Characterization and protein engineering of glycosyltransferases for the biosynthesis of diverse hepatoprotective cycloartane‐type saponins in Astragalus membranaceus

Abstract

Although plant secondary metabolites are important source of new drugs, obtaining these compounds is challenging due to their high structural diversity and low abundance. The roots of Astragalus membranaceus are a popular herbal medicine worldwide. It contains a series of cycloartane-type saponins (astragalosides) as hepatoprotective and antivirus components. However, astragalosides exhibit complex sugar substitution patterns which hindered their purification and bioactivity investigation. In this work, glycosyltransferases (GT) from A. membranaceus were studied to synthesize structurally diverse astragalosides. Three new GTs, AmGT1/5 and AmGT9, were characterized as 3-O-glycosyltransferase and 25-Oglycosyltransferase of cycloastragenol respectively. AmGT1 G146V/I variants were obtained as specific 3-O-xylosyltransferases by sequence alignment, molecular modelling and site-directed mutagenesis. A combinatorial synthesis system was established using AmGT1/5/9, AmGT1 G146V/S and the reported AmGT8 and AmGT8 A394F . The system allowed the synthesis of 13 astragalosides in Astragalus root with conversion rates from 22.6% to 98.7%, covering most of the sugar-substitution patterns for astragalosides. In addition, AmGT1 exhibited remarkable sugar donor promiscuity to use 10 different donors, and was used to synthesize three novel astragalosides and ginsenosides. Glycosylation remarkably improved the hepatoprotective and SARS-CoV-2 inhibition activities for triterpenoids. This is one of the first attempts to produce a series of herbal constituents via combinatorial synthesis. The results provided new biocatalytic tools for saponin biosynthesis.

Construction of a UDP-Arabinose Regeneration System for Efficient Arabinosylation of Pentacyclic Triterpenoids

“…The sugar donor activity is not the same for the different acceptors OA, BA, and GA, which may be because both the acceptor and donor participate in the glycosylation reaction, so that the sugar donor activity will be affected by the acceptor. This situation has also been reported for other UGTs. , Based on these results, UGT99D1 was highly specific for UDP-Ara and showed minor activity toward UDP-Xyl, which depended on the acceptor. Subsequently, all the arabinosylated products were verified using liquid chromatography–mass spectrometry (LC–MS; Figures S2–S10), indicating that one moiety of arabinose was transferred to compounds 1 , 2 , and 3 .…”

“…This situation has also been reported for other UGTs. 30,31 Based on these results, UGT99D1 was highly specific for UDP-Ara and showed minor activity toward UDP-Xyl, which depended on the acceptor. Subsequently, all the arabinosylated products were verified using liquid chromatography−mass spectrometry (LC−MS; Figures S2−S10), indicating that one moiety of arabinose was transferred to compounds 1, 2, and 3.…”

Progress in Identification of UDP-Glycosyltransferases for Ginsenoside Biosynthesis

“…AmGT1/5 and AmGT8 derived from A. membranaceus (Leguminosae) are responsible for 3- O - and 2′- O -glycosylation of astragalus saponins. , Interestingly, when expanding the range of substrate screening, AmGT1 and AmGT5 were found to also catalyze specific C-3 OH glycosylation of 20­( S / R )-PPT and 20­( S / R )-PPD (Table , Figures and ). In addition, a new compound, 3- O -xyloside of 20­( R )-PPD, was purified and characterized from the scaled-up reaction of AmGT1.…”

Exploring in vitro Oryzalin-Induced Polyploidy in Astragalus membranaceus: Implications for Gene Expression

“…The roots (Radix Astragali) are rich in bioactive compounds (esp. triterpene glycosides, flavonoids, saponins and alkaloids; (Zhang et al 2021), and are recognised for their immunomodulatory (Qader et al 2021), antioxidant (Cui et al 2022), antibacterial (Samuel et al 2021), hepatoprotective (Chen et al 2023b) diuretic (Jia et al 2021), antidiabetic (Chen et al 2022), anticancer (Liu et al 2023), and anti-inflammatory properties (Chen et al 2023a), making it a staple in traditional medicine (Md Ashif Ikbal et al 2022). Nevertheless, there is an inherent need to increase productivity and phytochemical content to meet the increasing demands of the pharmaceutical and nutraceutical industries (Wu et al 2003).…”