(671b) Prospecting Thiamine Diphosphate-Dependent Carboligases and Characterizing Their Promiscuity to Create Novel Metabolic Pathways from Primary Metabolites

Recent studies have demonstrated that enzyme promiscuity, the ability of an enzyme to accept non-native substrates and perform non-native chemistries, is widespread in nature. This provides an opportunity for biological engineers to both leverage this capacity for valuable unnatural transformations and to hone desired activities. One particularly interesting family of enzymes to this end is thiamine-diphosphate dependent carboligases, which condense two α-ketoacids (or aldehydes) to form new carbon-carbon bonds. Because of an abundance of α-ketoacids in the central metabolism of common metabolic engineering hosts like Escherichia coli, this allows for possible assembly of new and favorable biochemical pathways to targets of interest. One-step condensations could generate more efficient routes to desired targets and access to novel molecules, including chiral compounds. Here, we demonstrate our characterization workflow with proof-of-concept enzymes and reactions, assay previously uncharacterized enzymes, and map the promiscuity and catalytic capacity of this family of enzymes.