Functional identification of the terpene synthase family involved in diterpenoid alkaloids biosynthesis in Aconitum carmichaelii

Aconitum carmichaelii is a high-value medicinal herb widely used across China, Japan,and other Asian countries. Aconitine-type diterpene alkaloids(DAs) are the characteristic compounds in Aconitum. Although six transcriptomes, based on short-read next generation sequencing technology, have been reported from the Aconitum species, the terpene synthase(TPS) corresponding to DAs biosynthesis remains unidentified. We apply a combination of Pacbio isoform sequencing and RNA sequencing to provide a comprehensive view of the A. carmichaelii transcriptome. Nineteen TPSs and five alternative splicing isoforms belonging to TPS-b, TPS-c, and TPS-e/f subfamilies were identified. In vitro enzyme reaction analysis functional identified two sesqui-TPSs and twelve di TPSs.Seven of the TPS-c subfamily genes reacted with GGPP to produce the intermediate ent-copalyl diphosphate. Five Ac KSLs separately reacted with ent-CPP to produce ent-kaurene, ent-atiserene,and ent-13-epi-sandaracopimaradie: a new diterpene found in Aconitum. Ac TPSs gene expression in conjunction DAs content analysis in different tissues validated that ent-CPP is the sole precursor to all DAs biosynthesis, with Ac KSL1, Ac KSL2 s and Ac KSL3-1 responsible for C20atisine and napelline type DAs biosynthesis, respectively. These data clarified the molecular basis for the C20-DAs biosynthetic pathway in A. carmichaelii and pave the way for further exploration of C19-DAs biosynthesis in the Aconitum species.