Exploring Novel ‘Split’ Transketolases: From Discovery to Biocatalytic Scope

Biocatalysis is a major resource for biomanufacturing, offering the possibility to develop more efficient and sustainable processes than purely chemical routes. Transketolases (TKs) are thiamine diphosphate (ThDP)-dependent enzymes that catalyse the stereoselective formation of C-C bonds. Beyond the canonical 600-residue TKs, many prokaryotes encode a split variant made of two 300-residue subunits. It has been hypothesised that this split architecture could confer increased conformational flexibility and allow the accommodation of bulkier substrates, yet split TKs remain largely unexplored from a biocatalytic perspective, and the crystal structure of only one split TK has been solved. To advance our understanding, in this work in-house metagenomic libraries and public genomes from mesophiles and thermophiles were mined, and almost 500 split TK gene pairs were retrieved. Ten representatives were successfully cloned, purified, and assayed, doubling the number of putative split TKs ever produced and tested. These enzymes were investigated for activity with different substrates, stability, and enantioselectivity. sTK2 from the mesophile Fusobacterium nucleatum gave the highest conversion with glycolaldehyde and hydroxypyruvate, and was further biochemically characterised. Site-saturation libraries of its active site residues were prepared, of which some were preliminary tested, indicating residues with potential for future engineering. Thermostability studies showed that sTK4 and sTK7 were able to retain activity after 1 hour at 90 °C and 100 °C, respectively, exceeding the performance of thermostable TKs from the literature. Overall, the thermostable sTK4 and the metagenome-derived sTK23 were the best performing, displaying activity on various nonhydroxylated aliphatic and aromatic acceptors as wild-type. The latter also displayed excellent enantioselectivities. Unlike Escherichia coli TK, sTK23 and two additional metagenomic enzymes showed specificity for cross-coupling when tested with the alternative donor pyruvate. This study shed light on the diversity and biocatalytic potential of split TKs, identifying promising biocatalysts and providing a solid foundation for future optimisation.