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Introduction of the Early Pathway to Taxol Biosynthesis in Yeast by Means of Biosynthetic Gene Cluster Construction Using SOE-PCR and Homologous Recombination

  • Pia Dahm
  • Stefan Jennewein
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 643)

Abstract

Metabolic engineering of plant natural product pathways in heterologous systems requires the highly concerted action of several biosynthetic genes. Besides the functional heterologous expression of the genes encoding the natural product biosynthetic pathway, often additional extensive modifications in the host primary metabolism are also needed, in order to obtain efficient supply of the required biosynthetic building blocks to support the engineered natural product biosynthesis. Selection markers in heterologous expression systems, like baker’s yeast (Saccharomyces cerevisiae), are often limited and the chromosomal insertion prevents later modifications of engineered pathway, e.g. exchange of gene promoters, or the introduction of additional genetic regulatory elements in a timely manner. Thus the construction of biosynthetic gene clusters on episomal expression vectors seems a logical solution for this dilemma. Although manipulation of long DNA fragments still represents a challenge, by using PCR and in vitro homologous recombination, we assembled a biosynthetic gene cluster for the concerted heterologous expression of three important genes for the metabolic engineering of taxoid biosynthesis in yeast.

Key words

Biosynthetic gene clusters assembling of DNA fragments engineering of taxoid biosynthesis in yeast 

References

  1. 1.
    Wink, M. (1999) Plant secondary metabolites: biochemistry, function and biotechnology, in Biochemistry of Plant Secondary Metabolism, Wink, M., ed., CRC Press, Boca Raton, pp. 1–16.Google Scholar
  2. 2.
    Chang, M. C. Y., and Keasling, J. D. (2006) Production of isoprenoid pharmaceuticals by engineered microbes. Nat. Chem. Biol. 2, 674–681.PubMedCrossRefGoogle Scholar
  3. 3.
    Ketchum, R. E. B., Gibson, D. M., Croteau, R. B., and Shuler, M. L. (1999) The kinetics of taxoid accumulation in cell suspension cultures of Taxus following elicitation with methyl jasmonate. Biotechnol. Bioeng. 62, 97–105.PubMedCrossRefGoogle Scholar
  4. 4.
    Kumar, S., and Srivastava, S. (2005) Establishment of artemisinin combination therapy as first line treatment for combating malaria: Artemisia annua cultivation in India needed for providing sustainable supply chain of artemisinin Curr. Sci. India 89, 1097–1102.Google Scholar
  5. 5.
    Rao, K. V. (1995) A new large-scale process for taxol and related taxanes from Taxus brevifolia. Pharm. Res. 10, 521–524.CrossRefGoogle Scholar
  6. 6.
    Roth, R. J., and Acton, N. (1989) A simple conversion of artemisinic acid into artemisinin. J. Nat. Prod. 52, 1183–1185.PubMedCrossRefGoogle Scholar
  7. 7.
    Dejong, J. M., Liu, Y., Bollon, A. P., Long, R. M., Jennewein, S., Williams, D., and Croteau, R. B. (2005) Genetic engineering of Taxol biosynthetic genes in Saccharomyces cerevsiae. Biotechnol. Bioeng. 93, 212–224.CrossRefGoogle Scholar
  8. 8.
    Engels, B., Dahm, P., and Jennewein, S. (2008) Metabolic engineering of taxadiene biosynthesis in yeast as a first step towards Taxol production. Metab. Eng. 10, 201–206.PubMedCrossRefGoogle Scholar
  9. 9.
    Mertz, J. E., and Davis, R. W. (1972) Cleavage of DNA by R1 restriction endonuclease generates cohesive ends. P. Nat. Acad. Sci. U.S.A. 69, 3370–3374.CrossRefGoogle Scholar
  10. 10.
    Horton, R. M., Hunt, M. D., Ho, S. N., Pullen, J. K., and Pease, L. R. (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61–68.PubMedCrossRefGoogle Scholar
  11. 11.
    Chen, G. Q., Choi, I., Ramachandran, B., and Gouaux, E. (1994) Total gene synthesis: novel single-step and convergent strategies applied to the construction of a 779 base pair bacteriorhodopsin gene. J. Am. Chem. Soc. 116, 8799–8800.CrossRefGoogle Scholar
  12. 12.
    Stemmer, W. P. C., Crameri, A., Ha, K. D., Brennan, T. M., and Heyneker, H. L. (1995) Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164, 49–53.PubMedCrossRefGoogle Scholar
  13. 13.
    Itaya, M., Fujita, K., Kuroki, A., and Tsuge, K. (2008) Bottom-up genome assembly using the Bacillus subtilis genome vector. Nature Methods 5, 41–43.PubMedCrossRefGoogle Scholar
  14. 14.
    Gibson, D. G., Benders, G. A., Andrews-Pfannkoch, C., Denisova, E. A., Baden-Tillson, H., Zaveri, J., Stcokwell, T. B., Brownley, A., Thomas, D. W., Algire, M. A., Merryman, C., Young, L., Noskov, V. N., Glass, J. I., Venter, C., Hutchison, C. A., and Smith, H. O. (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319, 1215–1220.PubMedCrossRefGoogle Scholar
  15. 15.
    Shao, Z., Zhao, H., and Zhao, H. (2008) DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res. 37, 1–10.Google Scholar
  16. 16.
    Metzger, D., and Feil, R. (1990) Engineering the mouse genome by site-specific recombination. Curr. Opin. Biotechnol. 10, 470–476.CrossRefGoogle Scholar
  17. 17.
    Nash, H. A. (1975) Integrative recombination of bacteriophage Lambda DAN in vitro. Proc. Nat. Acad. Sci. U.S.A. 72, 1072–1076.CrossRefGoogle Scholar
  18. 18.
    Li, M. Z., and Elledge, S. J. (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nature Methods 4, 251–256.PubMedCrossRefGoogle Scholar
  19. 19.
    Bunny, K. (2005) In-Fusion™ PCR universal cloning kits. Clontechniques 1–3. (http://www.clontech.com/images/ctq/OCT05UPD/CR682071_US_InFusion.pdf) (access in August 31st, 2009).
  20. 20.
    Gilfoyle, A. P., and Mabbutt, B. C. (2007) Evaluation of the In-Fusion™ Cloning system for structural genomics. Clontechniques 20–21. (http://clontech.takara-bio.co.jp/archive/200711/pdf/200711_20.pdf) (access in August 31st, 2009).
  21. 21.
    Bitinaite, J., Rubino, M., Varma, K. H., Schildkraut, I., Vaislavila, R., and Vaiskunaite, R. (2007) USER™ friendly DNA engineering and cloning method by uracil excision. Nucleic Acids Res. 35, 1992–2002.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Pia Dahm
    • 1
  • Stefan Jennewein
    • 1
    • 2
  1. 1.Institute of Organic Chemistry and BiochemistryTechnical University of DarmstadtDarmstadtGermany
  2. 2.Fraunhofer Institute for Molecular Biology and Applied EcologyAachenGermany

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