Much effort has been devoted to the metabolic engineering of Klebsiella pneumoniae; however, our knowledge of the actual expression level of promoters used in K.pneumoniae is limited. In this study, the expression levels of three promoters were compared systematically by using the lacZ reporter gene with different carbon sources in K.pneumoniae. The results showed that, although promoters PT5 and Ptac designed for Escherichia coli were functional, PT5 appeared more efficient and the induction/repression ratio of Ptac was decreased extremely in K.pneumoniae. The basal level of Ptac for lacZ expression reached 396.5U/mg, which was 9.5-fold higher compared with PT5 in LB medium, indicating Ptac can be used as an efficient constitutive promoter as well as an efficient induced promoter in K.pneumoniae. In different carbon sources medium, a newly constructed endogenous constitutive Pbud proved to be a stable and weak promoter. On the basis of our data, a set of Pbud and Ptac promoters could meet the broad range (about 1,000 orders of magnitude) of gene expression needed for engineered K.pneumoniae in glycerol-based medium. (C) 2015 International Union of Biochemistry and Molecular Biology, Inc.