Klebsiella pneumoniae HR526, a new isolated 1,3-Propanediol (1,3-PD) producer, exhibited great productivity. However, the accumulation of lactate in the late-exponential phase remained an obstacle of 1,3-PD industrial scale production. Hereby, mutants lacking D-lactate pathway were constructed by knocking out the ldhA gene encoding fermentative D-lactate dehydrogenase (LDH) of HR526. The mutant K. pneumoniae LDH526 with the lowest LDH activity was studied in aerobic fed-batch fermentation. In experiments, conversion; and productivity increased from 95.39 gL(-1), 0.48 and 1.98 gL(-1)h(-1) to 102.06 gL(-1,) 0.52 mol mol(-1) and 2.13 gL(-1)h(-1), respectively. The diol (1,3-PD and 2,3-butanediol) conversion increased from 0.55 mol mol(-1) to a maximum of 0.65 mol mol(-1). Lactate would not accumulate until 1,3-PD exceeded 84 gL(-1), and the final lactate concentration decreased dramatically from more than 40 gL(-1) to <3 gL(-1). Enzymic measurements showed LDH activity decreased by 89-98% during fed-batch fermentation, and other related enzyme activities were not affected. NADH/NAD(+) enhanced more than 50% in the late-exponential phase as the D-lactate pathway was cut off, which might be the main reason for the change of final metabolites concentrations. The ability to utilize crude glycerol from biodiesel process and great genetic stability demonstrated that K. pnemoniae LDH526 was valuable for 1,3-PD industrial production. Biotechnol Bioeng. 2009;104: 965-972. (C) Wiley Periodicals, Inc.