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

BackgroundIt 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.ResultsWe 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.ConclusionsOur 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.

[1]  H. Mori,et al.  Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection , 2006, Molecular systems biology.

[2]  J. Cronan,et al.  Molecular cloning of the gene (poxB) encoding the pyruvate oxidase of Escherichia coli, a lipid-activated enzyme , 1984, Journal of bacteriology.

[3]  H. Shimizu,et al.  Evaluating 13C enrichment data of free amino acids for precise metabolic flux analysis , 2011, Biotechnology journal.

[4]  D. Koshland,et al.  Compensatory phosphorylation of isocitrate dehydrogenase. A mechanism for adaptation to the intracellular environment. , 1985, The Journal of biological chemistry.

[5]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[6]  J. Bautista,et al.  Evidence suggesting that the NADPH/NADP ratio modulates the splitting of the isocitrate flux between the glyoxylic and tricarboxylic acid cycles, in Escherichia coli , 1979, FEBS letters.

[7]  H. Kitano.  Systems Biology: A Brief Overview , 2002, Science.

[8]  K. Nakahigashi,et al.  Catabolic regulation analysis of Escherichia coli and its crp, mlc, mgsA, pgi and ptsG mutants , 2011, Microbial cell factories.

[9]  P. De Bièvre,et al.  Atomic weights of the elements. Review 2000 (IUPAC Technical Report) , 2009 .

[10]  M. Araúzo-Bravo,et al.  Metabolic flux analysis of pykF gene knockout Escherichia coli based on 13C-labeling experiments together with measurements of enzyme activities and intracellular metabolite concentrations , 2004, Applied Microbiology and Biotechnology.

[11]  P. De Bièvre,et al.  ATOMIC WEIGHTS OF THE ELEMENTS: REVIEW 2000 , 2003 .

[12]  H. Kornberg,et al.  The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli. , 1977, Journal of general microbiology.

[13]  Ka-Yiu San,et al.  Characterization of the Acetate‐Producing Pathways in Escherichia coli , 2008, Biotechnology progress.

[14]  Nobuyoshi Ishii,et al.  13C‐metabolic flux analysis for batch culture of Escherichia coli and its pyk and pgi gene knockout mutants based on mass isotopomer distribution of intracellular metabolites , 2010, Biotechnology progress.

[15]  R. Tait,et al.  Isolation and characterization of the phosphoglucose isomerase gene from Escherichia coli , 1989, Molecular and General Genetics MGG.

[16]  T. Coplen.  Atomic Weights of the Elements , 2003 .

[17]  Sang Yup Lee,et al.  Systems biotechnology for strain improvement. , 2005, Trends in biotechnology.

[18]  C. Wittmann,et al.  Metabolic flux analysis using mass spectrometry. , 2002, Advances in biochemical engineering/biotechnology.

[19]  Bernhard O Palsson,et al.  Latent Pathway Activation and Increased Pathway Capacity Enable Escherichia coli Adaptation to Loss of Key Metabolic Enzymes* , 2006, Journal of Biological Chemistry.

[20]  Tetsuya Yomo,et al.  Construction of Escherichia coli gene expression level perturbation collection. , 2009, Metabolic engineering.

[21]  Pei Yee Ho,et al.  Multiple High-Throughput Analyses Monitor the Response of E. coli to Perturbations , 2007, Science.

[22]  Bernhard Ø. Palsson,et al.  Genetic Basis of Growth Adaptation of Escherichia coli after Deletion of pgi, a Major Metabolic Gene , 2010, PLoS genetics.

[23]  M. Araúzo-Bravo,et al.  Effect of a pyruvate kinase (pykF-gene) knockout mutation on the control of gene expression and metabolic fluxes in Escherichia coli. , 2004, FEMS microbiology letters.

[24]  Peng Wang,et al.  Biochemical properties and physiological roles of NADP-dependent malic enzyme in Escherichia coli , 2011, The Journal of Microbiology.

[25]  Christoph Wittmann,et al.  Metabolic fluxes and beyond—systems biology understanding and engineering of microbial metabolism , 2010, Applied Microbiology and Biotechnology.

[26]  Sahm,et al.  13C tracer experiments and metabolite balancing for metabolic flux analysis: comparing two approaches , 1998, Biotechnology and bioengineering.

[27]  H. Mori,et al.  Responses of theCentral Metabolism in Escherichia coli to PhosphoglucoseIsomerase and Glucose-6-Phosphate DehydrogenaseKnockouts , 2003, Journal of bacteriology.

[28]  D. Laporte,et al.  Glyoxylate bypass operon of Escherichia coli: cloning and determination of the functional map , 1988, Journal of bacteriology.

[29]  G. Stephanopoulos,et al.  Metabolic Engineering: Principles And Methodologies , 1998 .

[30]  S. Ichihara,et al.  Identification and characterization of the ackA (acetate kinase A)-pta (phosphotransacetylase) operon and complementation analysis of acetate utilization by an ackA-pta deletion mutant of Escherichia coli. , 1994, Journal of biochemistry.

[31]  G. Sprenger,et al.  Cloning, nucleotide sequence, and functional expression of the Escherichia coli enolase (eno) gene in a temperature-sensitive eno mutant strain. , 1996, DNA sequence : the journal of DNA sequencing and mapping.

[32]  Pei Yee Ho,et al.  Effect of lpdA gene knockout on the metabolism in Escherichia coli based on enzyme activities, intracellular metabolite concentrations and metabolic flux analysis by 13C-labeling experiments. , 2006, Journal of biotechnology.

[33]  C. Wittmann,et al.  Response of the central metabolism of Escherichia coli to modified expression of the gene encoding the glucose‐6‐phosphate dehydrogenase , 2007, FEBS letters.