Skip to main content
SpringerLink
Log in

Efficient cell surface display of Lip2 lipase using C-domains of glycosylphosphatidylinositol-anchored cell wall proteins of Yarrowia lipolytica

  • Biotechnologically Relevant Enzymes and Proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The cell surface display of enzymes is of great interest because of its simplified purification stage and the possibility for recycling in industrial processes. In this study, we have focused on the cell wall immobilization of Yarrowia lipolytica Lip2 protein—an enzyme that has a wide technological application. By genome analysis of Y. lipolytica in addition to already characterized Ylcwp1, we identified five putative open reading frames encoding glycosylphosphatidylinositol-anchored proteins. Lip2 translation fusion with the carboxyl termini of these proteins revealed that all proteins were capable of immobilizing lipase in active form on the cell surface. The highest level of cell-bound lipase activity was achieved using C-domains encoded by YlCWP1, YlCWP3 (YALI0D27214g) and YlCWP6 (YALI0F18282g) comprising 16,173 ± 1,800, 18,785 ± 1,130 and 17,700 ± 2,101 U/g dry cells, respectively. To the best of our knowledge, these results significantly exceed the highest cell-bound lipase activity previously reported for engineered Saccharomyces cerevisiae and Pichia pastoris strains. Furthermore, the lyophilized biomass retained the activity and was robust to collecting/resuspending procedures. Nevertheless, in most cases, a substantial amount of lipase activity was also found in the growth medium. Further work will be necessary to better understand the nature of this phenomenon.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Alloue WA, Destain J, El Medjoub T, Ghalfi H, Kabran P, Thonart P (2008) Comparison of Yarrowia lipolytica lipase immobilization yield of entrapment, adsorption, and covalent bond techniques. Appl Biochem Biotechnol 150(1):51–63

    Article  CAS  Google Scholar 

  • Barth G, Gaillardin C (1997) Physiology and genetics of the dimorphic fungus Yarrowia lipolytica. FEMS Microbiol Rev 19(4):219–237

    Article  CAS  Google Scholar 

  • Dujon B, Sherman D, Fischer G, Durrens P, Casaregola S, Lafontaine I, De Montigny J, Marck C, Neuveglise C, Talla E, Goffard N, Frangeul L, Aigle M, Anthouard V, Babour A, Barbe V, Barnay S, Blanchin S, Beckerich JM, Beyne E, Bleykasten C, Boisrame A, Boyer J, Cattolico L, Confanioleri F, De Daruvar A, Despons L, Fabre E, Fairhead C, Ferry-Dumazet H, Groppi A, Hantraye F, Hennequin C, Jauniaux N, Joyet P, Kachouri R, Kerrest A, Koszul R, Lemaire M, Lesur I, Ma L, Muller H, Nicaud JM, Nikolski M, Oztas S, Ozier-Kalogeropoulos O, Pellenz S, Potier S, Richard GF, Straub ML, Suleau A, Swennen D, Tekaia F, Wesolowski-Louvel M, Westhof E, Wirth B, Zeniou-Meyer M, Zivanovic I, Bolotin-Fukuhara M, Thierry A, Bouchier C, Caudron B, Scarpelli C, Gaillardin C, Weissenbach J, Wincker P, Souciet JL (2004) Genome evolution in yeasts. Nature 430(6995):35–44

    Article  Google Scholar 

  • Fickers P, Benetti PH, Wache Y, Marty A, Mauersberger S, Smit MS, Nicaud JM (2005a) Hydrophobic substrate utilisation by the yeast Yarrowia lipolytica, and its potential applications. FEMS Yeast Res 5(6–7):527–543

    Article  CAS  Google Scholar 

  • Fickers P, Fudalej F, Le Dall MT, Casaregola S, Gaillardin C, Thonart P, Nicaud JM (2005b) Identification and characterisation of LIP7 and LIP8 genes encoding two extracellular triacylglycerol lipases in the yeast Yarrowia lipolytica. Fungal Genet Biol 42(3):264–274

    Article  CAS  Google Scholar 

  • Jaafar L, Zueco J (2004) Characterization of a glycosylphosphatidylinositol-bound cell-wall protein (GPI-CWP) in Yarrowia lipolytica. Microbiology 150(Pt 1):53–60

    Article  CAS  Google Scholar 

  • Kapteyn JC, Van Den Ende H, Klis FM (1999) The contribution of cell wall proteins to the organization of the yeast cell wall. Biochim Biophys Acta 1426(2):373–383

    CAS  Google Scholar 

  • Le Dall MT, Nicaud JM, Gaillardin C (1994) Multiple-copy integration in the yeast Yarrowia lipolytica. Curr Genet 26(1):38–44

    Article  Google Scholar 

  • Liu W, Zhao H, Jia B, Xu L, Yan Y (2010a) Surface display of active lipase in Saccharomyces cerevisiae using Cwp2 as an anchor protein. Biotechnol Lett 32(2):255–260

    Article  CAS  Google Scholar 

  • Liu WS, Pan XX, Jia B, Zhao HY, Xu L, Liu Y, Yan YJ (2010b) Surface display of active lipases Lip7 and Lip8 from Yarrowia lipolytica on Saccharomyces cerevisiae. Appl Microbiol Biotechnol 88(4):885–891

    Article  CAS  Google Scholar 

  • Liu XY, Chi Z, Liu GL, Wang F, Madzak C, Chi ZM (2010c) Inulin hydrolysis and citric acid production from inulin using the surface-engineered Yarrowia lipolytica displaying inulinase. Metab Eng 12(5):469–476

    Article  CAS  Google Scholar 

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

    CAS  Google Scholar 

  • Madzak C, Gaillardin C, Beckerich JM (2004) Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: a review. J Biotechnol 109(1–2):63–81

    Article  CAS  Google Scholar 

  • Matsumoto T, Fukuda H, Ueda M, Tanaka A, Kondo A (2002) Construction of yeast strains with high cell surface lipase activity by using novel display systems based on the Flo1p flocculation functional domain. Appl Environ Microbiol 68(9):4517–4522

    Article  CAS  Google Scholar 

  • Ni X, Yue L, Chi Z, Li J, Wang X, Madzak C (2009) Alkaline protease gene cloning from the marine yeast Aureobasidium pullulans HN2-3 and the protease surface display on Yarrowia lipolytica for bioactive peptide production. Mar Biotechnol (NY) 11(1):81–89

    Article  CAS  Google Scholar 

  • Nicaud JM, Madzak C, van den Broek P, Gysler C, Duboc P, Niederberger P, Gaillardin C (2002) Protein expression and secretion in the yeast Yarrowia lipolytica. FEMS Yeast Res 2(3):371–379

    CAS  Google Scholar 

  • Oberdoerffer P, Otipoby KL, Maruyama M, Rajewsky K (2003) Unidirectional Cre-mediated genetic inversion in mice using the mutant loxP pair lox66/lox71. Nucleic Acids Res 31(22):e140

    Article  Google Scholar 

  • Pandey A, Benjamin S, Soccol CR, Nigam P, Krieger N, Soccol VT (1999) The realm of microbial lipases in biotechnology. Biotechnol Appl Biochem 29(Pt 2):119–131

    CAS  Google Scholar 

  • Papanikolaou S, Muniglia L, Chevalot I, Aggelis G, Marc I (2002) Yarrowia lipolytica as a potential producer of citric acid from raw glycerol. J Appl Microbiol 92(4):737–744

    Article  CAS  Google Scholar 

  • 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(10):2802–2810

    Article  CAS  Google Scholar 

  • Roth R, Moodley V, van Zyl P (2009) Heterologous expression and optimized production of an Aspergillus aculeatus endo-1,4-beta-mannanase in Yarrowia lipolytica. Mol Biotechnol 43(2):112–120

    Article  CAS  Google Scholar 

  • Sambrook J, Maniatis T, Fritsch E (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  • Shimma Y, Saito F, Oosawa F, Jigami Y (2006) Construction of a library of human glycosyltransferases immobilized in the cell wall of Saccharomyces cerevisiae. Appl Environ Microbiol 72(11):7003–7012

    Article  CAS  Google Scholar 

  • Su GD, Zhang X, Lin Y (2010) Surface display of active lipase in Pichia pastoris using Sed1 as an anchor protein. Biotechnol Lett 32(8):1131–1136

    Article  CAS  Google Scholar 

  • Tanino T, Aoki T, Chung WY, Watanabe Y, Ogino C, Fukuda H, Kondo A (2009) Improvement of a Candida antarctica lipase B-displaying yeast whole-cell biocatalyst and its application to the polyester synthesis reaction. Appl Microbiol Biotechnol 82(1):59–66

    Article  CAS  Google Scholar 

  • Thompson A, Gasson MJ (2001) Location effects of a reporter gene on expression levels and on native protein synthesis in Lactococcus lactis and Saccharomyces cerevisiae. Appl Environ Microbiol 67(8):3434–3439

    Article  CAS  Google Scholar 

  • Ueda M, Tanaka A (2000) Genetic immobilization of proteins on the yeast cell surface. Biotechnol Adv 18(2):121–140

    Article  CAS  Google Scholar 

  • Van der Vaart JM, Caro LH, Chapman JW, Klis FM, Verrips CT (1995) Identification of three mannoproteins in the cell wall of Saccharomyces cerevisiae. J Bacteriol 177(11):3104–3110

    Google Scholar 

  • Van der Vaart JM, te Biesebeke R, Chapman JW, Toschka HY, Klis FM, Verrips CT (1997) Comparison of cell wall proteins of Saccharomyces cerevisiae as anchors for cell surface expression of heterologous proteins. Appl Environ Microbiol 63(2):615–620

    Google Scholar 

  • Washida M, Takahashi S, Ueda M, Tanaka A (2001) Spacer-mediated display of active lipase on the yeast cell surface. Appl Microbiol Biotechnol 56(5–6):681–686

    Article  CAS  Google Scholar 

  • Yue L, Chi Z, Wang L, Liu J, Madzak C, Li J, Wang X (2008) Construction of a new plasmid for surface display on cells of Yarrowia lipolytica. J Microbiol Methods 72(2):116–123

    Article  CAS  Google Scholar 

  • Yuzbashev TV, Yuzbasheva EY, Sobolevskaya TI, Laptev IA, Vybornaya TV, Larina AS, Matsui K, Fukui K, Sineoky SP (2010) Production of succinic acid at low pH by a recombinant strain of the aerobic yeast Yarrowia lipolytica. Biotechnol Bioeng 107(4):673–682

    Article  CAS  Google Scholar 

  • Yuzbasheva EY, Yuzbashev TV, Konstantinova TK, Laptev IA, PN I, Sineoky SP (2010) Capacity of the N- and C-domains of the cell wall protein Flo1p homologue in yeast Yarrowia lipolytica to display lipase Lip2 on the cell surface. Biotechnologiya 6:23–36, Article in Russian

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Russian State Collection of Industrial Microorganisms (VKPM), State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow, 117545, Russia

    Evgeniya Y. Yuzbasheva, Tigran V. Yuzbashev, Ivan A. Laptev, Tatiana K. Konstantinova & Sergey P. Sineoky

Authors
  1. Evgeniya Y. Yuzbasheva
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tigran V. Yuzbashev
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ivan A. Laptev
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tatiana K. Konstantinova
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sergey P. Sineoky
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Evgeniya Y. Yuzbasheva.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yuzbasheva, E.Y., Yuzbashev, T.V., Laptev, I.A. et al. Efficient cell surface display of Lip2 lipase using C-domains of glycosylphosphatidylinositol-anchored cell wall proteins of Yarrowia lipolytica . Appl Microbiol Biotechnol 91, 645–654 (2011). https://doi.org/10.1007/s00253-011-3265-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-011-3265-8

Keywords

Search

Navigation