Characterization of cinnamate 4-hydroxylase (CYP73A) and p -coumaroyl 3′-hydroxylase (CYP98A) from Leucojum aestivum , a source of Amaryllidaceae alkaloids

Biosynthesis of Amaryllidaceae alkaloids (AA) starts with the condensation of tyramine with 3,4-dihydroxybenzaldehyde. The latter derives from the phenylpropanoid pathway that involves modifications of trans -cinnamic acid, p -coumaric acid, caffeic acid, and possibly 4-hydroxybenzaldehyde, all potentially catalyzed by hydroxylase enzymes. Leveraging bioinformatics, molecular biology techniques, and cell biology tools, this research identifies and characterizes key enzymes from the phenylpropanoid pathway in Leucojum aestivum. Notably, we focused our work on trans -cinnamate 4-hydroxylase ( Lae C4H) and p -coumaroyl shikimate/quinate 3ʹ-hydroxylase ( Lae C3′H), two key cytochrome P450 enzymes, and on the ascorbate peroxidase/4-coumarate 3-hydroxylase ( Lae APX/C3H). Although Lae APX/C3H consumed p -coumaric acid, it did not result in the production of caffeic acid. Yeasts expressing Lae C4H converted trans -cinnamate to p- coumaric acid, whereas Lae C3′H catalyzed specifically the 3-hydroxylation of p -coumaroyl shikimate, rather than of free p -coumaric acid or 4-hydroxybenzaldehyde. In vivo assays conducted in planta in this study provided further evidence for the contribution of these enzymes to the phenylpropanoid pathway. Both enzymes demonstrated typical endoplasmic reticulum membrane localization in Nicotiana benthamiana adding spatial context to their functions. Tissue-specific gene expression analysis revealed roots as hotspots for phenylpropanoid-related transcripts and bulbs as hubs for AA biosynthetic genes, aligning with the highest AAs concentration. This investigation adds valuable insights into the phenylpropanoid pathway within Amaryllidaceae, laying the foundation for the development of sustainable production platforms for AAs and other bioactive compounds with diverse applications.