Beta-hydroxy non-standard amino acids (ꞵ-OH nsAAs) are a class of molecules with a broad range of applications such as precursors for ꞵ-lactone antibiotics, building blocks for antimicrobial peptides and use as small molecule therapeutics. However, their sustainable production is often hindered by conventional chemical synthesis strategies involving harsh chemicals, multi-step processes and racemic products. Biosynthetic approaches, like the use of the enzyme class L-threonine transaldolases (TTAs), offer a more efficient alternative for the synthesis of these compounds with broad substrate scope, high stereoselectivity and reduced environmental impact. This class of enzymes has demonstrated the thermodynamically favorable conversion of aldehydes and L-threonine to produce ꞵ-OH nsAAs and acetaldehyde. A key hindrance to their application in live cells is their low L-threonine affinity, requiring the supplementation of excess L-threonine. Therefore, the theoretical advantage of the thermodynamic favorability of the TTA-catalyzed reaction relative to other reactions cannot be realized until there is no longer a need to supply large excesses of L-threonine. This requires identification or engineering of a TTA with sub-millimolar affinity for L-threonine.
This work focuses on the characterization of an L-threonine transaldolase with enhanced affinity for L-threonine. Our approach combines bioprospecting for promising TTA candidates, with structure- and conservation-guided mutagenesis of a critical loop region to better determine its contributions to L-Thr co-substrate affinity. Through this, we have yielded sub-millimolar TTA L-threonine Km values, improving enzyme performance and reducing the need to supplement excess L-threonine to live E. coli cultures. By overcoming the limitations of low L-threonine affinity in TTAs, ꞵ-OH nsAAs can be synthesized more efficiently, paving the way for broader applications in cell-free and cellular biocatalytic contexts.
