Development of an Engineered Sugar Aminotransferase with Simultaneously Improved Stability and Non-Natural Substrate Activity to Synthesize the Glucosidase Inhibitor Valienamine  

作　　者：Runxi Wang Lu Qiao Mufei Liu Yanpeng Ran Jun Wang Wupeng Yan Yan Feng Li Cui 

机构地区：[1]State Key Laboratory of Microbial Metabolism&Joint International Research Laboratory of Metabolic and Developmental Sciences,School of Life Science and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China

出　　处：《Engineering》2024年第11期185-195,共11页工程（英文）

基　　金：supported by the National Key Research and Development Program of China(2018YFE0200501 and 2020YFA0907700);the National Natural Science Foundation of China(32271306 and 21977067).

摘　　要：Sugar aminotransferases(SATs)catalyze the installation of chiral amines onto specific keto sugars,pro-ducing bioactive amino sugars.Their activity has been utilized in artificial reactions,such as using the SAT WecE to transform valienone into the valuable a-glucosidase inhibitor valienamine.However,the low thermostability and limited activity on non-natural substrates have hindered their applications.Simultaneously improving stability and enzyme activity is particularly challenging owing to the acknowledged inherent trade-off between stability and activity.A customized combinatorial active-site saturation test-iterative saturation mutagenesis(CAST-ISM)strategy was used to simultaneously enhance the stability and activity of WecE toward valienone.Fourteen hotspots related to improving the stability-\activity trade-off were identified based on evolutionary conservation and the average mutation folding energy assessment of 57 residues in the active site of WecE.Positive mutagenesis and combinatorial mutations of these specific residues were accomplished via site-directed saturation mutagenesis(SSM)and iterative evolution cycles.Compared with those of the wild-type(WT)WecE,the quadruple mutant M4(Y321F/K209F/V318R/F319V)displayed a 641.49-fold increase in half-life(t_(1/2))at 40℃ and a 31.37-fold increase in activity toward the non-natural substrate valienone.The tri-ple mutant M3(Y321F/K209F/V318R)demonstrated an 83.04-fold increase in(t_(1/2))at 40℃and a 37.77-fold increase in activity toward valienone.The underlying mechanism was dependent on the strengthened interface interactions and shortened transamination reaction catalytic distance,compared with those of the WT,which improved the stability and activity of the obtained mutants.Thus,we accomplished a general target-oriented strategy for obtaining stable and highly active SATs for artificial amino-sugar biosynthesis applications.

关 键 词：Sugar aminotransferase Stability-activity trade-off Combinatorial active-site saturation test Iterative saturation mutagenesis Artificial reaction Valienamine 

分 类 号：TQ426.97[化学工程] Q814[生物学—生物工程]