The Escherichia coli Amino Acid Uptake Protein CycA: Regulation of Its Synthesis and Practical Application in l-Isoleucine Production

Amino acid transport systems perform important physiological functions; their role should certainly be considered in microbial production of amino acids. Typically, in the context of metabolic engineering, efforts are focused on the search for and application of specific amino acid efflux pumps. However, in addition, importers can also be used to improve the industrial process as a whole. In this study, the protein CycA, which is known for uptake of nonpolar amino acids, was characterized from the viewpoint of regulating its expression and range of substrates. We prepared a cycA-overexpressing strain and found that it exhibited high sensitivity to branched-chain amino acids and their structural analogues, with relatively increased consumption of these amino acids, suggesting that they are imported by CycA. The expression of cycA was found to be dependent on the extracellular concentrations of substrate amino acids. The role of some transcription factors in cycA expression, including of Lrp and Crp, was studied using a reporter gene construct. Evidence for the direct binding of Crp to the cycA regulatory region was obtained using a gel-retardation assay. The enhanced import of named amino acids due to cycA overexpression in the l-isoleucine-producing strain resulted in a significant reduction in the generation of undesirable impurities. This work demonstrates the importance of uptake systems with respect to their application in metabolic engineering.

Automated summary

Amino acid transport systems play important roles in microbial production of amino acids, and their significance in metabolic engineering should be acknowledged. This study characterizes the protein CycA, which is responsible for the uptake of nonpolar amino acids, and investigates its expression and substrate range. The overexpression of CycA enhances the import of specific amino acids and leads to a reduction in undesirable impurities. The expression of cycA is regulated by extracellular amino acid concentrations, with Lrp and Crp identified as potential transcription factors involved in its expression.