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Applied Microbiology and Biotechnology

, Volume 63, Issue 2, pp 136–142 | Cite as

Overproduction of lipase by Yarrowia lipolytica mutants

  • P. Fickers
  • J. M. NicaudEmail author
  • J. Destain
  • P. Thonart
Original Paper

Abstract

Non-genetically modified mutants with increased capacities of extracellular lipase production were obtained from Yarrowia lipolytica strain CBS6303 by chemical mutagenesis. Of the 400 mutants isolated, LgX64.81 had the highest potential for the development of an industrial lipase production process. This mutant exhibits lipase production uncoupled from catabolite repression by glucose, and a 10-fold increased productivity upon addition of oleic acid. Using a LIP2-LacZ reporter gene, we demonstrate that the mutant phenotype originates from a trans-acting mutation. The glucose uptake capacity of LgX64.81 is reduced 2.5-fold compared to the wild-type-strain, and it exhibits high lipase production on glucose medium. A trans-acting mutation in a gene involved in glucose transport could thus explain this mutant phenotype.

Keywords

Lipase Oleic Acid Lipase Production Tributyrin Extracellular Lipase 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

We thank Dr. Ongena for careful reading of the manuscript. This work was supported by the Institut National de la Recherche Scientifique and by the Centre National de la Recherche Scientifique. P. Fickers is recipient of a fellowship from the Fond pour la Formation à la Recherche dans l'Industrie et l'Agriculture.

References

  1. Barth G, Gaillardin C (1997) Physiology and genetics of the dimorphic fungus Yarrowia lipolytica. FEMS Microbiol Rev 19:219–237CrossRefPubMedGoogle Scholar
  2. Choupina A, Gonzalez F, Morin M, Burguillo F, Ferminan E, Dominguez A (1999) The lipase system of Yarrowia lipolytica. Curr Genet 35:297Google Scholar
  3. Corzo G, Revah S (1999) Production and characteristics of lipase from Yarrowia lipolytica 681. Bioresour Technol 70:173–180CrossRefGoogle Scholar
  4. Dear S, Staden R (1991) A sequence assembly and editing program for efficient management of large sequence projects. Nucleic Acids Res 19:3907–3911PubMedGoogle Scholar
  5. De Felice B, Pontecorvo G, Carfagna M (1997) Degradation of wastewaters from olive oil mills by Yarrowia lipolytica ATCC 20255 and Pseudomonas putida. Acta Biotechnol 3:231–239Google Scholar
  6. Destain J (1998) Production, purification et caractérisation de la lipase de Yarrowia lipolytica. PhD thesis, Faculté Universitaire des Sciences Agronomiques de Gembloux, Gembloux, BelgiumGoogle Scholar
  7. Destain J, Roblain D, Thonart P (1997) Improvement of lipase production from Yarrowia lipolytica. Biotechnol Lett 19:105–107CrossRefGoogle Scholar
  8. Frings C, Dunn R (1970) A colorimetric method for determination of total serum lipids based on the sulfo-phospho-vanillin reaction. Am J Clin Pathol 53:89–91PubMedGoogle Scholar
  9. Gancedo JM (1998) Yeast carbon catabolite repression. Microbiol Mol Biol Rev 62:334–361PubMedGoogle Scholar
  10. Jaeger K, Ransac S, Dijkstra B, Colson C, van Heuvel M, Misset O (1994) Bacterial lipases. FEMS Microbiol Rev 15:29–63PubMedGoogle Scholar
  11. Juretzek T, Wang H, Nicaud JM, Mauersberger S, Barth G (2000) Comparison of promoter suitable for regulated overexpression of β-galactosidase in the alkane-utilizing yeast Yarrowia lipolytica. Biotechnol Bioprocess Eng 5:320–326Google Scholar
  12. Juretzek T, Le Dall M, Mauersberger S, Gaillardin C, Barth G, Nicaud J (2001) Vectors for gene expression and amplification in the yeast Yarrowia lipolytica. Yeast 18:97–113CrossRefPubMedGoogle Scholar
  13. Kotting J, Eibl H (1994) Lipases and phospholipases in organic synthesis. In: Wooley P, Petersen S (eds) Lipase. Cambridge University Press, pp 289–313Google Scholar
  14. Le Dall MT, Nicaud JM, Gaillardin C (1994) Multiple-copy integration in the yeast Yarrowia lipolytica. Curr Genet 26:38–44PubMedGoogle Scholar
  15. Lie E, Persson A, Molin G (1991) Screening for lipase-producing microorganisms with a continuous cultivation system. Appl Microbiol Biotechnol 35:19–20Google Scholar
  16. Mauersberger S, Wang HJ, Gaillardin C, Barth G, Nicaud JM (2001) Insertional mutagenesis in the n-alkane-assimilating yeast Yarrowia lipolytica: generation of tagged mutations in genes involved in hydrophobic substrate utilization. J Bacteriol 17:5102–5109CrossRefGoogle Scholar
  17. Nicaud JM, Madzak C, van den Broek P, Gysler C, Duboc P, Neiderberger P, Gaillardin C (2002) Protein expression and secretion in the yeast Yarrowia lipolytica. FEMS Yeast Res 2:371–379CrossRefPubMedGoogle Scholar
  18. Novotny C, Dolezalova L, Musil P, Novak M (1988) The production of lipase by some Candida and Yarrowia yeast. J Basic Microbiol 4:221–227Google Scholar
  19. Ota Y, Gomi K, Kato S, Sugiura T, Minoda Y (1982) Purification and some properties of cell-bound lipase from Saccharomycopsis lipolytica. Agric Biol Chem 46:2885–2893Google Scholar
  20. Ota Y, Oikawa S, Morimoto Y, Minoda Y (1984) Nutritional factors causing mycelial development of Saccharomycopsis lipolytica. Agric Biol Chem 48:1933–1940Google Scholar
  21. Pandey A, Benjamin S, Soccol CR, Nigam P, Krieger N, Soccol VT (1999) The realm of microbial lipases in biotechnology. Biotechnol Appl Biochem 29:119–131PubMedGoogle Scholar
  22. Pereira-Meirelles FV, Rocha Leao NHM, Sant'Anna GL Jr (1997) A stable lipase from Candida lipolytica: cultivation conditions and crude enzyme characteristics. Appl Biochem Biotechnol 63–65:73–85Google Scholar
  23. Peters I, Nelson F (1948a) Factors influencing the production of lipase by Mycotorula lipolytica. J Bacteriol 55:581–591Google Scholar
  24. Peters I, Nelson F (1948b) Preliminary characterization of lipase of Mycotorula lipolytica. J Bacteriol 55:593–600Google Scholar
  25. Pignede G, Wang H, Fudalej F, Gaillardin C, Seman M, Nicaud JM (2000) Characterization of an extracellular lipase encoded by LIP2 in Yarrowia lipolytica. J Bacteriol 182:2802–2810CrossRefPubMedGoogle Scholar
  26. Sambrook J, Fritsch E, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.Google Scholar
  27. Sarda L, Desnuelle P (1958) Action de la lipase pancréatique sur les esters en émulsion. Biochim Biophys Acta 30:513–521Google Scholar
  28. Shahani M (1975) In: Reed K (ed) Enzymes in food processing, 2nd edn. Academic Press, New York, pp 181–217Google Scholar
  29. Thonart P, Destain J, Zgouli S, Antoine P, Godefroid J, Evrard P (1997) Les bacs à graisse: une solution aux problèmes des matières grasses pour les PME. Trib Eau 50:41–46Google Scholar
  30. Vulfson E (1994) Industrial application of lipases. In: Wooley P, Petersen S (eds) Lipases. Cambridge University Press, pp 271–288Google Scholar
  31. Wang H, LeDall MT, Wache Y, Laroche C, Belin JM, Gaillardin C, Nicaud JM (1999) Evaluation of acyl coenzyme A oxidase (Aox) isozyme function in the n-alkane-assimilating yeast Yarrowia lipolytica. J Bacteriol 181:5140–5148PubMedGoogle Scholar
  32. Wickerham L, Kurtzman C, Herman A (1970) Sexual reproduction in Candida lipolytica. Science 167:1141Google Scholar
  33. Zentler-Munro P, Assoufi A, Balasubramanian K, Cornell D, Benoliel D, Northfield T, Hodson M (1992) Therapeutic potential and clinical efficacy of acid resistant fungal lipase in the treatment of pancreatic steatorrhoea due to cystic fibrosis. Pancreas 7:311–319PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • P. Fickers
    • 1
    • 2
  • J. M. Nicaud
    • 1
    Email author
  • J. Destain
    • 3
  • P. Thonart
    • 2
  1. 1.Laboratoire de Microbiologie et de Génétique Moléculaire, UMR2585, CNRS INRAInstitut National Agronomique Paris-GrignonThiverval-GrignonFrance
  2. 2.Centre Wallon de Biologie Industrielle, Service de Technologie MicrobienneUniversité de LiègeLiègeBelgium
  3. 3.Centre Wallon de Biologie Industrielle, Unité de Bio-industriesFaculté Universitaire des Sciences Agronomiques de GemblouxGemblouxBelgium

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