The current study investigates the impact of mutation of 2,3-butanediol (BDO) formation pathway on glycerol metabolism and 1,3-propanediol (PDO) production by lactate dehydrogenase deficient mutant of Klebsiella pneumoniae J2B. To this end, BDO pathway genes, budA, budB, budC and budO (whole-bud operon), were deleted from K. pneumoniae J2B Delta ldhA and the mutants were studied for glycerol metabolism and alcohols (PDO, BDO) production. Delta budO-mutant-only could completely abolish BDO production, but with reductions in cell growth and PDO production. By modifying the culture medium, the Delta budO mutant could recover its performance on the flask scale. However, in bioreactor experiments, the Delta budO mutant accumulated a significant amount of pyruvate (>73 mM) in the late phase and PDO production stopped concomitantly. Glycolytic intermediates of glycerol, especially glyceraldehyde-3-phosphate (G3P) was highly inhibitory to glycerol dehydratase (GDHt); its accumulation, followed by pyruvate accumulation, was assumed to be responsible for the Delta budO mutant's low PDO production. (C) 2016 Elsevier Ltd. All rights reserved.