Functional validation of Capsicum frutescens aminotransferase gene involved in vanillylamine biosynthesis using Agrobacterium mediated genetic transformation studies in Nicotiana tabacum and Capsicum frutescens calli cultures

Capsaicinoid biosynthesis involves the participation of two substrates viz. vanillylamine and C9-C11 fatty acid moieties. Vanillylamine which is a derivative of vanillin is synthesized through a transaminase reaction in the phenylpropanoid pathway of capsaicinoid synthesis. Here we report the functional validation of earlier reported putative aminotransferase gene for vanillylamine biosynthesis in heterologous system using Agrobacterium mediated genetic transformation studies in Nicotiana tabacum and Capsicum frutescens calli cultures. Molecular analysis tools comprising PCR and Southern blot analysis have shown the integration of the foreign gene in N. tabacum and C. frutescens calli cultures. The study shows the production of vanillylamine in transformed N. tabacum callus cultures and also the reduction of vanillylamine production when whole gene based antisense binary vector construct was used in transformation of C. frutescens callus cultures. Vanillylamine production, aminotransferase assay with Western blot analysis for crude proteins of transformants established the production of putative aminotransferase (pAMT) protein in alternate plant. The result is a clear evidence of involvement of the reported putative aminotransferase responsible for vanillylamine biosynthesis in capsaicinoid biosynthesis pathway, confirming the gene function through functional validation.

► Vanillylamine is immediate precursor for all capsaicinoids. ► pAMT is involved in vanillylamine biosynthesis is unique to Capsicum sp. ► pAMT gene expressed in transgenic N. tabacum calli cultures produced vanillylamine. ► pAMt gene silenced restricted capsaicinoid production in C. frutesens calli cultures. ► Transformation experiments clearly reveal functional attributions of pAMT.