(693f) Profiling of Putative Threonine Transaldolases for Biosynthesis of Non-Standard Amino Acids

Beta-hydroxy non-standard amino acids (β-OH-nsAAs) have broad utility as small molecule drugs, precursors for beta-lactam antibiotics and building blocks for introducing novel functionality into proteins. Despite the valuable applications for these non-standard amino acids, organic synthesis routes are expensive and documented for only a limited number of β-OH-nsAA and existing biosynthesis approaches are limited by unfavorable equilibrium dynamics. A recently discovered enzyme class of threonine transaldolases can address the current limitations for β-OH-nsAA biosynthesis through a stereo-selective and thermodynamically favorable aldol condensation of an aromatic aldehyde and L-threonine. We have probed the substrate specificity of a TTA from Pseudomonas fluorescens to access novel β-OH-nsAAs. To explore the specificity of this enzyme class more broadly, we have expressed and characterized naturally occurring, putative TTAs. Through the screen of putative TTAs, we have identified different enzymes with broader substrate scope, lower threonine affinity, and faster initial reaction rates. To characterize the broad specificity of many TTAs, we optimized a high-throughput absorbance-based assay that couples an alcohol dehydrogenase with a TTA to produce a measurable output of enzyme activity. We have also produced β-OH-nsAAs in metabolically active, engineered E. coli cultures, a novel chassis for β-OH-nsAA production. Overall, our work helps identify new candidate enzymes that could enable biological synthesis of valuable small molecules and building blocks that expand protein functions.