Consequences of phosphoenolpyruvate:sugar phosphotranferase system and pyruvate kinase isozymes inactivation in central carbon metabolism flux distribution in Escherichia coli

BackgroundIn Escherichia coli phosphoenolpyruvate (PEP) is a key central metabolism intermediate that participates in glucose transport, as precursor in several biosynthetic pathways and it is involved in allosteric regulation of glycolytic enzymes. In this work we generated W3110 derivative strains that lack the main PEP consumers PEP:sugar phosphotransferase system (PTS-) and pyruvate kinase isozymes PykA and PykF (PTS-pykA- and PTS-pykF-). To characterize the effects of these modifications on cell physiology, carbon flux distribution and aromatics production capacity were determined.ResultsWhen compared to reference strain W3110, strain VH33 (PTS-) displayed lower specific rates for growth, glucose consumption and acetate production as well as a higher biomass yield from glucose. These phenotypic effects were even more pronounced by the additional inactivation of PykA or PykF. Carbon flux analysis revealed that PTS inactivation causes a redirection of metabolic flux towards biomass formation. A cycle involving PEP carboxylase (Ppc) and PEP carboxykinase (Pck) was detected in all strains. In strains W3110, VH33 (PTS-) and VH35 (PTS-, pykF-), the net flux in this cycle was inversely correlated with the specific rate of glucose consumption and inactivation of Pck in these strains caused a reduction in growth rate. In the PTS- background, inactivation of PykA caused a reduction in Ppc and Pck cycling as well as a reduction in flux to TCA, whereas inactivation of PykF caused an increase in anaplerotic flux from PEP to OAA and an increased flux to TCA. The wild-type and mutant strains were modified to overproduce L-phenylalanine. In resting cells experiments, compared to reference strain, a 10, 4 and 7-fold higher aromatics yields from glucose were observed as consequence of PTS, PTS PykA and PTS PykF inactivation.ConclusionsMetabolic flux analysis performed on strains lacking the main activities generating pyruvate from PEP revealed the high degree of flexibility to perturbations of the central metabolic network in E. coli. The observed responses to reduced glucose uptake and PEP to pyruvate rate of conversion caused by PTS, PykA and PykF inactivation included flux rerouting in several central metabolism nodes towards anabolic biosynthetic reactions, thus compensating for carbon limitation in these mutant strains. The detected cycle involving Ppc and Pck was found to be required for maintaining the specific growth and glucose consumption rates in all studied strains. Strains VH33 (PTS-), VH34 (PTS-pykA-) and VH35 (PTS-pykF-) have useful properties for biotechnological processes, such as increased PEP availability and high biomass yields from glucose, making them useful for the production of aromatic compounds or recombinant proteins.

[1]  H. Westerhoff,et al.  The Glycolytic Flux in Escherichia coli Is Controlled by the Demand for ATP , 2002, Journal of bacteriology.

[2]  E. Waygood,et al.  The control of pyruvate kinases of Escherichia coli. II. Effectors and regulatory properties of the enzyme activated by ribose 5-phosphate. , 1975, Canadian journal of biochemistry.

[3]  Guillermo Gosset,et al.  Improvement of Escherichia coli production strains by modification of the phosphoenolpyruvate:sugar phosphotransferase system , 2005, Microbial cell factories.

[4]  F. Bolivar,et al.  Pathway engineering for the production of aromatic compounds in Escherichia coli , 1996, Nature Biotechnology.

[5]  H. Kornberg,et al.  Two types of pyruvate kinase in Escherichia coli K12. , 1969, Biochimica et biophysica acta.

[6]  J. Liao,et al.  Control of gluconeogenic growth by pps and pck in Escherichia coli , 1993, Journal of bacteriology.

[7]  W. Wackernagel,et al.  Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. , 1995, Gene.

[8]  P. Boyer.  The inhibition of pyruvate kinase by ATP: a Mg++ buffer system for use in enzyme studies. , 1969, Biochemical and biophysical research communications.

[9]  M. Saier,et al.  The catabolite repressor/activator (Cra) protein of enteric bacteria , 1996, Journal of bacteriology.

[10]  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.

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

[12]  C. Francke,et al.  How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria , 2006, Microbiology and Molecular Biology Reviews.

[13]  B. Bachmann,et al.  Pedigrees of some mutant strains of Escherichia coli K-12. , 1972, Bacteriological reviews.

[14]  M H Saier,et al.  Frur mediates catabolite activation of pyruvate kinase (pykF) gene expression in Escherichia coli , 1996, Journal of bacteriology.

[15]  M. Bolognesi,et al.  The allosteric regulation of pyruvate kinase , 1996, FEBS letters.

[16]  A. Ishihama,et al.  Novel Members of the Cra Regulon Involved in Carbon Metabolism in Escherichia coli , 2010, Journal of Bacteriology.

[17]  F. Bolivar,et al.  Metabolic engineering and protein directed evolution increase the yield of L‐phenylalanine synthesized from glucose in Escherichia coli , 2004, Biotechnology and bioengineering.

[18]  J. Liao,et al.  Engineering of Escherichia coli central metabolism for aromatic metabolite production with near theoretical yield , 1994, Applied and environmental microbiology.

[19]  G. Sprenger.  From scratch to value: engineering Escherichia coli wild type cells to the production of l-phenylalanine and other fine chemicals derived from chorismate , 2007, Applied Microbiology and Biotechnology.

[20]  A. Cornish-Bowden.  Analysis of Progress Curves , 1976 .

[21]  E. Waygood,et al.  The control of pyruvate kinase of Escherichia coli. Binding of substrate and allosteric effectors to the enzyme activated by fructose 1,6-bisphosphate. , 1976, Biochemistry.

[22]  Francisco Bolívar,et al.  Replacement of the glucose phosphotransferase transport system by galactose permease reduces acetate accumulation and improves process performance of Escherichia coli for recombinant protein production without impairment of growth rate. , 2006, Metabolic engineering.

[23]  B D Sanwal,et al.  The control of pyruvate kinases of Escherichia coli: further studies of the enzyme activated by ribose-5-phosphate. , 1978, Canadian journal of biochemistry.

[24]  R. A. Cook,et al.  Characterization of the specific pyruvate transport system in Escherichia coli K-12 , 1987, Journal of bacteriology.

[25]  P. Dewick,et al.  Medicinal Natural Products: A Biosynthetic Approach , 1997 .

[26]  Alfredo Martínez,et al.  Expression of galP and glk in a Escherichia coli PTS mutant restores glucose transport and increases glycolytic flux to fermentation products , 2003, Biotechnology and bioengineering.

[27]  R. Jensen,et al.  Comparative Regulation of Isoenzymic 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthetases in Microorganisms , 1968, Journal of bacteriology.

[28]  H. Holmsen,et al.  The adenosine triphosphate inhibition of the pyruvate kinase reaction and its dependence on the total magnesium ion concentration. , 1969, The Biochemical journal.

[29]  Uwe Sauer,et al.  The PEP-pyruvate-oxaloacetate node as the switch point for carbon flux distribution in bacteria. , 2005, FEMS microbiology reviews.

[30]  L. Ingram,et al.  Characterization of the Zymomonas mobilis glucose facilitator gene product (glf) in recombinant Escherichia coli: examination of transport mechanism, kinetics and the role of glucokinase in glucose transport , 1995, Molecular microbiology.

[31]  H. Kornberg,et al.  Two tpes of pyruvate kinase in Escherichia coli K12 , 1969 .

[32]  Christoph Wittmann,et al.  Comparative Metabolic Flux Analysis of Lysine-Producing Corynebacterium glutamicum Cultured on Glucose or Fructose , 2004, Applied and Environmental Microbiology.

[33]  P. Dewick.  Medicinal Natural Products: A Biosynthetic Approach, 3Rd Edition , 2011 .

[34]  F. Bolivar,et al.  Determination of 3-deoxy-D-arabino-heptulosonate 7-phosphate productivity and yield from glucose in Escherichia coli devoid of the glucose phosphotransferase transport system. , 2001, Biotechnology and bioengineering.

[35]  A. D. de Graaf,et al.  Analysis of carbon metabolism in Escherichia coli strains with an inactive phosphotransferase system by (13)C labeling and NMR spectroscopy. , 2002, Metabolic Engineering.

[36]  Francisco Bolívar,et al.  Acetate Metabolism in Escherichia coli Strains Lacking Phosphoenolpyruvate: Carbohydrate Phosphotransferase System; Evidence of Carbon Recycling Strategies and Futile Cycles , 2008, Journal of Molecular Microbiology and Biotechnology.

[37]  G. Ferri,et al.  Divergent binding sites in pyruvate kinases I and II from Escherichia coli. , 1993, Biological chemistry Hoppe-Seyler.

[38]  B. Wanner,et al.  One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[39]  B. Hess,et al.  Analysis of progress curves. Interaction of pyruvate kinase from Escherichia coli with fructose 1,6-bisphosphate and calcium ions. , 1983, The Biochemical journal.

[40]  P. Maitra,et al.  A kinetic study of glycolytic enzyme synthesis in yeast. , 1971, The Journal of biological chemistry.

[41]  F. Bolivar,et al.  Metabolic engineering of Escherichia coli for improving l-3,4-dihydroxyphenylalanine (l-DOPA) synthesis from glucose , 2011, Journal of Industrial Microbiology & Biotechnology.

[42]  G R Jacobson,et al.  Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria. , 1993, Microbiological reviews.

[43]  G. Gosset,et al.  A direct comparison of approaches for increasing carbon flow to aromatic biosynthesis inEscherichia coli , 1996, Journal of Industrial Microbiology.

[44]  M. F. Drincovich,et al.  Functional dissection of Escherichia coli phosphotransacetylase structural domains and analysis of key compounds involved in activity regulation , 2010, The FEBS journal.

[45]  A Martinez,et al.  Cloning of the two pyruvate kinase isoenzyme structural genes from Escherichia coli: the relative roles of these enzymes in pyruvate biosynthesis , 1995, Journal of bacteriology.

[46]  F. Bolivar,et al.  Metabolic engineering for improving anthranilate synthesis from glucose in Escherichia coli , 2009, Microbial cell factories.

[47]  K. Izui,et al.  Regulation of Escherichia coli phosphoenolpyruvate carboxylase by multiple effectors in vivo. Estimation of the activities in the cells grown on various compounds. , 1980, Journal of biochemistry.

[48]  H. Umezawa,et al.  Pyruvate kinase of Escherichia coli. Its role in supplying nucleoside triphosphates in cells under anaerobic conditions. , 1974, Journal of biochemistry.

[49]  Guillermo Gosset,et al.  Production of aromatic compounds in bacteria. , 2009, Current opinion in biotechnology.

[50]  E. Ponce,et al.  Pyruvate kinase: current status of regulatory and functional properties. , 2003, Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology.

[51]  Alvaro R. Lara,et al.  Enhanced production of plasmid DNA by engineered Escherichia coli strains. , 2012, Journal of biotechnology.

[52]  U. Sauer,et al.  Metabolic Flux Responses to Pyruvate Kinase Knockout in Escherichia coli , 2002, Journal of bacteriology.

[53]  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.

[54]  M. Kumar,et al.  The commercial production of chemicals using pathway engineering. , 2000, Biochimica et biophysica acta.

[55]  Katy C. Kao,et al.  Global Expression Profiling of Acetate-grown Escherichia coli * , 2002, The Journal of Biological Chemistry.

[56]  A. Galizzi,et al.  The Allosteric Regulation of Pyruvate Kinase , 2000, The Journal of Biological Chemistry.

[57]  Masaru Tomita,et al.  Inhibitory effect of phosphoenolpyruvate on glycolytic enzymes in Escherichia coli. , 2007, Research in microbiology.

[58]  J. W. Frost,et al.  Altered Glucose Transport and Shikimate Pathway Product Yields in E.coli , 2003, Biotechnology progress.

[59]  P. Knowles,et al.  On the mechanism of 5-enolpyruvylshikimate 3-phosphate synthetase. , 1971, The Journal of biological chemistry.

[60]  W. Leuchtenberger,et al.  Biotechnological production of amino acids and derivatives: current status and prospects , 2005, Applied Microbiology and Biotechnology.

[61]  Francisco Bolívar,et al.  Metabolic engineering for the production of shikimic acid in an evolved Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system , 2010, Microbial cell factories.