Recombinant protein expression from lac derived promoters by the autoinduction regime is based on diauxic growth of Escherichia coli on glucose and lactose. Glycerol is used as a supporting carbon source during the lactose-induced expression. While this glycerol-based formulation usually provides high cell densities, successful protein expression by autoinduction is often very dependent on correct aeration level. This complicates the reproducibility and scalability of the cultures. In this study we investigate the use of an alternative autoinduction formulation, in which the supporting carbon source is provided by fed-batch-like slow glucose feed from a biocatalytically degraded polysaccharide. The glucose feed as supporting carbon source allowed for high level of autoinduced target protein expression from T7lac promoter in E. coli BL21(DE3) and from T5lac promoter in E. coil K-12 RB791(lacI(q)) with lactose concentrations of 0.5-2 g l(-1). Cell densities and protein yields per culture volume were similar to or higher than in the glycerol-based ZYM-5052 medium. In the glycerol-based medium, protein production was adversely influenced by high aeration level, resulting in 75-90% reduction in protein yield per cell compared to more moderately aerated conditions. The glucose fed-batch medium attenuated this oxygen-sensitivity and provided robust high-yield expression also under high aeration rates. It is concluded that the slow glucose feed as supporting carbon source mitigates aeration-related scale differences in autoinduced protein expression, and combined with the benefit of high product yields this makes the fed-batch autoinduction medium ideal for high-throughput screening and scale-up of the production process. (C) 2013 Elsevier Inc. All rights reserved.