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<p>Abstract</p> <p>Background</p> <p>It is quite important to understand how the central metabolism is regulated under nitrogen (N)- limitation as well as carbon (C)- limitation. In particular, the effect of C/N ratio on the metabolism is of practical interest for the heterologous protein production, PHB production, etc. Although the carbon and nitrogen metabolisms are interconnected and the overall mechanism is complicated, it is strongly desirable to clarify the effects of culture environment on the metabolism from the practical application point of view.</p> <p>Results</p> <p>The effect of C/N ratio on the metabolism in <it>Escherichia coli </it>was investigated in the aerobic continuous culture at the dilution rate of 0.2 h^-1based on fermentation data, transcriptional RNA level, and enzyme activity data. The glucose concentration was kept at 10 g/l, while ammonium sulfate concentration was varied from 5.94 to 0.594 g/l. The resultant C/N ratios were 1.68 (100%), 2.81(60%), 4.21(40%), 8.42(20%), and 16.84(10%), where the percentage values in brackets indicate the ratio of N- concentration as compared to the case of 5.94 g/l of ammonium sulfate. The mRNA levels of <it>crp </it>and <it>mlc </it>decreased, which caused <it>ptsG </it>transcript expression to be up-regulated as C/N ratio increased. As C/N ratio increased <it>cra </it>transcript expression decreased, which caused <it>ptsH, pfkA</it>, and <it>pykF </it>to be up-regulated. At high C/N ratio, transcriptional mRNA level of <it>soxR/S </it>increased, which may be due to the activated respiratory chain as indicated by up-regulations of such genes as <it>cyoA, cydB, ndh </it>as well as the increase in the specific CO₂production rate. The <it>rpoN </it>transcript expression increased with the increase in C/N ratio, which led <it>glnA, L, G </it>and <it>gltD </it>transcript expression to change in similar fashion. The <it>nac </it>transcript expression showed similar trend as <it>rpoN</it>, while <it>gdhA </it>transcript expression changed in reverse direction. The transcriptional mRNA level of <it>glnB</it>, which codes for P_II, <it>glnD </it>and <it>glnK </it>increased as C/N ratio increases. It was shown that GS-GOGAT pathway was activated for <it>gdhA </it>mutant under N- rich condition. In the case of <it>glnL </it>mutant, GOGAT enzyme activity was reduced as compared to the wild type under N- limitation. In the case of <it>gltB, D </it>mutants, GDH and GS enzymes were utilized under both N- rich and N- limited conditions. In this case, the transcriptional mRNA level of <it>gdhA </it>and corresponding GDH enzyme activity was higher under N- limitation as compared to N- rich condition.</p> <p>Conclusion</p> <p>The metabolic regulation of <it>E.coli </it>was clarified under both carbon (C)- limitation and nitrogen (N)- limitation based on fermentation, transcriptional mRNA level and enzyme activities. The overall regulation mechanism was proposed. The effects of knocking out N- assimilation pathway genes were also clarified.</p>