Genome-Wide Identification and Functional Verification of Core BBEL Genes for BIA Biosynthesis in Corydalis saxicola Bunting

Benzylisoquinoline alkaloids (BIAs) are significant defensive and pharmaceutical metabolites found in medicinal plants of the Papaveraceae family. Among these, dehydrocavidine is a distinctive bioactive constituent of Corydalis saxicola Bunting, an endangered medicinal plant endemic to karst habitats, known for its definite hepatoprotective, anti-inflammatory, and antiviral pharmacological properties. Berberine Bridge Enzyme-Like (BBEL) proteins serves as key rate-limiting enzymes catalyzing core skeletal oxidation in BIA biosynthesis. However, their functions in C. saxicola. remain uncharacterized. In this study, we performed a comprehensive functional genomic analysis of the BBEL family in C. saxicola (CsBBELs). A total of 22 CsBBEL members were identified from the genome of C. saxicola, all of which possess the conserved FAD-binding domain and BBE functional domain characteristic of the BBEL family. Phylogenetic analysis revealed that the CsBBEL family formed three clades closely clustered with homologs from related Papaveraceae species, indicating high conservation. Expression profiles demonstrated significant tissue specificity and Ca²⁺ stress response of the CsBBEL genes, with CsBBEL3, CsBBEL16, and CsBBEL17 being particularly highly expressed in roots. Protein structure prediction and molecular docking suggested that these candidates bind cavidine and tetrahydrocolumbamine. In vitro enzymatic assays confirmed that CsBBEL3/16/17 specifically catalyzed the four-electron oxidation of cavidine and tetrahydrocolumbamine to produce dehydrocavidine and columbamine, respectively, exhibiting oxidase activity in the BIA biosynthetic pathway. These findings not only contribute to germplasm conservation but also lay a foundation for the synthetic biology-based improvement of C. saxicola.