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Metabolic flux distribution and thermodynamic analysis of green fluorescent protein production in recombinant Escherichia coli: The effect of carbon source and CO 2 partial pressure

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Abstract

Increasing recombinant protein production yields from bacterial cultures remains an important challenge in biotechnology. Acetate accumulation due to high dissolved carbon dioxide (pCO2) concentrations in the medium has been identified as a factor that negatively affects such yields. Under appropriate culture conditions, acetate could be re-assimilated by bacterial cells to maintain heterologous proteins production. In this work, we developed a simplified metabolic network aiming to establish a reaction rate analysis for a recombinant Escherichia coli when producing green fluorescent protein (GFP) under controlled pCO2 concentrations. Because E. coli is able to consume both glucose and acetate, the analysis was performed in two stages. Our results indicated that GFP synthesis is an independent process of cellular growth in some culture phases. Additionally, recombinant protein production is influenced by the available carbon source and the amount of pCO2 in the culture medium. When growing on glucose, the increase in the pCO2 concentration produced a down-regulation of central carbon metabolism by directing the carbon flux toward acetate accumulation; as a result, cellular growth and the overall GFP yield decreased. However, the maximum specific rate of GFP synthesis occurred with acetate as the main available carbon source, despite the low activity in the other metabolic pathways. To maintain cellular functions, including GFP synthesis, carbon flux was re-distributed toward the tricarboxylic acid cycle and the pentose phosphate pathway to produce ATP and NADH. The thermodynamic analysis allowed demonstrating the feasibility of the simplified network for describing the metabolic state of a recombinant system.

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References

  1. Lee, S., H. Kim, J. Park, J. Park, and T. Kim (2009) Metabolic engineering of microorganisms: General strategies and drug production. Drug. Discov. Today 14: 78–88.

    Article  CAS  Google Scholar 

  2. Stephanopoulos, G. and J. Vallino (1991) Network rigidity and metabolic engineering in metabolite overproduction. Sci. 252: 1675–1681.

    Article  CAS  Google Scholar 

  3. Swartz, J. R. (2001) Advances in Escherichia coli production of therapeutic proteins. Curr. Opin. Biotechnol. 12: 195–201.

    Article  CAS  Google Scholar 

  4. Carneiro, S., S. Villas-Bôas, I. Rocha, and E. Ferreira (2010) Applying a metabolic foot printing approach to characterize the impact of the recombinant protein production in Escherichia coli. pp. 193–200. In: M. P. Rocha (eds.). Advances in Bioinformatics. Springer Berlin Heidelberg.

    Chapter  Google Scholar 

  5. Dixon, N. M. and D. Kell (1989) The inhibition by CO2 of the growth and metabolism of micro-organisms. J. Appl. Bacteriol. 67: 109–136.

    Article  CAS  Google Scholar 

  6. Bäumchen, C., A. Knoll, B. Husemann, J. Seletzky, B. Maier, C. Dietrich, G. Amoabediny, and J. Büchs (2007) Effect of dissolved carbon dioxide concentrations on growth of Corynebacterium glutamicum on D-glucose and L-lactate. J. Biotechnol. 128: 868–874.

    Article  Google Scholar 

  7. Oh, M. K. and J. Liao (2000) DNA microarray detection of metabolic responses to protein overproduction in Escherichia coli. Metabol. Eng. 2: 201–209.

    Article  CAS  Google Scholar 

  8. Richins, R. and W. Chen (2001) Effects of FIS over expression on cell growth, rRNA synthesis, and ribosome content in Escherichia coli. Biotechnol. Prog. 17: 252–257.

    Article  CAS  Google Scholar 

  9. Causey, T., K. Shanmugam, L. Yomano, and L. Ingram (2004) Engineering Escherichia coli for efficient conversion of glucose to pyruvate. Proc. Natl. Acad. Sci. USA. 101: 2235–2240.

    Article  CAS  Google Scholar 

  10. Kim, Y., L. Ingram, and K. Shanmugan (2007) Construction of an Escherichia coli K-17 mutant for homoethanologenic fermentation of glucose of xylose without foreign genes. Appl. Environ. Microbiol. 73: 1766–1771.

    Article  CAS  Google Scholar 

  11. Atsumi, S., A. Cann, M. Connor, C. Shen, K. Smith, M. Brynildsen, M. Chou, T. Hanai, and J. Liao (2008) Metabolic engineering of E. coli for 1-butanol production. Metabol. Eng. 10: 305–311.

    Article  CAS  Google Scholar 

  12. Henry, C., M. Jankowski, L. Broadbelt, and V. Hatzimanikatis (2006) Genome-scale thermodynamic analysis of Escherichia coli metabolism. Biophysic. J. 90: 1453–1461.

    Article  CAS  Google Scholar 

  13. Henry, C., L. Broadbelt, and V. Hatzimanikatis (2007) Thermodynamics-based metabolic flux analysis. Biophysic. J. 92: 1792–1805.

    Article  CAS  Google Scholar 

  14. Mavrovouniotis, M. L., G. Stephanopoulos, and G. Stephanopoulos (1990) Computer-aided synthesis of biochemical pathways. Biotechnol. Bioeng. 36: 1119–1132.

    Article  CAS  Google Scholar 

  15. Liu, L., R. Agren, S. Bordel, and J. Nielsen (2010) Use of genome-scale metabolic models for understanding microbial physiology. FEBS Lett. 584: 2556–2564.

    Article  CAS  Google Scholar 

  16. Stephanopoulos, G., A. Aristos, and J. Nielsen (1998) Metabolic engineering: principles and methodologies. pp. 285–307; 326–330. Academic Press, USA.

    Book  Google Scholar 

  17. Garg, S., L. Yang, and R. Mahadevan (2010) Thermodynamic analysis of regulation on metabolic networks using constrained-based modeling. BMC Res. Notes. 3: 125.

    Article  Google Scholar 

  18. Mavrovouniotis, M. L. (1990) Group contributions for estimating standard Gibbs energies of formation of biochemical compounds in aqueous solution. Biotechnol. Bioeng. 36: 1070–1082.

    Article  CAS  Google Scholar 

  19. Mavrovouniotis, M. L. (1991) Estimation of standard Gibbs energy changes of biotransformations. J. Biol. Chem. 266: 14440–14445.

    CAS  Google Scholar 

  20. Albe, K. R., M. Butler, and B. Wright (1990) Cellular concentrations of enzymes and their substrates. J. Theor. Biol. 143: 163–195.

    Article  CAS  Google Scholar 

  21. Baez, A., N. Flores, F. Bolivar, and O. Ramírez (2009) Metabolic and transcriptional response of recombinant Escherichia coli to elevated dissolved carbon dioxide concentrations. Biotechnol. Bioeng. 104: 102–110.

    Article  CAS  Google Scholar 

  22. Bearson, B. L., I. S. Lee, and T. A. Casey (2009) Escherichia coli O157: H7 glutamate- and arginine-dependent acid-resistance systems protect against oxidative stress during extreme acid challenge. Microbiol. 155: 805–812.

    Article  CAS  Google Scholar 

  23. Nielsen, J. (2001) Metabolic engineering. Appl. Microbiol. Biot. 55: 263–283.

    Article  CAS  Google Scholar 

  24. Vallino, J. J. (1991) Identification of branch-point restrictions in microbial metabolism through metabolic flux analysis and local network perturbations. Ph.D. Thesis. Massachusetts Institute of Technology, Cambridge, MA, USA.

    Google Scholar 

  25. Nielsen, J., J. Villadsen, and G. Liden (1994) Bioreaction Engineering Principles. 1st ed., pp. 24–27. Plenum Press, NY, USA.

    Book  Google Scholar 

  26. Shojaosadati, S. A., S. Varedi-Kolaei, V. Babaeipur, and A. M. Farnoud (2008) Recent advances in high cell density cultivation for production of recombinant protein. Iran J. Biotechnol. 6: 63–84.

    CAS  Google Scholar 

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Authors and Affiliations

  1. Bioengineering Department, Unidad Profesional Interdisciplinaria de Biotecnología, Instituto Politécnico Nacional, UPIBI-IPN, CP 07340, Mexico, Mexico

    R. Axayácatl González-García, Edgar Salgado-Manjarrez & Juan S. Aranda-Barradas

  2. Bioprocesses Department, Unidad Profesional Interdisciplinaria de Biotecnología, Instituto Politécnico Nacional, UPIBI-IPN, CP 07340, Mexico, Mexico

    E. Ines Garcia-Peña

Authors
  1. R. Axayácatl González-García
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  2. E. Ines Garcia-Peña
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  3. Edgar Salgado-Manjarrez
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  4. Juan S. Aranda-Barradas
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Correspondence to Juan S. Aranda-Barradas.

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González-García, R.A., Garcia-Peña, E.I., Salgado-Manjarrez, E. et al. Metabolic flux distribution and thermodynamic analysis of green fluorescent protein production in recombinant Escherichia coli: The effect of carbon source and CO 2 partial pressure. Biotechnol Bioproc E 18, 1049–1061 (2013). https://doi.org/10.1007/s12257-013-0277-5

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  • DOI: https://doi.org/10.1007/s12257-013-0277-5

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