In this study, the engineered E. coli was constructed for efficient transformation of glycerol to 1,3-propanediol (1,3-PDO). To regenerate NADPH, the key bottleneck in 1,3-PDO production, heterologous NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDN, encoded by gapN) pathway was introduced, and the gapN expression level was fine-tuned with specific 5'-untranslated regions (5'-UTR) to balance the carbon flux distribution between the metabolic pathways of NADPH regeneration and 1,3-PDO biosynthesis. Additionally, glucose was added to the medium to promote glycerol utilization and cell growth. To elevate the utilization of glycerol in the presence of glucose, E. coli JA11 was constructed through destroying PEP-dependent glucose transport system while strengthening the ATP-dependent transport system. Subsequent optimization of nitrogen sources further improved 1,3-PDO production. Finally, under the optimal fermentation condition, E. coli JA11 produced 13.47 g/L 1,3-PDO, with a yield of 0.64 mol/mol, increased by 325% and 100% compared with the original engineered E. coli JA03, respectively.