通过定向进化改变糖基转移酶SbGT34供体特异性应用于灯盏花乙素的生物合成

Natural glycosides (such as scutellarin) exhibit good pharmacological activities, but the current limited plant resource insufficiently meets clinical demand and the development of innovative drug research. Previously, we reported a glycosyltransferase SbGT34 cloned from Scutellaria baicalensis Georgi, which exhibited a high substrate specificity and catalysis activity toward scutellarein. An engineered yeast strain was constructed by combining the expression of SbGT34 and the enzyme participate in the biosynthesis of UDPG, approximately 1.2 g/L of scutellarein-7-O-glucoside was produced. Based on the highly conserved structure of glycosyltransferase (GT), and guided by catalytic activity, the donor-binding region of SbGT34 was reconstructed by rational evolutionary technology, so that the sugar donor specificity was converted from UDPG to UDPGA. Then we will introduce SbGT34m into the chassis cells Y1U, which dramatically increases the endogenous level of sugar donor UDPGA, further to construct the engineered yeast Y1UT, and the yield of the scutellarin (scutellarein-7-O-glucuronide) was greater than 1 g/L. This project based on the structure and activity to solve the common question of GTs donor specificity. This project will not only provide a basis for the study on the swapping of donor substrate specificity of glycosyltransferase, but also promote the molecular and drug innovation study on the medicinal glycosides. It also paves the way for the development of an economical process for biosynthesis of scutellarin.

某些植物来源的天然糖苷（如灯盏花乙素）具有良好的药理活性，但严重的资源依赖限制了其更广泛的临床应用和发展。前期从黄芩中获得了一个对野黄芩素具有高选择性和催化效率的糖基转移酶（GT）SbGT34，转化至工程菌催化获得单一产物野黄芩素-7-O-葡萄糖苷产量达1.2g/L。本项目以GTs的供体结合区的结构保守性为基础，结合催化活性，利用理性进化技术改构SbGT34的供体结构区，使其特异性识别的糖供体由UDP-葡萄糖转为UDP-葡萄糖醛酸（UDPGA），获得高酶活突变体SbGT34m；转化至可胞内合成UDPGA的酵母底盘细胞Y1U，获得工程菌株Y1UT，定向生物合成灯盏花乙素（野黄芩素-7-O-葡萄糖醛酸苷）产量大于1 g/L。本项目以结构和活性为导向解决植物GTs供体单一的共性科学难题，为利用GTs合成不同糖基取代的糖苷类药物提供理论和实验借鉴；为灯盏花乙素的来源提供了绿色可持续的生物技术策略。

某些植物来源的天然糖苷（如灯盏花乙素）具有良好的药理活性，但严重的资源依赖限制了其更广泛的临床应用和发展。前期从黄芩中获得了一个对野黄芩素具有高选择性和催化效率的糖基转移酶（GT）SbGT34，转化至工程菌催化获得单一产物野黄芩素-7-O-葡萄糖苷产量达1 .2g/L。本项目以GTs的供体结合区的结构保守性为基础，结合催化活性，利用理性进化技术改构SbGT34的供体结构区，使其特异性识别的糖供体由UDP-葡萄糖转为UDP-葡萄糖醛酸（UDPGA）；构建胞内合成UDPGA的酵母底盘细胞Y1U，将光紫黄芩葡萄糖醛酸转移酶SlUGT88D5转化至Y1U，获得具有广泛底物谱的酿酒酵母工程菌Y1UT-UGT88D5，催化野黄芩素生物合成灯盏花乙素的滴度约为0.1 g/L。本项目构建的糖基供体合成的酵母底盘细胞和糖苷化工程菌，为糖苷类活性化合物的合成提供了绿色可持续的生物技术策略。