Although plasmid-dependent microbial breeding is predominant in manufacturing bio-based chemicals, it shows pitfall of genetic instability and thus hinders industrial production. Alternatively, chromosome engineering free from plasmid enables genetic stability and thus represents the new trend of microbial breeding. 3-Hydroxypropionic acid (3-HP) is an economically important platform compound as the versatile precursor of a suite of C3 compound, such as 1, 3-PDO. Klebsiella pneumoniae is regarded as a promising host strain due to both its dha regulon and exceptional glycerol fermentation ability. To produce 3-HP in K. pneumoniae, the IS1 region in chromosome was replaced with the AD DNA cassette containing aldH gene from E.coli through homologous recombination approach. The engineered recombinant converted glycerol into 3-HPA and then 3-HP when 40 g/L of initial glycerol was added. The novelties of this study comprise (i) the genetic stability of plasmid-free strains (ii) without using any inducer and antibiotics and thus more applicable than plasmid-based strains.