Mining Of Threonine Transaldolase And Synthesis Of Unnatural Amino Acids By Cascade Reaction

β-Hydroxy-α-amino acids,a class of important non-natural amino acids,are essential for the synthesis of various biologically active products.In this study,a novel L-threonine transaldolase（Pm LTTA）was identified from Pseudomonas sp.through genome mining.Pm LTTA exhibited high activity and diastereoselectivity in the synthesis of L-threo-phenylserine from L-threonine and benzaldehyde.However,the practical application of Pm LTTA was impeded by the low conversion ratio and instable diastereoselectivity,which were caused by the toxicity of aldehydes and the complex kinetic/thermodynamic controls in the transaldehyde reaction.To solve these issues,alcohol dehydrogenase was employed to remove the by-product acetaldehyde,and then carboxylic acid reductase was introduced to alleviate the inhibition of benzaldehyde.A multi-enzyme cascade reaction,comprising of Pm LTTA,carboxylic acid reductase,alcohol dehydrogenase and glucose dehydrogenase,was constructed to prepare L-threo-phenylserine from cheap benzoic acid,in which alleviated inhibition of aldehydes and desirable diastereoselectivity were achieved.Finally,this study improved the conversion ratio and diastereoselectivity of the multi-enzyme cascade reaction by optimizing the influencing factors of the multi-enzyme cascade reaction.The main research contents are as follows:（1）Gene mining for L-threonine transaldolase.Through gene mining,Pm LTTA,which can be used for synthesis of chiralβ-hydroxy-α-amino acids,was obtained from the database.Purification and characterization were conducted.Specific activity of crude extract of Pm LTTA was 0.33 U·mg^-1,and specific activity of purified Pm LTTA was 5.48 U·mg^-1.The optimum p H and temperature were p H 8.0 and 70°C,respectively.Pm LTTA was a metal-independent and thermostable transaldolase.By measuring the effect of Pm LTTA amount and reaction time on the L-threonine transaldolase reaction,it was found that the conversion ratio was always lower than 25%,and the diastereoselectivity decreased with the prolongation of reaction time.（2）Removal of the bottleneck of the L-threonine transaldolase reaction.In order to solve the bottleneck of the transaldehyde reaction,alcohol dehydrogenase（Sc ADH）was introduced to convert the by-product acetaldehyde into ethanol,the equilibrium is shifted toward the direction of product formation,overcoming the thermodynamic limitation.Moreover,the diastereoselectivity was kept stable with increasing of reaction time.After the introduction of Sc ADH,the conversion ratio of the multi-enzyme cascade reaction reached 29.4%,and the de value reached 93.4%and remained stable.Carboxylic acid reductase（Anc CAR-A）could convert sodium benzoate into benzaldehyde,which served as a substrate for Pm LTTA,so that the benzaldehyde in the reaction system was kept at a relatively low level,thereby reducing the inhibitory effect of benzaldehyde on Pm LTTA.In addition,the use of water-soluble sodium benzoate as the starting substrate can avoid the addition of organic co-solvents such as DMSO that are toxic to enzymes.After the introduction of Anc CAR-A,the reaction conversion ratio reached 25.8%,and the de value reached 96.5%.（3）Optimization of multi-enzyme cascades.A multi-enzyme cascade reaction system containing Anc CAR-A,Pm LTTA,Sc ADH,and Bm GDH was successfully constructed,by using the cheap and readily available industrial by-product sodium benzoate as the starting substrate,to synthesize chiral phenylserine.The effects of ATP concentration,p H,temperature,enzyme loading ratio of CAR and LTTA,and L-threonine addition on the multi-enzyme cascade reaction were investigated,and the optimal conditions for the cascade reaction were determined:the reaction buffer was p H 8.0 HEPES,the ATP concentration was 10 m M,the reaction temperature was 25°C,the enzyme loading ratio of Anc CAR-A to Pm LTTA was 1:10,and the ratio of L-threonine to benzaldehyde was 2.5:1.Under the optimum conditions and 10 m M substrate concentration,the conversion ratio and de value reached 57.1%and 95.3%,respectively.This study provides an efficient cascade reaction system for the synthesis of valuable chiralβ-hydroxy-α-amino acids from industrial byproduct,and provides guidance for optimization of cascade reactions influenced by toxicity of aldehydes and complicated kinetic and thermodynamic controls.