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 generate β-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.