Advertisement

Biotechnology and Bioprocess Engineering

, Volume 17, Issue 6, pp 1196–1204 | Cite as

Production of β-carotene and acetate in recombinant Escherichia coli with or without mevalonate pathway at different culture temperature or pH

  • Anh Do Quynh Nguyen
  • Seon-Won Kim
  • Sung Bae Kim
  • Yang-Gon Seo
  • In-Young Chung
  • Dae Hwan Kim
  • Chang-Joon KimEmail author
Research Paper

Abstract

Natural β-carotene has received much attention as consumers have become more health conscious. Its production by various microorganisms including metabolically engineered Escherichia coli or Saccharomyces cerevisiae has been attempted. We successfully created a recombinant E. coli with an engineered whole mevalonate pathway in addition to β-carotene biosynthetic genes and evaluated the engineered cells from the aspects of metabolic balance between central metabolism and β-carotene production by comparison with conventional β-carotene producing recombinant E. coli (control) utilizing a native methylerythritol phosphate (MEP) pathway using bioreactor cultures generated at different temperatures or pHs. Better production of β-carotene was obtained in E. coli cultured at 37°C than at 25°C. A two-fold higher titer and 2.9-fold higher volumetric productivity were obtained in engineered cells compared with control cells. Notably, a marginal amount of acetate was produced in actively growing engineered cells, whereas more than 8 g/L of acetate was produced in control cells with reduced cell growth at 37°C. The data indicated that the artificial operon of the whole mevalonate pathway operated efficiently in redirecting acetyl-CoA into isopentenyl pyrophosphate (IPP), thereby improving production of β-carotene, whereas the native MEP pathway did not convert a sufficient amount of pyruvate into IPP due to endogenous feedback regulation. Engineered cells also produced lycopene with a reduced amount of β-carotene in weak alkaline cultures, consistent with the inhibition of lycopene cyclase.

Keywords

recombinant Escherichia coli engineered whole mevalonate pathway bioreactor culture β-carotene acetate 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Burri, B. J. (1997) Beta-carotene and human health: A review of current research. Neutri. Res. 17: 547–580.CrossRefGoogle Scholar
  2. 2.
    Jaramillo-Flores, M. E., J. J. Lugo-Martinez, E. Ramirez-Sanjuan, H. Montellano-Rosales, L. Dorantes-Alvarez, and H. Hernandez-Sanchez (2005) Effect of sodium chloride, acetic acid, and enzymes on carotene extraction in carrots (Daccus carota L.). J. Food Sci. 70: 136–142.CrossRefGoogle Scholar
  3. 3.
    Garcia-Gonzalez, M., J. Moreno, J. C. Manzano, F. J. Florencio, and M. G. Guerrero (2005) Production of Dunaliella salina biomass rich in 9-cis-β-carotene and lutein in a closed tubular photobioreactor. J. Biotechnol. 115: 81–90.CrossRefGoogle Scholar
  4. 4.
    Kim, S. -W., J. -B. Kim, W. -H. Jung, J. -H. Kim, and J. -K. Jung (2006) Over-production of β-carotene from metabolically engineered Escherichia coli. Biotechnol. Lett. 28: 897–904.CrossRefGoogle Scholar
  5. 5.
    Mantzouridou, F., T. Roukas, and P. Kotzekidou (2005) Production of beta-carotene from synthetic medium by Blakeslea trispora in fed-batch culture. Food Biotechnol. 18: 343–361.CrossRefGoogle Scholar
  6. 6.
    Malisorn, C. and W. Suntornsuk (2008) Optimization of β-carotene production by Rhodotorula glutinis DM28 in fermented radish brine. Biores. Technol. 99: 2281–2287.CrossRefGoogle Scholar
  7. 7.
    Saenge, C., B. Cheirsilp, T. T. Suksaroge, and T. Bourtoom (2011) Efficient concomitant production of lipids and carotenoids by oleaginous red yeast Rhodotorula glutinis cultured in palm oil mill effluent and application of lipids for biodiesel production. Biotechnol. Bioproc. Eng. 16: 23–33.CrossRefGoogle Scholar
  8. 8.
    Verwaal, R., J. Wang, J. -P. Meijnen, H. Visser, G. Sandmann, J. A. V. D. Berg, and A. J. J. V. Ooyen (2007) High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous. Appl. Environ. Microbiol. 73: 4342–4350.CrossRefGoogle Scholar
  9. 9.
    Yoon, S. -H, S. -H. Lee, A. Das, H. -K. Ryu, H. -J. Jang, J. -Y. Kim, D. -K. Oh, J. D. Keasling, and S. -W. Kim (2009) Combinatorial expression of bacterial whole mevalonate pathway for the production of β-carotene in E. coli. J. Biotechnol. 140: 218–226.CrossRefGoogle Scholar
  10. 10.
    Kim, J. H., S. -W. Kim, D. Q. A. Nguyen, H. Li, S. B. Kim, Y. -G. Seo, J. -K. Yang, I. -Y. Chung, D. H. Kim, and C. -J. Kim (2009) Production of β-carotene by recombinant Escherichia coli with engineered whole mevalonate pathway in batch and fed-batch cultures. Biotechnol. Bioproc. Eng. 14: 559–564.CrossRefGoogle Scholar
  11. 11.
    Das, A., S. -H. Yoon, S. -H. Lee, J. -Y. Kim, D. -K. Oh, and S. -W. Kim (2007) An update on microbial carotenoid production: Application of recent metabolic engineering tools. Appl. Micro-biol. Biotechnol. 77: 505–512.CrossRefGoogle Scholar
  12. 12.
    Eiteman, M. A. and E. Altman (2006) Overcoming acetate in Escherichia coli recombinant protein fermentations. Trends Biotechnol. 24: 530–536.CrossRefGoogle Scholar
  13. 13.
    Selvarasu, S., D. S. -W. Ow, S. Y. Lee, M. M. Lee, S. K. -W. Oh, I. A. Karimi, and D. -Y. Lee (2009) Characterizing Escherichia coli DH5α growth and metabolism in a complex medium using genome-scale flux analysis. Biotechnol. Bioeng. 102: 923–934.CrossRefGoogle Scholar
  14. 14.
    Vemuri, G. N., T. A. Mining, E. Altman, and M. A. Eiteman (2005) Physiological response of central metabolism in Escherichia coli to deletion of pyruvate oxidase and introduction of heterologous pyruvate carboxylase. Biotechnol. Bioeng. 90: 64–76.CrossRefGoogle Scholar
  15. 15.
    Won, W., C. Park, C. Park, S. Y. Lee, K. S. Lee, and J. Lee (2011) Parameter estimation and dynamic control analysis of central carbon metabolism in Escherichia coli. Biotechnol. Bioproc. Eng. 16: 216–228.CrossRefGoogle Scholar
  16. 16.
    Martin, V. J. J., D. J. Pitera, S. T. Withers, J. D. Newman, and J. D. Keasling (2003) Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat. Biotechnol. 21: 796–802.CrossRefGoogle Scholar
  17. 17.
    Pitera, D. J., C. J. Paddon, J. D. Newman, and J. D. Keasling (2007) Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli. Metab. Eng. 9: 193–207.CrossRefGoogle Scholar
  18. 18.
    Sambrook, J. and D. W. Russel (2010) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.Google Scholar
  19. 19.
    Schierle, J., B. Pietsch, A. Ceresa, and C. Fizet (2004) Method for the determination of β-carotene in supplements and raw materials by reversed-phase liquid chromatography: Single laboratory validation. J. AOAC Int. 87: 1070–1082.Google Scholar
  20. 20.
    Torrecilla, J. S., M. Camara, V. Fernandez-Ruiz, G. Piera, and J. O. Caceres (2008) Solving the spectroscopy interference effects of β-carotene and lycopene by neural networks. J. Agric. Food. Chem. 56: 6261–6266.CrossRefGoogle Scholar
  21. 21.
    Sandmann, G., M. Albrecht, G. Schnurr, O. Knorzer, and P. Boger (1999) The biotechnological potential and design of novel carotenoids by gene combination in Escherichia coli. Trends Biotechnol. 17: 233–237.CrossRefGoogle Scholar
  22. 22.
    Vemuri, G. N., E. Altman, D. P. Sangurdekar, A. B. Khodursky, and M. A. Eiteman (2006) Overflow metabolism in Escherichia coli during steady-state growth: Transcriptional regulation and effect of redox ratio. Appl. Env. Microbiol. 72: 3653–3661.CrossRefGoogle Scholar
  23. 23.
    Mey, M. D., S. D. Maeseneire, W. Soetaert, and E. Vandamme (2007) Minimizing acetate formation in E. coli fermentations. J. Ind. Microbiol. Biotechnol. 34: 689–700.CrossRefGoogle Scholar
  24. 24.
    Roe, A. J., C. O’Byrns, D. Mclaggan, and I. R. Booth (2002) Inhibition of Escherichia coli growth by acetic acid: A problem with methionine biosynthesis and homocystein toxicity. Microbiol. 148: 2215–2222.Google Scholar
  25. 25.
    Warnecke, T. and R. T. Gill (2005) Organic acid toxicity, tolerance, and production in Escherichia coli biorefining applications. Microb. Cell Fact. 4: 25.CrossRefGoogle Scholar
  26. 26.
    Raja, N., M. Goodson, D. G. Smith, and R. J. Rowbury (1991) Decrease DNA damage by acid and increased repair of aciddamaged DNA in acid-habituated Escherichia coli. J. Appl. Bacteriol. 70: 507–511.CrossRefGoogle Scholar
  27. 27.
    Tabata, K. and S. -I. Hashimoto (2004) Production of mevalonate by a metabolically-engineered Escherichia coli. Biotechnol. Lett. 26: 1487–1491.CrossRefGoogle Scholar
  28. 28.
    Dahlgren, M. E., A. L. Powell, R. L. Greasham, and H. A. George (1993) Development of scale-down techniques for investigation of recombinant Escherichia coli fermentations: Acid metabolites in shake flasks and stirred bioreactors. Biotechnol. Prog. 9: 580–586.CrossRefGoogle Scholar
  29. 29.
    Soini, J., K. Ukkonen, and P. Neubauer (2008) High cell density media for Escherichia coli are generally designed for aerobic cultivations-consequences for large-scale bioprocesses and shake flask cultures. Microb. Cell Fact. 7: 26.CrossRefGoogle Scholar
  30. 30.
    Anguelova, T. and J. Warthesen (2000) Lycopene stability in tomato powders. J. Food. Sci. 65: 67–70.CrossRefGoogle Scholar
  31. 31.
    Stancik, L. M., D. M. Stancik, B. Schmidt, D. M. Barnhart, Y. N. Yoncheva, and J. L. Slonczewski (2002) pH-dependent expression of periplasmic proteins and amino acid catabolism in Escherichia coli. J. Bacteriol. 184: 4246–4258.CrossRefGoogle Scholar
  32. 32.
    Krulwich, T. A., G. Sachs, and E. Padan (2011) Molecular aspects of bacterial pH sensing and homeostasis. Nat. Rev. Micro. 9: 330–343.CrossRefGoogle Scholar
  33. 33.
    Zilberstein, D., V. Agmon, S. Schuldiner, and E. Padan (1984) Escherichia coli intracellular pH, membrane potential, and cell growth. J. Bacteriol. 158: 246–252.Google Scholar
  34. 34.
    Alper, H., K. Miyaoku, and G. Stephanopoulos (2006) Characterization of lycopene-overproducing E. coli strains in high cell density fermentations. Appl. Microbiol. Biotechnol. 72: 968–974.CrossRefGoogle Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Anh Do Quynh Nguyen
    • 1
  • Seon-Won Kim
    • 2
  • Sung Bae Kim
    • 1
  • Yang-Gon Seo
    • 1
  • In-Young Chung
    • 3
  • Dae Hwan Kim
    • 4
  • Chang-Joon Kim
    • 1
    Email author
  1. 1.Department of Chemical & Biological Engineering and ERIGyeongsang National UniversityJinjuKorea
  2. 2.Division of Applied Life Science (BK21), PMBBRCGyeongsang National UniversityJinjuKorea
  3. 3.Department of Electronic and Communications EngineeringKwangwoon UniversitySeoulKorea
  4. 4.School of Electrical EngineeringKookmin UniversitySeoulKorea

Personalised recommendations