Abstract For recombinant protein production in E. coli fed-batch cultures, post-induction conditions have great influence in the quantity and quality of the product. The present paper covers the effect of different factors affecting the cellular environment in recombinant aldolase (rhamnulose-1-phosphate aldolase, RhuA) production. An operational mode employing an exponential addition profile for constant specific growth rate has been analyzed, in order to understand and define possible modifications with influence on post-induction cellular behavior. A constant addition profile has been demonstrated to render higher specific aldolase production than the exponential addition profile, probably due to a more constant environment for the cells. On the other hand, amino acid (leucine) supplementation has proven to increase protein quality in terms of activity units (U) per unit mass of RhuA (U mg−1 RhuA), alleviating metabolic overload. Based on the above, a production process was set up and scaled up to pilot plant. Resulting production was double that of a standard laboratory operation, 45,000 U L−1, and almost all the protein retained the 6xHis-tag with the highest quality, 11.3 U mg−1 RhuA.