Improving Activity and C β -Stereoselectivity of l -Threonine Transaldolase via Structure-Guided Protein Engineering and a Visual High-Throughput Screening Method

Chiral β-hydroxy-α-amino acids are key chiral building blocks of many natural products and drug molecules, but their asymmetric synthesis remains a notable challenge. l-Threonine transaldolase (LTTA) exhibits favorable stereoselectivity in β-hydroxy-α-amino acid synthesis, but its relatively low activity restricts its widespread application. In this study, we established a visual high-throughput screening method to improve the activity and Cβ-stereoselectivity of a novel LTTA from Pseudomonas sp. EMN2 (LTTA11), which relied on the conversion of the byproduct acetaldehyde with a thiamine pyrophosphate (ThDP)-dependent enzyme. A triple mutant M3 (F59G/M65N/F70T) was identified by saturation mutagenesis and iterative saturation mutagenesis, which exhibited a 160-fold improvement in the catalytic efficiency relative to that of wild-type enzyme and enhanced Cβ-stereoselectivity (97.3% de) toward 4-methylsulfonylbenzaldehyde. This mutant also exhibited excellent catalytic performance toward various substituted aromatic aldehydes, particularly para-substituted substrates, with good to excellent de values (92%–97.3%). Gram-scale preparation of l-threo-4-methylsulfonylphenylserine and l-threo-4-nitrophenylserine was performed with a high space-time yield (20 g L–1 h–1 and 23 g L–1 h–1), representing the highest levels reported to date. This work offers valuable guidance for further engineering of LTTA for the industrial production of β-hydroxy-α-amino acids.