Metabolic engineering of Klebsiella pneumoniae for the de novo production of 2-butanol as a potential biofuel

Butanol isomers are important bulk chemicals and promising fuel substitutes. The inevitable toxicity of n-butanol and isobutanol to microbial cells hinders their final titers. In this study, we attempt to engineer Klebsiella pneumoniae for the de novo production of 2-butanol, another butanol isomer which shows lower toxicity than n-butanol and isobutanol. 2-Butanol synthesis was realized by the extension of the native meso-2,3-butanediol synthesis pathway with the introduction of diol dehydratase and secondary alcohol dehydrogenase. By the screening of different secondary alcohol dehydrogenases and diol dehydratases, 320 mg/L of 2-butanol was produced by the best engineered K. pneumoniae. The production was increased to 720 mg/L by knocking out the ldhA gene and appropriate addition of coenzyme B12. Further improvement of 2-butanol to 1030 mg/L was achieved by protein engineering of diol dehydratase. This work lays the basis for the metabolic engineering of microorganism for the production of 2-butanol as potential biofuel.