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Background  It has long been recognized that analyzing the behaviour of the complex intracellular biological networks is important for breeding industrially useful microorganisms. However, because of the complexity of these biological networks, it is currently not possible to obtain all the desired microorganisms. In this study, we constructed a system for analyzing the effect of gene expression perturbations on the behavior of biological networks in  Escherichia coli . Specifically, we utilized  13  C metabolic flux analysis ( 13  C-MFA) to analyze the effect of perturbations to the expression levels of  pgi  and  eno  genes encoding phosphoglucose isomerase and enolase, respectively on metabolic fluxes.    Results  We constructed gene expression-controllable  E. coli  strains using a single-copy mini F plasmid. Using the  pgi  expression-controllable strain, we found that the specific growth rate correlated with the  pgi  expression level.  13  C-MFA of this strain revealed that the fluxes for the pentose phosphate pathway and Entner-Doudoroff pathway decreased, as the  pgi  expression lelvel increased. In addition, the glyoxylate shunt became active when the  pgi  expression level was almost zero. Moreover, the flux for the glyoxylate shunt increased when the  pgi  expression level decreased, but was significantly reduced in the  pgi -knockout cells. Comparatively,  eno  expression could not be decreased compared to the parent strain, but we found that increased  eno  expression resulted in a decreased specific growth rate.  13  C-MFA revealed that the metabolic flux distribution was not altered by an increased  eno  expression level, but the overall metabolic activity of the central metabolism decreased. Furthermore, to evaluate the impact of perturbed expression of  pgi  and  eno  genes on changes in metabolic fluxes in  E. coli  quantitatively, metabolic sensitivity analysis was performed. As a result, the perturbed expression of  pgi  gene had a great impact to the metabolic flux changes in the branch point between the glycolysis and pentose phosphate pathway, isocitrate dehydrogenase reaction, anaplerotic pathways and Entner-Doudoroff pathway. In contrast, the impact of perturbed  eno  expression to the flux changes in  E. coli  metabolic network was small.    Conclusions  Our results indicate that the response of metabolic fluxes to perturbation to  pgi  expression was different from that to  eno  expression; perturbations to  pgi  expression affect the reaction related to the Pgi protein function, the isocitrate dehydrogenase reaction, anaplerotic reactions and Entner-Doudoroff pathway. Meanwhile,  eno  expression seems to affect the overall metabolic activity, and the impact of perturbed  eno  expression on metabolic flux change is small. Using the gene expression control system reported here, it is expected that we can analyze the response and adaptation process of complex biological networks to gene expression perturbations.

microbial cell factories

Investigating the effects of perturbations to pgi and eno gene expression on central carbon metabolism in Escherichia coli using 13 C metabolic flux analysis