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

A proposed model showing the role of MeJA and GA regulated transcript levels of BpCYP450 in triterpenoids biosynthesis. MeJA and GA regulate the expression of key enzymes of BpW , CYP89S1 , CYP97B62 and CYP86A182 at the transcriptional level. BpMYB21 binds to the MBS region of CYP86A182 promoter and activates the transcriptional process of CYP86A182 ; and BpW , CYP89S1 , CYP97B62 and CYP86A182 jointly regulate the triterpenoids biosynthesis, and most possibly located in the same gene cluster. In addition, CYP89S1 has the ability to increase the resistance of plants to abiotic stress. The genes shown by the purple dotted line connection may be located in the same gene cluster, and the orange dotted line shows that MeJA and GA may be involved in other pathways of triterpenoids synthesis. • CYP450 and BpW displayed similar expression patterns by hormones. • Three CYP450 new genes catalyze the conversion of lupeol to betulinic acid. • CYP89S1 can enhance salt and alkali resistance in yeast. 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.