Summary
We have developed a vector system for high-copy-number integration into the ribosomal DNA of the yeast Kluyveromyces lactis. This system is analogous to the pMIRY-system developed for Saccharomyces cerevisiae. Plasmids containing a portion of K. lactis rDNA for targeted homologous recombination, as well as the S. cerevisiae TRP1 gene with various promoter deletions, were constructed and, after transformation to K. lactis, analyzed for both copy number and stability. These plasmids were found to be present in about 60 copies per cell and were stably maintained during growth under non-selective conditions. Using this vector system, we expressed a fusion construct containing the S. cerevisiae GAL7 promoter, the SUC2 (invertase) signal sequence and the gene coding for α-galactosidase from the plant Cyamopsis tetragonoloba. Although the maximum copy number of these integrated plasmids was only about 15, we nevertheless obtained a high level of α-galactosidase production (250 mg/l) with a secretion efficiency of about 95%. When compared to extrachromosomal K. lactis vectors containing the same fusion construct, the multicopy integrants showed a much higher α-galactosidase production level and a considerably higher stability under non-selective conditions.
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References
Beggs JD (1978) Nature 275: 104–109
Bianchi MM, Falcone C, Chen XJ, Wéslowki-Louvel M, Frontali L, Fukuhara H (1987) Curr Genet 12: 185–192
Bianchi MM, Santarelli R, Frontali L (1991) Curr Genet 19: 155–161
Braus G, Paravicini G, Hütter R (1988) Mol Gen Genet 212: 495–504
Broach JR (1982) Cell 28: 203–204
Chen XJ, Saliola M, Falcone C, Bianchi MM, Fukuhara H (1986) Nucleic Acids Res 14: 4471–4481
Chen XJ, Bianchi MM, Suda K, Fukuhara H (1989) Curr Genet 16: 95–98
Das S, Hollenberg CP (1982) Curr Genet 6: 123–128
Erhart E, Hollenberg CP (1983) J Bacteriol 156: 83–89
Falcone C, Saliola M, Chen XJ, Frontali L, Fukuhara H (1986) Plasmid 15: 248–252
Fellinger AJ, Verbakel JMA, Veale RA, Sudbery PE, Bom IJ, Overbeeke N, Verrips CT (1991) Yeast 7: 463–473
Gietz RD, Sugino A (1988) Gene 74: 527–534
Hughes SG, Overbeeke N, Robinson S, Pollock K, Smeets FLM (1988) Plant Mol Biol 11: 783–789
Lopes TS, Klootwijk J, Veenstra AE, Van der Aar PC, Van Heerikhuizen H, Raué HA, Planta RJ (1989) Gene 79: 199–206
Lopes TS, Hakkaart GAJ, Koerts BL, Raué HA, Planta RJ (1991) Gene 105: 83–90
Maleszka R, Clark-Walker GD (1990) Mol Gen Genet 223: 342–344
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Meier H, Reid JSG (1982) Reserve polysaccharides other than starch in higher plants. In: Loewus FA, Tanner W (eds) Encyclopedia of plant physiology, New, Series 13A. Springer-Verlag, New York, pp 418–471
Nogi Y, Fukasawa T (1983) Nucleic Acids Res 11: 8555–8568
Overbeeke N, Fellinger AJ, Toonen MY, Van Wassenaar PD, Verrips CT (1989) Plant Mol Biol 13: 541–550
Overbeeke N, Termorshuizen GHM, Giuseppin MLF, Underwood DR, Verrips CT (1990) Appl Environ Microbiol 56: 1429–1434
Pedersen MB (1983) Carlsberg Res Comm 48: 485–503
Petes TD (1979) Proc Natl Acad Sci USA 76: 410–414
Sierkstra LN, Verbakel JMA, Verrips CT (1991) Curr Genet 19: 81–87
Southern EM (1975) J Mol Biol 98: 503–517
Stark MJR, Milner JS (1989) Yeast 5: 35–50
Taussig R, Carlsson M (1983) Nucleic Acids Res 11: 1943–1954
Van den Berg JA, Van der Laken KJ, Van Ooyen AJJ, Renniers TCHM, Rietveld K, Schaap A, Brake AJ, Bishop RJ, Schultz K, Moyer D, Richman M, Shuster JR (1990) Bio/ Technology 8: 135–139
Verbakel JMA (1991) Heterologous gene expression in the yeast Saccharomyces cerevisiae. PhD thesis, Rijksuniversiteit Utrecht, The Netherlands
Verbeet MPh, Van Heerikhuizen H, Klootwijk J, Fontijn R, Planta RJ (1984) Mol Gen Genet 195: 116–125
Yanisch-Perron C, Vieira J, Messing J (1985) Gene, 33: 103–119
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Communicated by L.A. Grivell
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Bergkamp, R.J.M., Kool, I.M., Geerse, R.H. et al. Multiple-copy integration of the α-galactosidase gene from Cyamopsis tetragonoloba into the ribosomal DNA of Kluyveromyces lactis . Curr Genet 21, 365–370 (1992). https://doi.org/10.1007/BF00351696
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DOI: https://doi.org/10.1007/BF00351696