Engineering Diol Dehydratase for Enhanced Activity Towards Non-Natural Substrates

Abstract

Combinatorial biosynthesis has enabled the development of novel or artificial biosynthetic pathways for the generation of pharmaceuticals, chemicals and biofuels. The establishment of these pathways usually requires novel or engineered enzymes with desired functions. The coenzyme B12-dependent diol dehydratase from Klebsiella oxytoca was previously demonstrated to dehydrate its natural substrate 1,2-propanediol for 1-propanol production. In this work, we engineer the enzyme to catalyze a longer chain C4 triol (1,2,4-butanetriol) for the production of 1,4-butanediol via structure-based redesign. To achieve this, a systematic study of the active site is performed using its natural substrate 1,2-propanediol. Analysis of the enzyme in substrate-free and substrate-bound forms leads to the identification of key amino acids involved in substrate binding and orientation. A rational design strategy is then developed to increase the enzyme selectivity and activity towards 1,2,4-butanetriol. Following in silico screening, the mutants with the highest potential to interact with 1,2,4-butanetriol are selected for enzyme kinetics study. This approach results in mutants with increased activity towards 1,2,4-butanetriol as compared to the wild type enzyme. Whole cell conversion studies result in more efficient dehydration of 1,2,4-butanetriol into 4-hydroxybutyraldehyde and its subsequent native reduction into 1,4-butanediol in Escherichia coli.