Advertisement

High Isoprenoid Flux Escherichia coli as a Host for Carotenoids Production

  • Wonchul SuhEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 834)

Abstract

A noncarotenogenic microbe E. coli was engineered for high production of carotenoids. To increase the isoprenoid flux, the chromosomal native promoters of the rate-controlling steps (dxs, idi and ispDispF) in the isoprenoid pathway were replaced with a strong bacteriophage T5 promoter (PT5) by using the λ-Red recombinase system in combination with the Flp/FRT site-specific recombination system for marker excision and P1 transduction for gene trait stacking. The resulting high isoprenoid flux E. coli can be used as a starting strain to produce various carotenoids by introducing heterologous carotenoid genes. In this study, the high isoprenoid flux E. coli was transformed with a plasmid carrying the β-carotene biosynthetic genes from Pantoea stewartii for β-carotene production.

Key words

High isoprenoid flux Escherichia coli Chromosomal promoter replacement Metabolic engineering Isoprenoid pathway Carotenoids β-carotene 

Notes

Acknowledgments

The author thanks Professor Barry L. Wanner for providing λ-Red strains and plasmids used in this work and Wendy Suh for critical reading of the manuscript.

References

  1. 1.
    Sandmann, G., Albrecht, M., Schnurr, G., Knörzer, P., and Böger, P. (1999) The biotechnological potential and design of novel carotenoids by gene combination in Escherichia coli. Trends Biotechnol. 17, 233–237.PubMedCrossRefGoogle Scholar
  2. 2.
    Schmidt-Dannert, C., Lee, P. C., and Mijts, B. N. (2006) Creating carotenoid diversity in E. coli cells using combinatorial and directed evolution strategies. Phytochemistry Reviews 5, 67–74.CrossRefGoogle Scholar
  3. 3.
    Rohdich, F., Zepeck, F., Adam, P., Hecht, S., Kaiser, J., Laupitz, R., Gräwert, T., Amslinger, S., Eisenreich, W., Bacher, A., and Arigoni, D. (2003) The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: Studies on the mechanisms of the reactions catalyzed by IspG and IspH protein. Proc. Natl. Acad. Sci. USA. 100, 1586–1591.PubMedCrossRefGoogle Scholar
  4. 4.
    Ruther, A., Misawa, N., Boger, P., and Sandmann, G. (1997) Production of zeaxanthin in Escherichia coli transformation with different carotenogenic plasmids. Appl. Microbiol. Biotechnol. 48, 162–167.PubMedCrossRefGoogle Scholar
  5. 5.
    Matthews, P. D. and Wurtzel, E. T. (2000) Metabolic engineering of carotenoid accumulation in Escherichia coli by modulation of the isoprenoid precursor pool with expression of deoxyxylulose phosphate synthase. Appl. Microbiol. Biotechnol. 53, 396–400.PubMedCrossRefGoogle Scholar
  6. 6.
    Albrecht, M., Misawa, N., and Sandmann, G. (1999) Metabolic engineering of the terpenoid biosynthetic pathway of Escherichia coli for production of the carotenoids β-carotene and zeaxanthin. Biotechnol. Letters 21, 791–795.CrossRefGoogle Scholar
  7. 7.
    Noack, D., Roth, M., Geuther, R., Müller, G., Undisz, K., Hoffmeier., C., and Gáspár, S. (1981) Maintenance and genetic stability of vector plasmids pBR322 and pBR325 in Escherichia coli K12 strains grown in a chemostat. Mol. Gen. Genet. 184, 121–124.PubMedCrossRefGoogle Scholar
  8. 8.
    Bentley, W. E., Mirjalili, N., Andersen, D. C., Davis, R. H., and Kompala, D. S. (1990) Plasmid-encoded protein: the principal factor in the “metabolic burden” associated with recombinant bacteria. Biotechnol Bioeng. 35, 668–681.PubMedCrossRefGoogle Scholar
  9. 9.
    Yuan, L. Z., Rouvière, P. E., LaRossa, R. A., and Suh, W. (2006) Chromosomal promoter replacement of the isoprenoid pathway for enhancing carotenoid production in E. coli. Metab. Eng. 8, 79–90.PubMedCrossRefGoogle Scholar
  10. 10.
    Datsenko, K. A. and Wanner, B. L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA. 97, 6640–6645.PubMedCrossRefGoogle Scholar
  11. 11.
    Casadaban, M. J. and Cohen, S. N. (1980) Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J. Mol. Biol. 138, 179–207.PubMedCrossRefGoogle Scholar
  12. 12.
    Bachmann, B. J. (1996) Derivations and genotypes of some mutant derivatives of Escherichia coli K-12. InEscherichia coli and Salmonella typhimurium: Cellular and Molecular Biology” (F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger, Eds.), 2nd ed., pp. 2460–2488, ASM Press, Washington, DC.Google Scholar
  13. 13.
    Miller, J. H. (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  14. 14.
    Abe, T., Abboud, J.-L. M., Belio, F., Bosch, E., Garcia, J. I., Mayoral, J. A., Notario, R., Ortega, J., Roses, M. (1998) Empirical treatment of solvent-solute interactions: Medium effects on the electronic absorption spectrum of β-carotene. J. Phy. Org. Chem. 11, 193–200.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.DuPont Central Research and DevelopmentWilmingtonUSA

Personalised recommendations