Multi-enzyme cascade for sustainable synthesis of L-threo-phenylserine by modulating aldehydes inhibition and kinetic/thermodynamic controls

l-Threonine transaldolase could catalyze the transaldolation of l-threonine and aldehyde to generateβ-hydroxy-α-amino acids with high diastereoselectivity.A novel l-threonine transaldolase(PmLTTA)was identified from Pseudomonas sp.through genome mining.PmLTTA exhibited high activity in the synthesis of l-threo-phenylserine from l-threonine and benzaldehyde,with specific activity of 5.48 U mg-1.However,the application of PmLTTA was impeded by the low conversion ratio and variable diastereoselectivity,which were caused by the toxicity of aldehydes and kinetic/thermodynamic controls in the transaldolation reaction.To solve these issues,alcohol dehydrogenase was used to remove the by-product acetaldehyde,and then carboxylic acid reductase was introduced to alleviate the inhibition of benzaldehyde and toxicity of DMSO.Finally,a multi-enzyme cascade reaction,comprising of PmLTTA,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.Under the optimized conditions,the conversion ratio of 57.1%and de value of 95.3%were reached.This study provides an efficient and green approach for the synthesis of chiral l-threo-phenylserine from industrial byproduct,and provides guidance for the development of cascade reactions influenced by the toxic intermediates and complicated kinetic/thermodynamic controls.