Abstract: Glycerol can be transformed to 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae with accompanying ethanol accumulation.Aldehyde dehydrogenase (ALDH) is one of the key enzymes for biosynthesis of ethanol, which competes for reducing equivalents NADH with 1,3-propanediol dehydrogenase which catalyzes biosynthesis of 1,3-PD.Based on metabolic analysis, one reasonable method to improve 1,3-PD yield is to inhibit the catalysis of ALDH so that ethanol production could be restrained. In this paper, a homologous recombination vector pUCAT was constructed, in which the ALDH gene of K.pneumoniae was disrupted by inserting tetracycline resistance gene (Tcr).The amplified DNA fragment of 5′ALDH-Tcr-3′ALDH from pUCAT was used to transform K.pneumoniae M5aL and the ALDH gene knockout recombinants were obtained.Comparing with those of the wild type K.pneumoniae M5aL, the ALDH activity of the recombinants were undetected, cell growth was inhibited obviously, ethanol yields were decreased by 43%—53%, 1,3-PD yields and molar conversions from glycerol to 1,3-PD were increased by 27%—42% and 19%—24% respectively.