Functional Characterization of <i>Pl</i> C4H1 and <i>Pl</i> C3H1: Essential for Furofuran Lignan Biosynthesis in <i>Phryma leptostachya</i>

This study aimed to elucidate the enzymatic basis of furofuran lignan biosynthesis in <i>Phryma leptostachya</i>. By integration of bioinformatics, molecular biology, and metabolic engineering approaches, two critical cytochrome P450 enzymes, <i>Pl</i>C4H1 (<i>trans</i>-cinnamate 4-hydroxylase) and <i>Pl</i>C3H1 (<i>p</i>-coumarate 3-hydroxylase), were identified and functionally characterized. Bioinformatic analyses revealed conserved P450 motifs across candidate enzymes. Tissue-specific expression profiling showed that <i>Pl</i>C4H1 and <i>Pl</i>C3H1 were highly expressed in roots, aligning with regions of lignans accumulation. Yeast functional assays demonstrated that <i>Pl</i>C4H1 catalyzes the hydroxylation of <i>trans</i>-cinnamic acid to <i>p</i>-coumaric acid. Notably, <i>Pl</i>C3H1 exhibited broader substrate flexibility, hydroxylating 5-<i>O</i>-<i>p</i>-coumaroylquinic acid to 5-<i>O</i>-caffeoylquinic acid and <i>p</i>-coumaraldehyde to caffeic aldehyde, with a marked preference for 5-<i>O</i>-<i>p</i>-coumaroylquinic acid. In planta assays, further substantiation of the involvement of these enzymes in the phenylpropanoid pathway highlighted their enzymatic contribution to lignan biosynthesis. These findings not only uncover two key molecular players in furofuran lignan biosynthesis but also establish a foundation for engineering sustainable biosynthetic platforms to enhance lignan yields and develop novel bioactive metabolites.