甲酸脱氢酶催化活性的定向进化及其高效表达

Directed Evolution for Catalytic Activity of Formate Dehydrogenase and Its Overexpression

甲酸脱氢酶(formate dehydrogenase,FDH)属于D-2-羟基酸脱氢酶类,能催化甲酸氧化生成二氧化碳,同时能将氧化型辅酶I(Oxdized form of nicotinamide adenine dinucleotide,NAD+)还原成还原型辅酶I(Redued form of nicotinamide adenine dinucleotide,NADH),在NADH的再生中起重要作用。为了获得高活性的甲酸脱氢酶突变体,本研究以博伊丁假丝酵母甲酸脱氢酶(Candida boidinii formate dehydrogenases,CbFDH)突变体(CbFDHC23S)为亲本,进行了2轮定向进化,获得了一个比酶活性约为亲本4倍,且更适合于在生理条件下进行辅酶再生的突变体M2。然后,利用计算机辅助的手段初步阐明了其温度特性和催化效率改变的分子机制。最后,借助共表达策略进一步提高了突变体M2在大肠杆菌中的表达水平,超声裂解液中的甲酸脱氢酶活性达到45.85 U/mL,远远高于亲本单拷贝表达水平。本研究为增强NADH再生能力、降低NADH的再生成本,实现FDH偶联催化的手性醇及氨基酸衍生物等食品添加剂高效、廉价的绿色生物合成奠定理论基础。

Formate dehydrogenase(FDH)is a D-2-hydroxy acid dehydrogenase,and catalyzes the oxidation of formate to carbon dioxide,coupled with reduction of NAD+(oxidized nicotinamide adenine dinucleotide(NAD))to NADH(reduced NAD)that plays a key role in the process of NADH regeneration.In order to obtain highly active formate dehydrogenase mutants,the Candida boidinii formate dehydrogenase CbFDHC23 S was used as the parent to conduct two rounds of directional evolution,and a mutant M2 was obtained.The specific activity of M2 is about 4 times more than the parent and M2 and is more suitable for coenzyme regeneration under physiological conditions.Then,the molecular mechanism of the temperature characteristic and the catalytic efficiency change was preliminarily elucidated by the computer aided method.Finally,with the help of the co-expression strategy,the expression level of mutant M2 in Escherichia coli is further improved,and the formate dehydrogenase activity in the ultrasonic lysate reaches 45.85 U/mL,which is far higher than the expression level of the parent single copy.This study laid a theoretical foundation for the green biosynthesis of food additives such as chiral alcohols and amino acid derivatives catalyzed by FDH coupling to enhance the regeneration capacity of NADH,reduce the regeneration cost of NADH,and achieve high efficiency and low cost.