14-02: Diol dehydratase engineering for biosynthesis of methyl ethyl ketone

With a high energy density (31.5MJ/kg) and a low boiling point (79°C), methyl ethyl ketone (MEK, or butanone) matches DoE’s criteria for advanced liquid fuels/fuel additives very well.  However, MEK is a non-natural chemical and not produced in any microbial fermentation. This research aims to use 2,3-butanediol, a metabolite commonly present with high titers (e.g. 150g/L by Klebsiella pneumonia), as the immediate precursor to produce MEK.  Only one-step enzymatic dehydration is required to convert 2,3-butanediol to MEK through the catalysis of diol dehydratase (DDH). The native substrates of all characterized DDHs so far include glycerol and 1,2-propanediol, while 2,3-butanediol was recognized as a weak substrate with a k_cat value one or two magnitudes slower than propanediol. Here we report cloning, expression and characterization of a novel DDH from Lactobacillus brevis, and its mutations rationally designed according to structure information of DDHs. These engineered L. brevis DDHs demonstrated substrate specificity shifts with enhanced activities toward meso-2,3- butanediol. To our limited knowledge, this study is the first report of improvement of the critical rate-limiting enzyme for MEK biosynthesis.