College of Life and Environmental Sciences, Minzu University of China, 27 Zhong Guan Cun Southern Street, Beijing 100081, China.
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 1 Xian Nong Tan Street, Beijing, 100050, China.
Org Biomol Chem. 2019 Sep 21;17(35):8106-8114. doi: 10.1039/c9ob01352k. Epub 2019 Aug 28.
Icariin (1a), a 7-O-glycosylated flavonoid glycoside, is recognized as the major pharmacologically active ingredient of Epimedium plants, which have been used in traditional Chinese medicine for thousands of years. However, no glycosyltransferase (GT) responsible for the 7-O-glycosylation of flavonoids has been identified from Epimedium plants to date. Herein, a GT, Ep7GT, was identified from E. pseudowushanense B. L. Guo, which can regiospecifically transfer a glucose moiety to baohuoside (1) at 7-OH to form icariin (1a). Ep7GT showed a rare broad donor substrate spectrum, including UDP-glucose, UDP-xylose, UDP-N-acetylglucosamine, UDP-rhamnose, UDP-galactose, UDP-glucuronic acid and TDP-glucose. Moreover, two new derivatives of icariin (1a), 7-O-β-d-[2-(acetylamino)-2-deoxy-glucopyranosyl]-baohuoside (1b) and 7-O-β-d-xylosyl-baohuoside (1c), were biosynthesized by using Ep7GT in vitro. Engineered Escherichia coli harbouring Ep7GT was constructed, and 10.1 μg mL icariin (1a) was yielded by whole-cell biotransformation with baohuoside (1) as the substrate. The present work not only characterizes the GT responsible for the 7-O-glycosylation in the biosynthesis of icariin in Epimedium plants, but also indicates the significant potential of an enzymatic approach for the production of glycosylated baohuoside derivatives with different sugar moieties. What's more, these findings also provide a promising alternative for producing natural/unnatural bioactive flavonoid glycosides by metabolic engineering.
朝藿定 C（1a）是一种 7-O-糖基化的黄酮类糖苷，被认为是淫羊藿属植物中主要的具有药理活性的成分，这些植物在中国传统医学中已经使用了数千年。然而，迄今为止，尚未从淫羊藿属植物中鉴定出负责黄酮类 7-O-糖基化的糖基转移酶（GT）。在此，从 Epimedium pseudowushanense B. L. Guo 中鉴定出一种 GT，即 Ep7GT，它可以在 7-OH 位置将葡萄糖部分特异性地转移到宝藿苷（1）上，形成朝藿定 C（1a）。Ep7GT 表现出罕见的广泛供体底物谱，包括 UDP-葡萄糖、UDP-木糖、UDP-N-乙酰葡萄糖胺、UDP-鼠李糖、UDP-半乳糖、UDP-葡萄糖醛酸和 TDP-葡萄糖。此外，通过使用 Ep7GT 在体外合成了朝藿定 C（1a）的两种新衍生物，7-O-β-D-[2-（乙酰氨基）-2-脱氧-吡喃葡萄糖基]-宝藿苷（1b）和 7-O-β-D-木糖基宝藿苷（1c）。构建了含有 Ep7GT 的工程大肠杆菌，并以宝藿苷（1）为底物进行全细胞生物转化，可产生 10.1 μg mL 的朝藿定 C（1a）。本研究不仅鉴定了淫羊藿属植物中朝藿定 C 生物合成中负责 7-O-糖基化的 GT，而且还表明了酶法生产具有不同糖基的糖基化宝藿苷衍生物的巨大潜力。更重要的是，这些发现还为通过代谢工程生产天然/非天然生物活性黄酮类糖苷提供了一种有前途的替代方法。
