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Comparison of promoters suitable for regulated overexpression of β-galactosidase in the alkane-utilizing yeastYarrowia lipolytica

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Abstract

Promoters of the genesG3P, ICL1, POT1, POX1, POX2 andPOX5 of the yeastY. lipolytica were studied in respect to their regulations and activities during growth on different carbon sources. The aim of this study was to select suitable promoters for high expression of heterologous genes in this yeast. For this purpose the promoters were fused with the reporter genelacZ ofE. coli and integrated as single copies into the genome ofY. lipolytica strain PO1d. The measurement of expressed activities of β-galactosidase revealed thatpICL1, pPOX2 andpPOT1 are the strongest regulable promoters available forY. lipolytica, at present.pPOX2 andpPOT4 were highly induced during growth on oleic acid and were completely repressed by glucose and glycerol.pICL1 was strongly inducible by ethanol besides alkanes and fatty acids, however, not completely repressible by glucose or glycerol. Ricinoleic acid methyl ester appeared as a very strong inducer forpPOT1 andpPOX2, in spite of that it inhibited growth ofY. lipolytica transformants.

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References

  1. Barth, G. and C. Gaillardin (1996)Yarrowia lipolytica. pp. 313–388. In: K. Wolf (ed.):Nonconventional yeasts in biotechnology. A Handbook. Springer-Verlag, Berlin, Germany.

    Google Scholar 

  2. Barth, G. and C. Gaillardin (1997) Physiology and genetics of the dimorphic fungusYarrowia lipolytics.FEMS Micobiol. Rev. 19: 219–237.

    Article  CAS  Google Scholar 

  3. Gaillardin, C. and H. Heslot (1988) Genetic engineering inYarrowia lipolytica.J. Basic Microbiol. 28: 161–174.

    Article  CAS  Google Scholar 

  4. Romanos, M., C. Scorer, and J. Clare (1992) Foreign gene expression in yeast.Yeast 8: 423–488.

    Article  CAS  Google Scholar 

  5. Sudbery, P. (1994) Non-Saccharomyces yeasts.Yeast 10: 1707–1726.

    Article  CAS  Google Scholar 

  6. Müller, S., T. Sandal, P. Kamp-Hansen, and H. Dalboge (1998) Comparison of expression systems in the yeastsSaccharomyces cerevisiae, Hansenula polymorpha, Klyuveromyces lactis, Schizosaccharomyces pombe andYarrowia lipolyca.Yeast 14: 1267–1283.

    Article  Google Scholar 

  7. Fournier, P., L. Guyaneux, M. Chasles, and C. Gaillardin (1991) Scarcity of ars sequences isolated in a morphogenesis mutant of the yeastYarrowia lipolytica.Yeast 7: 25–36.

    Article  CAS  Google Scholar 

  8. Fournier, P., A. Abbas, M. Chasles, B. Kudla, D. M. Ogrydziak, D. Yaver, J.-W. Xuan, A. Peito, A.-M. Ribet, C. Feynerol, F. He, and C. Caillardin (1993) Colocalization of centromeric and replicative functions on autonomously replicating sequences isolated from the yeastYarrowia lipolytica.Proc. Natl. Acad. Sci. USA 90: 4912–4916.

    Article  CAS  Google Scholar 

  9. Matsuoka, M., M. Matsubara, H. Daidoh, T. Imanaka, K. Uchida, and S. Aiba (1993) Analysis of regions essential for the function of chromosomal replicator sequences fromYarrowia lipolytica.Mol. Gen. Genet. 237: 327–333.

    CAS  Google Scholar 

  10. Vernis, L., A. Abbas, M. Chasles, G. M. Gaillardin, C. Brun, J. A. Huberman, and P. Fournier (1997) An origin of replication and a centromere are both needed to establish a replicative plasmid in the yeastYarrowia lipolytica.Mol. Cell. Biol. 17: 1995–2004.

    CAS  Google Scholar 

  11. Vernis, L., M. Chasles, P. Pasero, A. Lepingle, C. Caillardin, and F. Fournier (1999) Short DNA fragments without sequence similarity are initiation sites for replication in the chromosome of the yeastYarrowia lipolytica.Mol. Cell. Biol. 10: 757–769.

    CAS  Google Scholar 

  12. Le Dall, M.-T., J.-M. Nicaud, and C. Gaillardin (1994) Multi-copy integration in the yeastYarrowia lipolytica.Curr. Genet. 26: 38–44.

    Article  Google Scholar 

  13. Juretzek, T. M. T. Le Dall, S. Mauersberger, C. Gaillardin, G. Barth, and J. M. Nicaud (2000) Vectors for gene expression and amplification in the yeastYarrowia lipolytica. Yeast in press.

  14. Matoba, S., J. Fukuyama, R. A. Wing, and D. M. Ogrydziak (1988) Intracellular precursors and secrition of alkaline extracellular protease ofYarrowia lipolytica.Mol. Cell. Biol. 8: 4904–4916.

    CAS  Google Scholar 

  15. Fabre, E., J. M. Nicaud, M. C. Lopez, and C. Caillardin (1991) Role of the proregion in the production and secretion of theYarrowia lipolytica alkaline extracellular protease.J. Biol. Chem. 266: 3782–3790.

    CAS  Google Scholar 

  16. Matoba, S., K. A. Morano, D. J. Klionsky, K. Kim, and D. M. Ogrydziak (1997) Dipeptidyl aminopeptidase processing and biosynthesis of extracellular protease fromYarrowia lipolytica.Microbiology 143: 3263–3272.

    Article  CAS  Google Scholar 

  17. Blanchin-Roland, S., R. Cordero Otero, and C. Gaillardin (1994) Two upstream UAS control expression of theXPR2 gene encoding an extracellular alkaline protease in the yeastYarrowia lipolytica.Mol. Cell. Biol. 14: 327–338.

    CAS  Google Scholar 

  18. Park, G. S., C. C. Chang, J. Y. Kim, D. M. Ogrydziak, and D. D. Ryu (1997) Expression, secretion, and processing of rice a-amylase in the yeastYarrowia lipolytica.J. Biol. Chem. 272: 6876–6881.

    Article  CAS  Google Scholar 

  19. Chang, C. C., D. D. Ryu, C. S. Park, and J. Y. Kim (1998) Improvement of heterologous protein productivity using recombinantYarrowia lipolytica and cyclic fed-batch process strategy.Biotechnol. Bioeng. 59: 379–385.

    Article  CAS  Google Scholar 

  20. Madzak, C., S. Blanchin-Roland, R. R. Cordero-Otero, and C. Gaillardin (1999) Functional analysis of upstream regulating regions from theYarrowia lipolytica XPR2 promoter.Microbiology 145: 75–87.

    CAS  Google Scholar 

  21. Barth, G. and T. Scheuber (1993) Cloning of the isocitrate lyase gene (ICL1) fromYarrowia lipolytica and characterization of the deduced protein.Mol. Gen. Genet. 241: 422–430.

    Article  CAS  Google Scholar 

  22. Berninger, G., R. Schmidtchen, G. Casel, A. Knoerr, K. Rautenstrauss, W.-H. Kunau, and E. Schweizer (1993) Structure and metabolic control of theYarrowia lipolytica peroxisomal 3-oxoacyl-CoA-thiolase gene.Eur. J. Biochem. 216: 607–613.

    Article  CAS  Google Scholar 

  23. Wang, H., A. Le Clainche, M. T. Le Dall, Y. Wache, Y. Pagot, J. M. Belin, C. Gaillardin, and J. M. Nicaud (1998) Cloning and characterization of the peroxisomal acyl CoA oxidaseACO3 gene from the alkane-utilizing yeastYarrowia lipolytica.Yeast 14: 1373–1386.

    Article  CAS  Google Scholar 

  24. Wang, H. J., M. T. Le Dall, Y. Wach, C. Laroche, J. M. Belin, C. Gaillardin, and J. M. Nicaud (1999) Evaluation of acyl coenzyme A oxidase (Aox) isozyme function in the n-alkane-assimilating yeastYarrowia lipolytica.J. Bacteriol. 181: 5140–5148.

    CAS  Google Scholar 

  25. Sambrook, J., E. E. Fritsch, and T. Maniatis (1989)Molecular cloning. A laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.

    Google Scholar 

  26. Mauersberger, S., M. Ohkuma, W-H. Schunck, and M. Takagi (1996)Candida maltosa. pp 411–580. In: K. Wolf (ed.);Nonconventional yeasts in biotechnology. A Hand-book. Springer-Verlag, Berlin, Germany.

    Google Scholar 

  27. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. C. Seidman, J. A. Smith, and K. Struhl (1993)Current Protocols in Molecular Biology, Vol. 1, Harvard Medical School, Massachusetts, USA.

    Google Scholar 

  28. Juretzek, T. (1999)Entwicklung von Wirts-Vektor-Systemen zur heterologen Expression von Proteinen in der micinkonventionellen Hefe Yarrowia lipolyticaund ihre Anwendung für die Cytochrom P450-katalysierte Stoffumwandlung, PhD thesis. Technische Universität Dresden, Germany.

    Google Scholar 

  29. Schmidtchen, R. (1994)Untersuchungen zur Regulation des 3-Oxcacyl-CoA-Thio-lacs-1-Gens aus Yarrowia lipoly-tica, PhD thesis, Friedrich-Alexander-Universität, Erlangen, Germany.

    Google Scholar 

  30. Madzak, C., B. Tréton, and S. Blanchin-Roland (2000) Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression, of heterologous proteins in the yeastYarrowia lipolytic.J. Mol. Microbiol. Biotechnol. 2: 207–216.

    CAS  Google Scholar 

  31. Sanger, F., S. Nicklen, and A. R. Coulson (1977) DNA sequencing with chain-terminating inhibitors.Proc. Natl. Acad. Sci. USA 74: 5463–5467.

    Article  CAS  Google Scholar 

  32. Gaillardin, C. and A.-M. Ribet (1987)LEU2 directed expression of β-galactosidase activity and phleomycin resistance inYarrowia lipolytica.Curr. Genet. 11: 369–375.

    Article  CAS  Google Scholar 

  33. Pagot, Y., A. Endrizzi, J. M. Nicaud, and J. M. Belin (1997) Utilization of an auxotrophic strain of the yeastYarrowia lipolytica to improve gamma-decalactone production yields.Lett. Appl. Microbiol. 25: 113–116.

    Article  CAS  Google Scholar 

  34. Gerber, J. (1999)Untersuchungen zur Optimierung des Elektromentransportsystems für die Cytochrom P450 katalysierte Biotransformation von Steroiden in Yarrowia lipolytica. Diploma thesis, Technische Universität Dresden, Germany.

    Google Scholar 

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Juretzek, T., Wang, HJ., Nicaud, JM. et al. Comparison of promoters suitable for regulated overexpression of β-galactosidase in the alkane-utilizing yeastYarrowia lipolytica . Biotechnol. Bioprocess Eng. 5, 320–326 (2000). https://doi.org/10.1007/BF02942206

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