Abstract:

Threonine aldolases are valuable biocatalysts for carbon-carbon bond formation in the synthesis of β-hydroxy-α-amino acids. In this work, a threonine aldolase from Pseudomonas putida (PSALD) was engineered using the continuous directed evolution platform OrthoRep in a yeast strain auxotrophic for both glycine and threonine. Two evolution campaigns were carried out: one on L-threonine, which yielded variants several-fold more active in yeast, and another on the commercially available β-hydroxy-α-amino acid substrate DL-β-(2-thienyl)-serine, which produced variants with improved specificity toward aromatic substrates. Variants from the L-threonine evolution campaign were further characterized by high-throughput sequencing and competition-based fitness assays, which shed light on key groups of mutations responsible for improved activity in yeast. Kinetic analysis of variants evolved on DL-β-(2-thienyl)-serine revealed one variant, E3, to exhibit a 29-fold increase in specificity toward the aromatic amino acid L-phenylserine over L-threonine compared to PSALD. Further investigation using site-saturation mutagenesis and kinetic assays identified residue Y318 as a gatekeeper position governing the enzyme’s ability to catalyze efficient cleavage of L-threonine. Aldolase mutants were also profiled for activity toward bulky biphenyl β-hydroxy-α-amino acids, revealing additional variants with enhanced specificity. Together, these results establish a scalable selection system for studying threonine aldolases and their evolutionary trajectories across diverse substrates, while also outlining key considerations for adapting the platform to other β-hydroxy-α-amino acids.