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- Title
Investigating the effects of perturbations to pgi and eno gene expression on central carbon metabolism in Escherichia coli using <sup>13</sup> C metabolic flux analysis.
- Authors
Yuki Usui; Takashi Hirasawa; Chikara Furusawa; Tomokazu Shirai; Natsuko Yamamoto; Hirotada Mori; Hiroshi Shimizu
- Abstract
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.
- Subjects
ECOLOGICAL disturbances; GENE expression; METABOLISM; BIOCHEMISTRY; PHYSIOLOGY; MICROORGANISMS
- Publication
Microbial Cell Factories, 2012, Vol 11, Issue 1, p87
- ISSN
1475-2859
- Publication type
Article
- DOI
10.1186/1475-2859-11-87