Chiral β-hydroxy α-amino acid structural motifs are interesting and common synthons present in multiple APIs and drug candidates. To access these chiral building blocks either multistep chemical syntheses are required or the application of threonine aldolases, which catalyze aldol reactions between an aldehyde and glycine. Bioinformatics tools have been utilized to identify the gene encoding threonine aldolase from Vanrija humicola and subsequent preparation of its recombinant version from E. coli fermentation. We planned to implement this enzyme as a key step to access the synthesis of our target API. Beyond this specific application, the aldolase was purified, characterized and the substrate scope of this enzyme further investigated. A number of enzymatic reactions were scaled-up and the products recovered to assess the diastereoselectivity and scalability of this asymmetric synthetic approach towards β-hydroxy α-amino acid chiral building blocks.,Copyright ? 2019 Elsevier B.V. All rights reserved.