Abstract <p><i>Escherichia coli</i> strains are widely used in biotechnology for the production of compounds of great interest, such as L-amino acids and recombinant proteins. The aim of this study was to determine the effect of protein Nε-lysine acetylation processes on the metabolism of commonly used <i>E. coli</i> strains MG1655 and BL21(DE3), as well as strains producing the amino acids L-threonine and L-proline. We demonstrate that the acetylation profile of one of the key enzymes of glycolysis, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), depends on the strain in which this protein was synthesized. Deletion of genes involved in acetate metabolism and acetylation of the ε-amino group of protein lysine affects the metabolism of the producer strains and the productivity of the target amino acids. Approaches involving modulation of protein acetylation may be useful in metabolic engineering in creating more efficient producer strains.</p>