Converting 2,3-Butanediol to Butanone By Engineered Diol Dehydratase

High titers of 2,3-butanediol are commonly present as the major end product in anaerobic fermentation in various microbes (e.g. 150g/L by Klebsiella pneumonia), but with an excessively high boiling point of 177^oC, cost-effective separation is not feasible.  Diol dehydratase (DDH, EC 4.2.1.28 or 4.2.1.30) converts a diol molecule into its corresponding ketone or aldehyde.  Catalyzed by DDH, the dehydration of 2,3-butanediol yields butanone (methyl ethyl ketone or MEK),  a potential advanced liquid fuel/fuel additive due to its high energy density (31.5 MJ/kg) and a low heat of vaporization (0.45 MJ/Kg).  However, biosynthesis of butanone has not been demonstrated, due to (1) butanone is not a natural metabolite present in any microbial fermentation; and (2) the native substrates of all known diol dehydratases (DDHs) are 1,2-propanediol, glycerol and ethanediol.  2,3-butanediol, when used as a substrate for wild type DDHs to produce butanone, exhibits an activity 1 or 2 fold lower than the native substrates.  This research aims to not only construct and optimize metabolic pathways, but also more importantly, engineer the key enzymes of rate-limiting steps to improve the specific activities and substrate/co-factor preference desired for high productivity. Here we report our recent progresses on cloning, expression and characterization of DDHs from Lactobacillus brevis, and the creation of engineered DDHs using a rational designed approach based on known structures of DDHs. These engineered DDHs demonstrate substrate specificity shifts with enhanced activity toward meso-2,3-butanediol, thus paving a path for metabolic synthesis of MEK from low cost renewable biomass.