Structural and functional characterization of genes and enzymes involved in withanolide biosynthesis in Physalis alkekengi L

Physalis alkekengi L. is recognized as a significant source of various secondary metabolites, particularly c28 steroidal lactones known as withanolides and physalins, renowned for their therapeutic properties with a rich history in traditional medicine. In this study, we characterized the sequences of key downstream genes (PaFPPS, PaSQS, PaSQE, PaCAS, PaHYD1, and PaDWF5-1) involved in the biosynthesis of withanolides, marking the first characterization of these genes in P. alkekengi. Our findings revealed highly conserved amino acid sequences in P. alkekengi, with maximum similarity observed with Withania somnifera. Notably, essential domains crucial for enzyme function were preserved in P. alkekengi, indicating conserved enzyme activity. Comparative analysis of secondary structures, 3D topologies, and evolutionary studies supported ancestral homology. Investigations into the differential gene expression of these genes across seven tissues (young leaves, stems, roots, flowers, mature green fruit, breaker fruit, and red ripe fruit) highlighted higher expression levels in P. alkekengi leaves. These gene expression patterns were corroborated by phytochemical analyses using chromatographic techniques. High-Performance Liquid Chromatography (HPLC) confirmed the production of two key withanolides, withanolide A and withanone, in P. alkekengi, with maximum production observed in leaves and flowers. These findings suggest that P. alkekengi holds promise as an alternative to W. somnifera for large-scale industrial production of withanolides, particularly withanolide A. Using P. alkekengi eliminates the need to sacrifice the plant, which is typically required in traditional extraction methods from the roots of W. somnifera.