A High‐Throughput Visual Screen for the Directed Evolution of C β ‐stereoselectivity of L‐threonine Aldolase

L-Threonine aldolase (L-TA) is a pyridoxal phosphate-dependent enzyme that catalyzes the reversible condensation of glycine and aldehydes to form β-hydroxy-α-amino acids. The combination of directed evolution and efficient high-throughput screening methods is an effective strategy for enhancing the enzyme's catalytic performance. However, few feasible high-throughput methods exist for engineering the Cβ-stereoselectivity of L-TAs. Here, we present a novel method of screening for variants with improved Cβ-stereoselectivity; this method couples an L-threo-phenylserine dehydrogenase, which catalyzes the specific oxidation of L-threo-4-methylsulfonylphenylserine (L-threo-MTPS), with the concurrent synthesis of NADPH, which is easily detectable via 340-nm UV absorption. This enables the visual detection of L-threo-MTPS produced by L-TA through the measurement of generated NADPH. Using this method, we discover an L-TA variant with significantly higher diastereoselectivity, increasing from 0.98% de (for the wild-type) to 71.9% de.