Piperonal synthase from black pepper (Piper nigrum) synthesizes a phenolic aroma compound, piperonal, as a CoA-independent catalysis

Piperonal is a simple aromatic aldehyde compound with a characteristic cherry-like aroma and has been widely used in the flavor and fragrance industries. Despite piperonal being an important aroma in black pepper (Piper nigrum), its biosynthesis remains unknown. In this study, the bioinformatic analysis of the P. nigrum transcriptome identified a novel hydratase-lyase, displaying 72% amino acid identity with vanillin synthase, a member of the cysteine proteinase family. In in vivo substrate-feeding and in vitro enzyme assays, the hydratase-lyase catalyzed a side-chain cleavage of 3,4-methylenedioxycinnamic acid (3,4-MDCA) to produce 3,4-methylenedioxybenzaldehyde (piperonal) and thus was named piperonal synthase (PnPNS). The optimal pH for PnPNS activity was 7.0, and showed a K(m) of 317.2 μM and a k(cat) of 2.7 s(−1). The enzyme was most highly expressed in the leaves, followed by the fruit. This characterization allows for the implementation of PnPNS in various microbial platforms for the biological production of piperonal. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13765-022-00691-0.