An Engineered Glycerol Dehydratase With Improved Activity for the Conversion of meso-2,3-butanediol to Butanone

There is substantial interest in engineering microorganisms to produce industrial chemicals that are currently derived from petroleum. One of these petrochemicals is butanone, which could be produced from microbially synthesized 2,3-butanediol through the action of a suitable dehydratase enzyme. Unfortunately, however, there are no known enzymes that natively catalyze this reaction. In this work, the authors set out to engineer the B₁₂ -dependent glycerol dehydratase from Klebsiella pneumoniae (KpGDHt), in order to increase its activity for the conversion of meso-2,3-butanediol into butanone. The authors began by fusing the α and β subunits of the enzyme, to simplify downstream high-throughput screening protocols. Serendipitously, the fusion protein showed a 20°C increase in its temperature optimum. Using this stabilized scaffold as a starting point, the authors employed the combinatorial active site saturation test and consensus-guided mutagenesis to randomize 28 residues within 12 Å of the KpGDHt active site. By screening over 5500 variants, the authors discovered a single point mutation (T200S) that increased the catalytic efficiency of meso-2,3-butanediol dehydration by four-fold, to a value of k_cat /K_M  = 5.1 × 10³ M^-1 s^-1 . Thus the authors report what is, to date, the most comprehensive mutagenesis and the largest engineered increase in catalytic efficiency on the B₁₂ -dependent glycerol dehydratase scaffold.