Abstract
Multiple gene expression can be introduced in a yeast strain with using only two markers by means of the two new vectors described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3. The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette consisting of the strong PGK1 promoter and the GCY1 terminator. The gene of interest (YFG1) is cloned between the promoter and terminator of pB3 PGK. The pB3 PGK-YFG1 is integrated into the genome by a single restriction cut within the YFG1 gene and integrated in the YFG1 locus. The strain is further transformed with the pCRE3 vector. The CRE recombinase expressed from this vector removes the zeocin marker and makes it possible to use the pB3 PGK vector over again in the same strain after curing of the pCRE3 vector. The 2µ-based pCRE3 carries the aureobasidin A, zeocin and URA3 markers. pCRE3 is easily cured by growth in nonselective medium without active counterselection. The screening for loss of the chromosomal zeocin marker, as well as curing of the pCRE3 vector, is done in one step, by scoring zeocin sensitivity. This can be done because the zeocin marker is present in both the pB3 PGK and pCRE3. The S. cerevisiae pentose phosphate pathway genes RK11, RPE1, TAL1, and TKL1 were cloned in pB3 PGK and integrated in the locus of the respective gene, resulting in simultaneous overexpression of the genes in the xylose-fermenting S. cerevisiae strain TMB3001.
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
Oliver, S. G. (1996) From DNA sequence to biological function. Nature 379, 597–600.
Mewes, H. W., Albermann, K., Bahr, M., Frishman, D., Gleissner, A., Hani, J., et al. (1997) Overview of the yeast genome. Nature 387, 7–65.
Wolfe, K. H., and Shields, D. C. (1997) Molecular evidence for an ancient duplication of the entire yeast genome. Nature 387, 708–713.
Wieczorke, R., Krampe, S., Weierstall, T., Freidel, K., Hollenberg, C. P., and Boles, E. (1999) Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. FEBS Lett. 464, 123–128.
Bailey, J. E. (1991) Toward a science of metabolic engineering. Science 252, 1668–1675.
Kacser, H., and Burns, J. A. (1973) The control of flux. Symp. Soc. Exp. Biol. 27, 65–104.
Heinrich, R., and Rapoport, T. A. (1974) Alinear steady-state treatment of enzymatic chains: General properties, control and effect-or-strength. Eur. J. Biochem. 42, 89–95.
Schaaff, I., Heinisch, J., and Zimmermann, F. K. (1989) Overproduction of glycolytic enzymes in yeast. Yeast 5, 285–290.
Niederberger, P., Prasad, R., Miozzari, G., and Kacser, H. (1992) Astrategy for increasing an in-vivo flux by genetic manipulations. The tryptophan system of yeast. Biochem. J. 287, 473–479.
Hauf, J., Zimmermann, F. K., and Müller, S. (2000) Simultaneous genomic overexpression of seven glycolytic enzymes in the yeast Saccharomyces cerevisiae. Enzyme Microb. Technol. 26, 688–698.
Smits, H. P., Hauf, J., Müller, S., Hobley, T. J., Zimmermann, F. K., Hahn-Hägerdal, B., et al. (2000) Simultaneous overexpression of enzymes of the lower part of glycolysis can enhance the fermentative capacity of Saccharomyces cerevisiae. Yeast 16, 1325–1334.
Johansson, B., and Hahn-Hägerdal, B. (2002) The non-oxidative pentose phosphate pathway controls the fermentation rate of xylulose but not of xylose in Saccharomyces cerevisiae TMB3001. FEMS Yeast Research 2, 277–282.
Kacser, H., and Acerenza, L. (1993) A universal method for achieving increases in metabolite production. Eur. J. Biochem. 216, 361–367.
Fell, D. A., and Thomas, S. (1995) Physiological control of metabolic flux: the requirement for multi-site modulation. Biochem. J. 311, 35–39.
Johansson, B., and Hahn-Hägerdal, B. (2002) Overproduction of pentose phosphate pathway enzymes using a new CRE-loxP expression vector for repeated genomic integration in Saccharomyces cerevisiae. Yeast 19, 225–231.
Mellor, J., Dobson, M. J., Roberts, N. A., Tuite, M. F., Emtage, J. S., White, S., et al. (1983) Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae. Gene 24, 1–14.
Hermann, H., Hacker, U., Bandlow, W., and Magdolen, V. (1992) pYLZ vectors: Saccharomyces cerevisiae/Escherichia coli shuttle plasmids to analyze yeast promoters. Gene 119, 137–141.
Nieto, A., Prieto, J. A., and Sanz, P. (1999) Stable high-copy-number integration of Aspergillus oryzae alpha-AMYLASE cDNA in an industrial baker’s yeast strain. Biotechnol. Prog. 15, 459–466.
Mumberg, D., Müller, R., and Funk, M. (1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156, 119–122.
Labbé, S., and Thiele, D. J. (1999) Copper ion inducible and repressible promoter systems in yeast. Methods Enzymol. 306, 145–153.
Güldener, U., Heck, S., Fielder, T., Beinhauer, J., and Hegemann, J. H. (1996) Anew efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 24, 2519–2524.
Gietz, R. D. and Woods, R. A. (1994) High efficiency transformation in yeast, in Molecular Genetics of Yeast: Practical Approaches (Johnston, J. A., ed.), Oxford University Press, Oxford, UK, pp. 121–134.
Johansson, B. (2001) Metabolic engineering of the pentose phosphate pathway of xylose fermenting Saccharomyces cerevisiae, Lund University, Lund, Sweden.
Zaldivar, J., Borges, A., Johansson, B., Smits, H. P., Villas-Boas, S. G., Nielsen, J., et al. (2002) Fermentation performance and intracellular metabolite patterns in laboratory and industrial xylose-fermenting Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 59, 436–442.
Jeppsson, M., Johansson, B., Hahn-Hägerdal, B., and Gorwa-Grauslund, M. F. (2002) Reduced oxidative pentose phosphate pathway flux in recombinant xylose-utilizing Saccaromyces cerevisiae strains improves the ethanol yield from xylose. Appl. Environ. Microbiol. 68, 1604–1609.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Johansson, B., Hahn-Hägerdal, B. (2004). Multiple Gene Expression by Chromosomal Integration and CRE-loxP-Mediated Marker Recycling in Saccharomyces cerevisiae . In: Balbás, P., Lorence, A. (eds) Recombinant Gene Expression. Methods in Molecular Biology, vol 267. Humana Press. https://doi.org/10.1385/1-59259-774-2:287
Download citation
DOI: https://doi.org/10.1385/1-59259-774-2:287
Publisher Name: Humana Press
Print ISBN: 978-1-58829-262-9
Online ISBN: 978-1-59259-774-1
eBook Packages: Springer Protocols