The effect of fed-batch operation (FBO) strategy was investigated using pretreated-beet molasses, containing galactose that induces the lac promoter, on benzaldehyde lyase (BAL) production by recombinant Escherichia coli BL21(DE3)pLySs. After batch cultivation with 30 g l(-1) pretreated-beet molasses consisting of 7.5 g l(-1) glucose and 7.5 g l(-1) fructose, three FBO strategies were applied at dissolved oxygen (=40%) cascade to air-flow rate. In FBO1 when air-flow rate decreased considerably, feed was given to the system in pulses in such a way that pretreated-beet molasses concentration increased by 10 kg m(-3) (containing 2.5 g l(-1) glucose+2.5 g l(-1) fructose); however, decrease in air-flow rate demonstrated only the absence of glucose but not fructose. Thus, in FBO2 when fructose and glucose were completely utilized, pretreated-beet molasses was pulse-fed and its concentration increased by 10 g l(-1). In FBO3 with the decreased amount of pretreated-beet molasses (6 g l(-1)), shift response time from glucose to fructose consumption was avoided, and glucose and fructose consumptions were well correlated with air-flow rate, and the highest C (X) (8.04 g l(-1)) and BAL (2,315 U ml(-1)) production were obtained (t = 24 h) with the highest substrate yield on cell and product formation.