In the present work the impact of large production scale was investigated for Bacillus megaterium expressing green fluorescent protein (GFP). Specifically designed scale-down studies, mimicking the intermittent and continuous nutrient supply of large- and small-scale processes, were carried out for this purpose. The recombinant strain revealed a 40% reduced GFP yield for the large-scale conditions. In line with extended carbon loss via formation of acetate and carbon dioxide, this indicated obvious limitations in the underlying metabolism of B. megaterium under the large-scale conditions. Quantitative analysis of intracellular amino acids via validated fast filtration protocols revealed that their level strongly differed between the two scenarios. During cultivation in large-scale set-up, the availability of most amino acids, serving as key building blocks of the recombinant protein, was substantially reduced. This was most pronounced for tryptophan, aspartate, histidine, glutamine, and lysine. In contrast alanine was increased, probably related to a bottleneck at the level of pyruvate which also triggered acetate overflow metabolism. The pre-cursor quantifications could then be exploited to verify the presumed bottlenecks and improve recombinant protein production under large-scale conditions. Addition of only 5 mM tryptophan, aspartate, histidine, glutamine, and lysine to the feed solution increased the GFP yield by 100%. This rational concept of driving the lab scale productivity of recombinant microorganisms under suboptimal feeding conditions emulating large scale can easily be extended to other processes and production hosts. Biotechnol. Bioeng. 2012; 109:1538–1550. © 2012 Wiley Periodicals, Inc.