Exploring the biocatalytic potential of wild-type split transketolases.

Split transketolases (split TKs) are an underexplored class of carboligases with potential for application in biocatalysis. Here, we investigate ten split TKs and reveal substantial functional heterogeneity, with marked differences in substrate scope, activity, enantioselectivity, and robustness. The enzymes accepted diverse acceptors, aliphatic and aromatic, while retaining the canonical (S)-selectivity of full-length TKs. Pyruvate acceptance further revealed distinct catalytic behaviours, including cross-coupling and self-coupling specificity. Selected enzymes also displayed high organic solvent tolerance. Notably, pQR2829 combined high activity, strong stereocontrol, and cross-coupling specificity, whereas pQR2809 showed exceptional robustness and broad substrate acceptance. Overall, these findings establish split TKs as a diverse and promising class of carboligases for further biocatalytic development and engineering.