BACKGROUND: Previously, Escherichia coli NSK015 was developed for high yield of 1,3-propanediol (1,3-PDO) production. To further improve 1,3-PDO concentration, parameters including kLa values with different agitations and impeller numbers, concentrations of coenzyme B12 and feeding strategies during fed-batch fermentation were investigated. RESULTS: In this study, aerobic conditions at 300 rpm agitation and 1 vvm aeration with two Rushton turbine impellers (kLa = 33.6 h?1) and the concentration of coenzyme B12 at 7.5 ?mol L?1 were identified as the best optimized conditions to improve 1,3-PDO production by the strain. With a two-pulsed continuous feeding, E. coli NSK015 produced 1,3-PDO up to 60.3 g L?1 with the 1,3-PDO yield approaching a theoretical maximum of 0.97 g g?1 and productivity of 0.42 g L?1 h?1 in fed-batch fermentation, in which concentration was improved about 60% compared to that of batch fermentation. CONCLUSION: The result indicated the efficiency of E. coli NSK015 in producing 1,3-PDO under optimal aerobic conditions. The strain could even enhance growth and maintain enzymatic activities involved in the 1,3-PDO pathway without utilizing antibiotics, isopropyl ?-d-1-thiogalactopyranoside (IPTG) or enriching nutrients. Plasmid instability, high production cost related to medium preparation and purification, and waste disposal were not of concern. This may provide a new insight for large-scale 1,3-PDO production by E. coli NSK015. Additionally, E. coli NSK015 could be a microbial host model for further developing new 1,3-PDO-producing microorganisms regardless of plasmid, inducer, antibiotics and rich nutrients in fermentation medium. © 2023 Society of Chemical Industry (SCI). © 2023 Society of Chemical Industry (SCI).