Abstract
Phenylpropenes, such as isoeugenol and eugenol, are produced as defend compounds, floral attractants, and flavor constituents by phenylpropene synthases belonging to the PIP reductase family. Moreover, isoeugenol is proposed to be involved in the biosynthesis of dibenzocyclooctadiene lignans, the main active compounds of Schisandra chinensis (Turcz.) Baill. fruits (SCF). S. chinensis, a woody vine plant, is widely used for its medicinal, horticultural, edible, and economic values. In this study, nine ScPIP genes were identified and characterized from the transcriptome datasets of SCF. The expression profiles revealed that ScPIP genes were differentially expressed during different developmental stages of SCF. Three ScPIPs were selected and cloned as candidate genes encoding phenylpropene synthases according to phylogenetic analysis. ScPIP1 was proved to function as isoeugenol synthase (IGS) and designated as ScIGS1 through in vivo functional characterization in Escherichia coli. Subcellular localization analysis demonstrated that ScIGS1 was localized in both the cytoplasm and nucleus. The three-dimensional (3D) model of ScIGS1 was obtained using homology modeling. Site-directed mutagenesis experiments revealed that the substitution of residues at positions 110 and 113 impacted the product specificity of ScIGS1 and the mutation of Lys157 to Ala abolishing catalytic function. Moreover, the k(cat) values of mutants were lower than that of ScIGS1 using a deep learning approach. In conclusion, this study provides a basis for further research on PIP reductases and the biosynthetic pathway of dibenzocyclooctadiene lignans.