Enhancing and Reversing the Stereoselectivity of Escherichia coli Transketolase via Single‐Point Mutations

Chiral auxiliary methodology and chiral assays have been developed to establish the enantiomeric purities of erythrulose and 1,3‐dihydroxypentan‐2‐one generated using wild‐type (WT) Escherichia coli transketolase (TK). L ‐Erythrulose was formed in 95% ee and (3 S )‐1,3‐dihydroxypentan‐2‐one in 58% ee . Since the latter compound was formed in moderate ee , TK libraries were screened to identify higher performing mutants. A colorimetric screen and chiral assay were successfully applied to a 96‐well format, and new active TK mutants were identified, which gave 1,3‐dihydroxypentan‐2‐one in high stereoselectivities. Remarkably, active‐site single‐point mutants were identified that were able to both enhance and reverse the stereoselectivity of TK.