The production of 1,3-propanediol (1,3-PDO) from glucose was investigated using Klebsiella pneumoniae J2B, which converts glycerol to 1,3-PDO and synthesize an essential coenzyme B12. In order to connect the glycolytic pathway with the pathway of 1,3-PDO synthesis from glycerol, i.e., to directly produce diol from glucose, glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate phosphatase from Saccharomyces cerevisiae were overexpressed. Additionally, the effect of expression levels and the use of isoforms of these two enzymes on glycerol and 1,3-PDO production were studied. Furthermore, to prevent loss of produced glycerol, the glycerol oxidation pathways were disrupted. Finally, the conversion rate of glycerol to 1,3-PDO was increased via homologous overexpression of glycerol dehydratase and 1,3-PDO oxidoreductase. The resultant strain successfully produced 1,3-PDO from glucose at a yield of 0.27mol/mol along with glycerol at 0.52mol/mol. Improvement of the engineered K. pneumoniae J2B to further increase conversion of glycerol to 1,3-PDO is discussed.