Direct Production of 2-Butanol from Glucose by Recombinant Klebsiella pneumoniae Strains

2-Butanol is a promising biofuel due to its favorable properties and lower microbial toxicity compared to other butanol isomers. However, microbial production remains challenging due to the absence of a native biochemical pathway for directly converting sugars into 2-butanol. To achieve this goal, glucose should be directed through the 2,3-butanediol (2,3-BD) pathway, involving α-acetolactate synthase, α-acetolactate decarboxylase, and butanediol dehydrogenase for the formation of meso-2,3-BD, followed by diol dehydratase-catalyzed conversion of meso-2,3-BD to butanone and alcohol dehydrogenase-mediated reduction in butanone to 2-butanol. In this study, we report the development of six new recombinant strains based on Klebsiella pneumoniae G31, in which the metabolic pathway for converting glucose to meso-2,3-BD was extended to 2-butanol. All engineered strains harbored the vitamin B12-dependent diol dehydratase complex (pduCDEGH) from Lentilactobacillus diolivorans DSM 14421 under its native promoter control. In addition, pduQ from the same strain, and adh from Clostridium beijerinckii DSM 51 encoding alcohol dehydrogenases were expressed under native, T7, or Ptac promoters. The highest yield of 2-butanol from glucose was achieved by K. pneumoniae K6 carrying the adh gene under the control of the T7 promoter—437 mg/L. Using 2-butanone as a substrate, K6 again produced the highest titer of 2-butanol (3.9 g/L), followed by the recombinant K8 (with adh under the Ptac promoter), and notably, by the native K. pneumoniae strains. Therefore, although pduQ encodes a key alcohol dehydrogenase in L. diolivorans, it has weaker properties than adh for the K. pneumoniae host in all promoter configurations. As the high expression levels of adh under T7 promoter control were driven by the native bacterial RNA polymerase, this promoter–host combination appears particularly suitable for developing other strains of industrial relevance.