Engineering L-threonine transaldolase and a chaperone assisted cascade for the efficient biosynthesis of L-threo-p-methylsulfonylphenylserine.

The efficient asymmetric synthesis of L-threo-p-methylsulfonylphenylserine (L-threo-4-MTPS) is frequently hindered by the limited catalytic efficiency of natural transaldolases and severe feedback inhibition caused by acetaldehyde accumulation. In this study, we identified a novel L-threonine transaldolase from Chitiniphilus eburneus (CeLTTA) via systematic genome mining. To overcome its kinetic limitations, structure-guided semi-rational evolution was employed, yielding the triple mutant F70N/F59A/C57M with 4.46-fold increase in specific activity and maintained superior stereocontrol. Molecular dynamics simulations elucidated that these mutations enhanced catalytic performance by rigidifying the global structure and optimizing the hydrophobic core, thereby improving substrate confinement within the active site. Furthermore, to address the critical bottleneck of acetaldehyde toxicity, we engineered an artificial, cofactor-independent cascade by coupling CeLTTA with a formolase (FLS) scavenging module. This design efficiently converts the toxic byproduct into acetoin, alleviating feedback inhibition without external cofactor supplementation. Optimization of the genetic architecture and chaperone co-expression revealed that the GroES-GroEL system was pivotal for resolving protein folding issues and maximizing pathway flux. The final engineered E. coli strain achieved a titer of 18.8 mM with a conversion of 93.9% under optimized conditions. This work provides a robust biocatalytic platform for L-threo-4-MTPS production and demonstrates a versatile strategy integrating enzyme engineering with metabolic pathway assembly to overcome intrinsic enzymatic constraints.