Batch cultivation of recombinant bacteria in shake flasks typically results in low cell density due to nutrient depletion. Previous studies on high cell density cultivation in shake flasks have relied mainly on controlled release mechanisms. Here, we report a true fed-batch strategy to achieve high cell density of recombinant E. coli in shake flasks in 24 h by feeding a mixture of glycerol and yeast extract with a syringe pump. Feed composition and feed rate were obtained by cybernetic model-based, multi-objective optimization. Model parameters were estimated from time-course measurement of substrate, biomass, and dissolved oxygen levels. The optimized process yielded 20.7 g dry cell weight/L, in agreement with the model prediction. Volumetric protein productivity improved by 10-34-fold compared to batch cultivation with 2.8-fold further improvement when the fed-batch process was replicated in a 3 L bioreactor. The process has significance in the routine laboratory cultivations and in scaleup studies.