Identification of a key peptide cyclase for novel cyclic peptide discovery in Pseudostellaria heterophylla

Orbitides, also known as Caryophyllaceae-type cyclic peptides, from Traditional Chinese Medicine plant Pseudostellaria heterophylla (Mil.) Pax have great potential for improving memory and treating diabetes. Orbitides are ribosomally encoded and post-translationally modified peptides, but this key biosynthesis enzyme is still unknown in P. heterophylla. We investigated the orbitide distribution in P. heterophylla and mined novel precursor peptide genes and peptide cyclase from multiple omics data. The function of the key tailoring gene was elucidated using transient heterologous expression and virus-induced gene silencing systems. Our findings suggest that PhPCY3 is a unique gene involved in the cyclization of linear precursor peptides in planta. Molecular docking and multiple sequence alignment, followed by site-directed mutagenesis, showed that N500 and S502 were the key amino acid residues. More than 100 precursor peptide gene sequences were identified, and known active orbitides, such as heterophyllin B and pseudostellarin E/F/G, were successfully biosynthesized. Four novel orbitides, namely cyclo-[LDGPPPYF], cyclo-[WGSSTPHT], cyclo-[GLPIGAPWG], and cyclo-[FGDVGPVI], were identified using the heterologous expression platform. In this study, we describe a gene-guided approach for elucidating the biosynthesis pathway and discovering novel orbitides. Our work provides a strategy for mining and biosynthesizing novel orbitides in P. heterophylla and other plants to further investigate their activities.