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 <i>Klebsiella pneumoniae</i> 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 (<i>pduCDEGH</i>) from <i>Lentilactobacillus diolivorans</i> DSM 14421 under its native promoter control. In addition, <i>pduQ</i> from the same strain, and <i>adh</i> from <i>Clostridium beijerinckii</i> DSM 51 encoding alcohol dehydrogenases were expressed under native, T7, or P<i>tac</i> promoters. The highest yield of 2-butanol from glucose was achieved by <i>K. pneumoniae</i> K6 carrying the <i>adh</i> 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 <i>adh</i> under the P<i>tac</i> promoter), and notably, by the native <i>K. pneumoniae</i> strains. Therefore, although <i>pduQ</i> encodes a key alcohol dehydrogenase in <i>L. diolivorans</i>, it has weaker properties than <i>adh</i> for the <i>K. pneumoniae</i> host in all promoter configurations. As the high expression levels of <i>adh</i> 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.