Convergent Evolution and De Novo Reconstitution of Apiin Biosynthesis

Apiin, an active flavonoid apioside, occurs in phylogenetically distant plants, such as parsley and chilli pepper (Capsicum annuum L.). Its sporadic distribution suggests the existence of convergent apiin biosynthesis; however, the molecular mechanisms underlying this convergence remain unclear. In this study, apiin biosynthesis in chilli pepper was comprehensively clarified. The structural genes involved in the biosynthesis of apigenin 7-O-glucoside, a precursor of apiin, were conserved using a homology-based approach. An apiin-biosynthetic apiosyltransferase (ApiGT), CaApiGT1, was identified from Capsicum annuum using comparative transcriptomic technology and in vitro enzymatic assays. Despite its strict specificity for the UDP-apiose donor, CaApiGT1 recognised multiple acceptors, including flavonoid 7-, 4'-and 3-O-glycosides. The sequence identity between CaApiGT1 and other apiin-biosynthetic ApiGTs was below 30%. CaApiGT1 (UGT79) and other apiin-biosynthetic ApiGTs (UGT94) belong to different UDP-dependent glycosyltransferase (UGT) families. These evidences collectively confirmed that CaApiGT1 is a novel ApiGT. Thus, convergent apiin biosynthesis was essentially caused by the convergent evolution of ApiGTs, rather than by distinct biosynthetic pathways between phylogenetically distant plants. Subsequently, CaApiGT1 was expressed in Escherichia coli to construct an engineered strain (EAA3PF) for apiin synthesis from apigenin. Additionally, an engineered Saccharomyces cerevisiae strain (YA3P) was developed for de novo apigenin synthesis. Co-cultivation of EAA3PF and YA3P reconstitutes the apiin biosynthetic pathway. This study provides new insight into the molecular convergence and de novo reconstitution of apiin biosynthesis.