Disruption of a global regulatory gene to enhance central carbon flux intophenylalanine biosynthesis in Escherichia coli

Genetic engineering of microbes for commercial metabolite production tradit ionally has sought to alter the levels and/or intrinsic activities of key e nzymes in relevant biosynthetic pathway(s). Microorganisms exploit similar strategies for flux control, but also coordinate flux through sets of relat ed pathways by using global regulatory circuits. We have engineered a globa l regulatory system of Escherichia coli, Csr (carbon storage regulator), to increase precursor for aromatic amino acid biosynthesis. Disruption of csr A increases gluconeogenesis, decreases glycolysis, and thus elevates phosph oenolpyruvate, a limiting precursor of aromatics. A strain in which the aro matic (shikimate) pathway had been optimized produced twofold more phenylal anine when csrA was disrupted. Overexpression of tktA (transketolase) to in crease the other precursor, erythrose-4-phosphate, yielded similar to1.4-fo ld enhance ment, while both changes were additive. These effects of csrA we re not mediated by increasing the regulatory enzymes of phenylalanine biosy nthesis. This study introduces the concept of "global metabolic engineering " for second-generation strain improvement.