酵母细胞催化合成18α-甘草酸  

Synthesis of 18α-glycyrrhizic acid by yeast cells

作　　者：邢登雪 张良 李文强 梁建华 秦磊 张根林[1] 李春[1,2,3] XING Dengxue;ZHANG Liang;LI Wenqiang;LIANG Jianhua;QIN Lei;ZHANG Genlin;LI Chun(Key Laboratory for Green Processing of Chemical Engineering of Xinjiang Bingtuan,School of Chemistry and Chemical Engineering,Shihezi University,Shihezi 832003,Xinjiang,China;Key Laboratory of Medical Molecule Science and Pharmaceutics Engineering,Ministry of Industry and Information Technology,Institute of Biochemical Engineering,School of Chemistry and Chemical Engineering,Beijing Institute of Technology,Beijing 100081,China;Key Laboratory for Industrial Biocatalysis,Ministry of Education,Department of Chemical Engineering,Tsinghua University,Beijing 100084,China)

机构地区：[1]石河子大学化学化工学院,新疆兵团绿色化工过程重点实验室,新疆石河子832003 [2]北京理工大学化学与化工学院,生物化工研究所医药分子科学与制剂工信部重点实验室,北京100081 [3]清华大学化学工程系,工业生物催化教育部重点实验室,北京100084

出　　处：《化工学报》2024年第9期3266-3276,共11页CIESC Journal

基　　金：国家自然科学基金项目(22138006,22108154)。

摘　　要：18α-甘草酸(18α-GL)是一种齐墩果烷型皂苷,比其差向异构体18β-GL极性小、亲脂性好,更强的抗毒抗炎作用和更高的肝脏靶向性使18α-GL成为保肝护肝领域的主要药物成分。但目前18α-GL的制备方法污染大、效率低,亟需开发一种绿色简单地合成18α-GL的方法。利用酵母细胞异源表达糖基转移酶,通过全细胞催化方式鉴定出可催化18α-甘草次酸(18α-GA)特异性合成18α-单葡萄糖醛酸甘草次酸(18α-GAMG)的糖基转移酶cGuCSyGT和催化18α-GAMG特异性合成18α-GL的糖基转移酶GgUGT1。进一步运用蛋白质结构预测和分子动力学模拟探究cGuCSyGT对18α-GA的催化活性比18β-GA低的原因。最后采用优化底物添加浓度、底盘细胞、底物添加时间、反应时间、培养基成分补加和底物溶剂的策略构建了酵母催化合成18α-GAMG和18α-GL的最优工艺,使18α-GAMG和18α-GL的产量分别达到(36.38±1.87)mg/L和(39.32±0.75)mg/L。本研究实现了18α-GAMG和18α-GL的微生物催化合成,可为18α-GL的微生物全合成提供理论基础和技术支撑。18α-Glycyrrhizin(18α-GL)is an oleanane-type saponin.18α-GL is less polar and more lipophilic than its diastereomer 18β-GL.Its stronger anti-toxic and anti-inflammatory effects and higher liver targeting make 18α-GL a major drug ingredient in the field of liver protection.However,the current preparation method of 18α-GL is highly polluting and has low efficiency.Therefore,there is an urgent need to develop a green and simple method to synthesize 18α-GL.Glycosyltransferases were heterologously expressed in yeast cells.Through whole-cell catalysis,glycosyltransferases cGuCSyGT was identified as being able to catalyze the specific synthesis of 18α-glycyrrhetinic acid 3-O-monoglucuronide(18α-GAMG)from 18α-glycyrrhetinic acid(18α-GA)and GgUGT1 yeast cells[J].CIESC Journal,2024,75(9):3266-3276 was identified as being able to catalyze the specific synthesis of 18α-glycyrrhizin(18α-GL)from 18α-GAMG.We further employed protein structure prediction and molecular dynamics simulation to explore the reason why cGuCSyGT has lower catalytic activity for 18α-GA than 18β-GA.Finally,an optimal process for yeast catalytic synthesis of 18α-GAMG and 18α-GL was constructed by optimizing various parameters,including substrate addition concentration,chassis host cells,substrate addition time,catalytic time,medium component addition and substrate solvent.Thus,the production of 18α-GAMG and 18α-GL reached(36.38±1.87)mg/L and(39.32±0.75)mg/L,respectively.This research achieved the microbial catalytic synthesis of 18α-GAMG and 18α-GL,which will provide a theoretical basis and technical support for the total microbial synthesis of 18α-GL.

关 键 词：甘草 18Α-甘草酸 18α-单葡萄糖醛酸甘草次酸 糖基转移酶 酵母细胞催化 

分 类 号：Q814[生物学—生物工程]