene resources for the regulation of betulinic acid synthesis and the further analysis of the triterpene metabolic network.