Functional characterization of a cycloartenol synthase and four glycosyltransferases in the biosynthesis of cycloastragenol-type astragalosides from Astragalus membranaceus

Characterization of a Malabaricane-Type Triterpene Synthase from Astragalus membranaceus and Enzymatic Synthesis of Astramalabaricosides

“…AmUGT7 could catalyze a reaction of 1a with UDP-Glc to afford 1b at a 45% conversion rate (Figure D). Therefore, these results indicate that AmUGT14/15/7 are not only responsible for the biosynthesis of cycloastragenol-type astragalosides but also might be involved in the biosynthesis of malabaricane-type triterpenoid glycosides in A. membranaceus . , …”

“…AmOSC1 and AmOSC3 have been verified as the lupeol synthase (AmLUS) and cycloartenol synthase (AmCAS1), respectively, in previous work (Figure S1, Supporting Information). 24,26 To confirm the biochemical function of AmOSC2, an engineered yeast strain harboring the recombinant plasmid of pESC-His-AmOSC2 with both pESC-Ura-tHMG1 and pESC-Leu-ERG7 − was constructed. After 7 days of fermentation, the products were extracted and detected by GC-MS, in which an evident product (1) at t R = 27.5 min and a minor product (2) at t R = 23.3 min with MS ion peaks at m/z 444 and 426, respectively, were observed compared with that of the negative control (Figure 1).…”

“…To explore the glycosylation of malabaricane-type triterpenoids in the biosynthesis of astramalabaricosides, we initially screened 24 candidate glycosyltransferases from A. membranaceus. 24 To our delight, three glycosyltransferases (AmUGT14/15/7) were able to recognize 1 or arabidiol 3-O-β-D-glucoside (1a) to generate the corresponding glycosides with UDP-Glc or UDP-Xyl, respectively. HPLC-MS analysis revealed that AmUGT14 could not only convert 1 to 1a with a conversion rate of 68% but also utilize 1 as a substrate with a 36% conversion rate when UDP-Xyl was used as the sugar donor (Figures 5B and Figure S8).…”

“…Therefore, these results indicate that AmUGT14/15/7 are not only responsible for the biosynthesis of cycloastragenol-type astragalosides but also might be involved in the biosynthesis of malabaricane-type triterpenoid glycosides in A. membranaceus. 24,27 Subsequently, two glucosylated products, 1a and 1b, were prepared from scaled-up enzymatic reactions with yields of 44% and 39%, respectively. Their structures were determined by HRESIMS, 1D NMR and 2D NMR, and specific rotation analysis.…”

“…Restriction enzymes and DNA ligase were purchased from Takara Biotechnology Co. Ltd.. inserted into the BamH I restriction enzyme site of pESC-His vector (Table S1), resulting in the pESC-His-AmOSCs for heterologous expression in Saccharomyces cerevisiae INVSc1. 24,37,38 Additionally, the gene encoding truncated HMG-CoA reductase (tHMG1) was cloned into pESC-Leu vector, and the gene encoding antisense lanosterol synthase (ERG7 − ) was cloned into pESC-Ura vector. The three positive recombinant plasmids were transformed into the competent cells of S. cerevisiae INVSc1 simultaneously, instructed by the protocols of Frozen-EZ Yeast Transformation II Kit (Zymo Research).…”