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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
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.
Chang, M. C. Y., and Keasling, J. D. (2006) Production of isoprenoid pharmaceuticals by engineered microbes. Nat. Chem. Biol. 2, 674–681.
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.
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.
Rao, K. V. (1995) A new large-scale process for taxol and related taxanes from Taxus brevifolia. Pharm. Res. 10, 521–524.
Roth, R. J., and Acton, N. (1989) A simple conversion of artemisinic acid into artemisinin. J. Nat. Prod. 52, 1183–1185.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Metzger, D., and Feil, R. (1990) Engineering the mouse genome by site-specific recombination. Curr. Opin. Biotechnol. 10, 470–476.
Nash, H. A. (1975) Integrative recombination of bacteriophage Lambda DAN in vitro. Proc. Nat. Acad. Sci. U.S.A. 72, 1072–1076.
Li, M. Z., and Elledge, S. J. (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nature Methods 4, 251–256.
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).
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).
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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Dahm, P., Jennewein, S. (2010). Introduction of the Early Pathway to Taxol Biosynthesis in Yeast by Means of Biosynthetic Gene Cluster Construction Using SOE-PCR and Homologous Recombination. In: Fett-Neto, A. (eds) Plant Secondary Metabolism Engineering. Methods in Molecular Biology, vol 643. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-723-5_11
Download citation
DOI: https://doi.org/10.1007/978-1-60761-723-5_11
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-722-8
Online ISBN: 978-1-60761-723-5
eBook Packages: Springer Protocols