Characterization and protein engineering of glycosyltransferases for the biosynthesis of diverse hepatoprotective cycloartane-type saponins in Astragalus membranaceus |
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Authors: | Kuan Chen Meng Zhang Baihan Gao Aobulikasimu Hasan Junhao Li Yang'oujie Bao Jingjing Fan Rong Yu Yang Yi Hans Ågren Zilong Wang Haiyang Liu Min Ye Xue Qiao |
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Affiliation: | 1. State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China;2. State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China These authors contributed equally to this work.;3. Department of Physics and Astronomy, Uppsala University, Uppsala, Sweden;4. State Key Laboratory of Phytochemistry and Plant Resources in West China and Yunnan Key Laboratory of Natural Medicinal Chemistry, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China |
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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-O-glycosyltransferase of cycloastragenol respectively. AmGT1G146V/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, AmGT1G146V/S and the reported AmGT8 and AmGT8A394F. 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. |
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Keywords: | astragalosides biosynthesis glycosyltransferases protein engineering Astragalus membranaceus |
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