Engineering of the Thiamine Diphosphate-Dependent JanthE for the Synthesis of Tertiary Alcohols

The development of (bio)catalytic methods to chiral tertiary alcohols is in demand, yet the enantioselective synthesis of tertiary alcohols with sterically hindered moieties is problematic. JanthE is a novel thiamine diphosphate-dependent enzyme that catalyzes aldehyde-ketone cross-coupling reactions with a wide range of donor and acceptor substrates. However, product formation is low and precludes large-scale production. We investigated the conversion of 2-oxobutanoate and the bulky ketone phenoxy-2-propanone to the product 2-hydroxy-2-methyl-1-phenoxypentan-3-one as a model reaction for the synthetic capabilities of JanthE. As the reaction design did not significantly improve the yield, we proceeded to rational protein engineering. Docking experiments identified V121, Y268, P293, Y297, and K567 in the active site as important residues for catalysis. Remarkably, the single-point variant JanthE K567S led to a fivefold increase in product formation (90% ee) compared to the wild type enzyme (93% ee). The reaction was performed at preparative scale proving the direct possibility of application. Additionally, when the reaction was extended to longer 2-oxopentanoate, the double variant Y297P_K567S showed a 5-fold increase in product formation compared to the wild type. Our results demonstrate the evolvability and versatility of JanthE for the enantioselective synthesis of sterically hindered tertiary alcohols.