Versatile CYP98A enzymes catalyse meta‐hydroxylation reveals diversity of salvianolic acids biosynthesis

Summary Salvianolic acids (SA), such as rosmarinic acid (RA), danshensu (DSS), and their derivative salvianolic acid B (SAB), etc. widely existed in Lamiaceae and Boraginaceae families, are of interest due to medicinal properties in the pharmaceutical industries. Hundreds of studies in past decades described that 4‐coumaroyl‐CoA and 4‐hydroxyphenyllactic acid (4‐HPL) are common substrates to biosynthesize SA with participation of rosmarinic acid synthase (RAS) and cytochrome P450 98A (CYP98A) subfamily enzymes in different plants. However, in our recent study, several acyl donors and acceptors included DSS as well as their ester‐forming products all were determined in SA‐rich plants, which indicated that previous recognition to SA biosynthesis is insufficient. Here, we used Salvia miltiorrhiza , a representative important medicinal plant rich in SA, to elucidate the diversity of SA biosynthesis. Various acyl donors as well as acceptors are catalysed by Sm RAS to form precursors of RA and two Sm CYP98A family members, Sm CYP98A14 and Sm CYP98A75, are responsible for different positions' meta ‐hydroxylation of these precursors. Sm CYP98A75 preferentially catalyses C‐3′ hydroxylation, and Sm CYP98A14 preferentially catalyses C‐3 hydroxylation in RA generation. In addition, relative to C‐3′ hydroxylation of the acyl acceptor moiety in RA biosynthesis, Sm CYP98A75 has been verified as the first enzyme that participates in DSS formation. Furthermore, Sm CYP98A enzymes knockout resulted in the decrease and overexpression leaded to dramatic increase of SA accumlation. Our study provides new insights into SA biosynthesis diversity in SA‐abundant species and versatility of CYP98A enzymes catalytic preference in meta ‐hydroxylation reactions. Moreover, CYP98A enzymes are ideal metabolic engineering targets to elevate SA content.