Comprehensive screening strategy coupled with structure-guided engineering of l-threonine aldolase from Pseudomonas putida for enhanced catalytic efficiency towards l-threo-4-methylsulfonylphenylserine.

Abstract

l-Threonine aldolases (TAs) can catalyze aldol condensation reactions to form β-hydroxy-α-amino acids, but afford unsatisfactory conversion and poor stereoselectivity at the Cβ position. In this study, a directed evolution coupling high-throughput screening method was developed to screen more efficient l-TA mutants based on their aldol condensation activity. A mutant library with over 4000l-TA mutants from Pseudomonas putida were obtained by random mutagenesis. About 10% of mutants retained activity toward 4-methylsulfonylbenzaldehyde, with five site mutations (A9L, Y13K, H133N, E147D, and Y312E) showing higher activity. Iterative combinatorial mutant A9V/Y13K/Y312R catalyzed l-threo-4-methylsulfonylphenylserine with a 72% conversion and 86% diastereoselectivity, representing 2.3-fold and 5.1-fold improvements relative to the wild-type. Molecular dynamics simulations illustrated that additional hydrogen bonds, water bridge force, hydrophobic interactions, and π-cation interactions were present in the A9V/Y13K/Y312R mutant compared with the wild-type to reshape the substrate-binding pocket, resulting in a higher conversion and Cβ stereoselectivity. This study provides a useful strategy for engineering TAs to resolve the low Cβ stereoselectivity problem and contributes to the industrial application of TAs.