Advertisement

Applied Microbiology and Biotechnology

, Volume 103, Issue 7, pp 3085–3097 | Cite as

Identification and evaluation of novel anchoring proteins for cell surface display on Saccharomyces cerevisiae

  • Apisan Phienluphon
  • Wuttichai Mhuantong
  • Katewadee Boonyapakron
  • Pacharawan Deenarn
  • Verawat Champreda
  • Duangdao Wichadakul
  • Surisa SuwannarangseeEmail author
Applied genetics and molecular biotechnology

Abstract

The development of arming yeast strains as whole-cell biocatalysts involves a selection of effective anchoring proteins to display enzymes and proteins on yeast cell surface. To screen for novel anchoring proteins with improved efficiency, a bioinformatics pipeline for the identification of glycosylphosphatidylinositol-anchored cell wall proteins (GPI-CWPs) suitable for attaching passenger proteins to the cell surface of Saccharomyces cerevisiae has been developed. Here, the C-terminal sequences (CTSs) of putative GPI-CWPs were selected based on the criteria that the sequence must contain a serine/threonine-rich (S/T) region of at least 30% S/T content, a total threonine content of at least 10%, a continuous S/T stretch of at least 130 amino acids in length, and a continuous T-rich region of at least 10 amino acids in length. Of the predicted 790 proteins, 37 putative GPI-CWPs were selected from different yeast and fungal species to be evaluated for their performance in displaying yeast-enhanced green fluorescent protein and β-glucosidase enzyme. This led to the identification of five novel anchoring proteins with higher performance compared to α-agglutinin used as benchmark. In particular, the CTS of uncharacterized protein in Kluyveromyces lactis, namely 6_Kl, is the most efficient anchoring protein of the group. The CTS of 6_Kl protein provided a β-glucosidase activity of up to 23.5 U/g cell dry weight, which is 2.8 times higher than that of the CTS of α-agglutinin. These identified CTSs could be potential novel anchoring protein candidates for construction of efficient arming yeasts for biotechnology applications in the future.

Keywords

Cell surface display Anchoring protein GPI-anchored protein Saccharomyces cerevisiae β-Glucosidase Bioinformatics 

Notes

Acknowledgements

We thank Dr. Wananit Wimuttisuk and Mr. Christopher Keith Campbell for critically reading the manuscript.

Funding information

This work was financially supported by the National Center for Genetic Engineering and Biotechnology, Thailand (Grant no. P1300748).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with animals or human participants performed by any of the authors.

Supplementary material

253_2019_9667_MOESM1_ESM.pdf (285 kb)
ESM 1 (PDF 284 kb)

References

  1. Abe H, Shimma Y, Jigami Y (2003) In vitro oligosaccharide synthesis using intact yeast cells that display glycosyltransferases at the cell surface through cell wall-anchored protein Pir. Glycobiology 13:87–95CrossRefPubMedGoogle Scholar
  2. Andreu C, del Olmo M (2017) Development of a new yeast surface display system based on Spi1 as an anchor protein. Appl Microbiol Biotechnol 101:287–299CrossRefPubMedGoogle Scholar
  3. Andreu C, del Olmo M (2018) Yeast arming systems: pros and cons of different protein anchors and other elements required for display. Appl Microbiol Biotechnol 102:2543–2561CrossRefPubMedGoogle Scholar
  4. Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nature Biotechnol 15:553–557CrossRefGoogle Scholar
  5. Boisramé A, Cornu A, Da Costa G, Richard ML (2011) Unexpected role for a serine/threonine-rich domain in the Candida albicans Iff protein family. Eukaryot Cell 10:1317–1330CrossRefPubMedGoogle Scholar
  6. Caro LHP, Tettelin H, Vossen JH, Ram AFJ, Van Den Ende H, Klis FM (1997) In silicio identification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisiae. Yeast 13:1477–1489CrossRefPubMedGoogle Scholar
  7. Cheon SA, Jung J, Choo JH, Oh DB, Kang HA (2014) Characterization of putative glycosylphosphatidylinositol-anchoring motifs for surface display in the methylotrophic yeast Hansenula polymorpha. Biotechnol Lett 36:2085–2094CrossRefPubMedGoogle Scholar
  8. Cock PJA, Antao T, Chang JT, Chapman BA, Cox CJ, Dalke A, Friedberg I, Hamelryck T, Kauff F, Wilczynski B, de Hoon MJL (2009) Biopython: freely available Python tools for computational molecular biology and bioinformatics. Bioinform 25:1422–1423CrossRefGoogle Scholar
  9. Cormack BP, Bertram G, Egerton M, Gow NAR, Falkow S, Brown AJP (1997) Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans. Microbiology 143:303–311CrossRefPubMedGoogle Scholar
  10. De Groot PWJ, Brandt BW (2012) ProFASTA: a pipeline web server for fungal protein scanning with integration of cell surface prediction software. Fungal Genet Biol 49:173–179CrossRefPubMedGoogle Scholar
  11. De Groot PWJ, Ram AF, Klis FM (2005) Features and functions of covalently linked proteins in fungal cell walls. Fungal Genet Biol 42:657–675CrossRefPubMedGoogle Scholar
  12. Dujon B, Sherman D, Fischer G, Durrens P, Casaregola S, Lafontaine I, de Montigny J, Marck C, Neuvéglise C, Talla E, Goffard N, Frangeul L, Aigle M, Anthouard V, Babour A, Barbe V, Barnay S, Blanchin S, Beckerich J-M, Beyne E, Bleykasten C, Boisram 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 J-M, Nikolski M, Oztas S, Ozier-Kalogeropoulos O, Pellenz S, Potier S, Richard G-F, Straub M-L, Suleau A, Swennen D, Tekaia F, Wésolowski-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 J-L (2004) Genome evolution in yeasts. Nature 430:35–44CrossRefPubMedGoogle Scholar
  13. Eisenhaber B, Schneider G, Wildpaner M, Eisenhaber F (2004) A sensitive predictor for potential GPI lipid modification sites in fungal protein sequences and its application to genome-wide studies for Aspergillus nidulans, Candida albicans, Neurospora crassa, Saccharomyces cerevisiae and Schizosaccharomyces pombe. J Mol Biol 337:243–253CrossRefPubMedGoogle Scholar
  14. Frieman MB, Cormack BP (2004) Multiple sequence signals determine the distribution of glycosylphosphatidylinositol proteins between the plasma membrane and cell wall in Saccharomyces cerevisiae. Microbiology 150:3105–3114CrossRefPubMedGoogle Scholar
  15. Hamada K, Fukuchi S, Arisawa M, Baba M, Kitada K (1998) Screening for glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in Saccharomyces cerevisiae. Mol Gen Genet 258:53–59CrossRefPubMedGoogle Scholar
  16. Hamada K, Terashima H, Arisawa M, Yabuki N, Kitada K (1999) Amino acid residues in the ω -minus region participate in cellular localization of yeast glycosylphosphatidylinositol-attached proteins. J Bacteriol 181:3886–3889PubMedGoogle Scholar
  17. Harnpicharnchai P, Champreda V, Sornlake W, Eurwilaichitr L (2009) A thermotolerant β-glucosidase isolated from an endophytic fungi, Periconia sp., with a possible use for biomass conversion to sugars. Protein Expr Purif 67:61–69CrossRefPubMedGoogle Scholar
  18. Inokuma K, Hasunuma T, Kondo A (2014) Efficient yeast cell-surface display of exo- and endo-cellulase using the SED1 anchoring region and its original promoter. Biotechnol Biofuels 7:8CrossRefPubMedGoogle Scholar
  19. Jaafar L, Zueco J (2004) Characterization of a glycosylphosphatidylinositol-bound cell-wall protein (GPI-CWP) in Yarrowia lipolytica. Microbiology 150:53–60CrossRefPubMedGoogle Scholar
  20. Klis FM, Boorsma A, De Groot PWJ (2006) Cell wall construction in Saccharomyces cerevisiae. Yeast 23:185–202CrossRefPubMedGoogle Scholar
  21. Kollar R, Reinhold BB, Petrakova E, Yeh HJ, Ashwell G, Drgonova J, Kapteyn JC, Klis FM, Cabib E (1997) Architecture of the yeast cell wall. β(1→6)-glucan interconnects mannoprotein, β(1→3)-glucan, and chitin. J Biol Chem 272:17762–17775CrossRefPubMedGoogle Scholar
  22. Kondo A, Ueda M (2004) Yeast cell-surface display--applications of molecular display. Appl Microbiol Biotechnol 64:28–40CrossRefPubMedGoogle Scholar
  23. Krogh A, Larsson B, von Heijne G, Sonnhammer ELL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580CrossRefPubMedGoogle Scholar
  24. Kuroda K, Ueda M (2013) Arming technology in yeast-novel strategy for whole-cell biocatalyst and protein engineering. Biomolecules 3:632–650CrossRefPubMedGoogle Scholar
  25. Kuroda K, Matsui K, Higuchi S, Kotaka A, Sahara H, Hata Y, Ueda M (2009) Enhancement of display efficiency in yeast display system by vector engineering and gene disruption. Appl Microbiol Biotechnol 82:713–719CrossRefPubMedGoogle Scholar
  26. Laplaza JM, Torres BR, Jin YS, Jeffries TW (2006) Sh ble and Cre adapted for functional genomics and metabolic engineering of Pichia stipitis. Enzym Microb Technol 38:741–747CrossRefGoogle Scholar
  27. Li M, Borodina I (2015) Application of synthetic biology for production of chemicals in yeast Saccharomyces cerevisiae. FEMS Yeast Res 15:1–12CrossRefPubMedGoogle Scholar
  28. Liu W, Zhao H, Jia B, Xu L, Yan Y (2010) Surface display of active lipase in Saccharomyces cerevisiae using Cwp2 as an anchor protein. Biotechnol Lett 32:255–260CrossRefPubMedGoogle Scholar
  29. Matsuoka H, Hashimoto K, Saijo A, Takada Y, Kondo A, Ueda M, Ooshima H, Tachibana T, Azuma M (2014) Cell wall structure suitable for surface display of proteins in Saccharomyces cerevisiae. Yeast 31:67–76CrossRefPubMedGoogle Scholar
  30. Mayor S, Riezman H (2004) Sorting GPI-anchored proteins. Nat Rev Mol Cell Biol 5:110–120CrossRefPubMedGoogle Scholar
  31. Mergler M, Wolf K, Zimmermann M (2004) Development of a bisphenol A-adsorbing yeast by surface display of the Kluyveromyces yellow enzyme on Pichia pastoris. Appl Microbiol Biotechnol 63:418–421CrossRefPubMedGoogle Scholar
  32. Nakamura Y, Shibasaki S, Ueda M, Tanaka A, Fukuda H, Kondo A (2001) Development of novel whole-cell immunoadsorbents by yeast surface display of the IgG-binding domain. Appl Microbiol Biotechnol 57:500–505CrossRefPubMedGoogle Scholar
  33. Pepper LR, Cho YK, Boder ET, Shusta EV (2008) A decade of yeast surface display technology: where are we now? Comb Chem High Throughput Screen 11:127–134CrossRefPubMedGoogle Scholar
  34. Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786CrossRefPubMedGoogle Scholar
  35. Phillips GJ (2001) Green fluorescent protein--a bright idea for the study of bacterial protein localization. FEMS Microbiol Lett 204:9–18PubMedGoogle Scholar
  36. Pierleoni A, Martelli PL, Casadio R (2008) PredGPI: a GPI-anchor predictor. BMC Bioinformatics 9:392CrossRefPubMedGoogle Scholar
  37. Poisson G, Chauve C, Chen X, Bergeron A (2007) FragAnchor: a large-scale predictor of glycosylphosphatidylinositol anchors in eukaryote protein sequences by qualitative scoring. Genomics Proteomics Bioinformatics 5:121–130CrossRefPubMedGoogle Scholar
  38. Presnyak V, Alhusaini N, Chen YH, Martin S, Morris N, Kline N, Olson S, Weinberg D, Baker KE, Graveley BR, Coller J (2015) Codon optimality is a major determinant of mRNA stability. Cell 160:1111–1124CrossRefPubMedGoogle Scholar
  39. Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  40. Sato N, Matsumoto T, Ueda M, Tanaka A, Fukuda H, Kondo A (2002) Long anchor using Flo1 protein enhances reactivity of cell surface-displayed glucoamylase to polymer substrates. Appl Microbiol Biotechnol 60:469–474CrossRefPubMedGoogle Scholar
  41. Schoffelmeer EAVJ, Van Doorn AA, Cornelissen BJ, Haring MA (2001) FEM1, a Fusarium oxysporum glycoprotein that is covalently linked to the cell wall matrix and is conserved in filamentous fungi. Mol Gen Genomics 265:143–152CrossRefGoogle Scholar
  42. Schreuder MP, Brekelmans S, Van Den Ende H, Klis FM (1993) Targeting of a heterologous protein to the cell wall of Saccharomyces cerevisiae. Yeast 9:399–409CrossRefPubMedGoogle Scholar
  43. Shibasaki S, Ueda M, Iizuka T, Hirayama M, Ikeda Y, Kamasawa N, Osumi M, Tanaka A (2001) Quantitative evaluation of the enhanced green fluorescent protein displayed on the cell surface of Saccharomyces cerevisiae by the fluorometric and confocal laser scanning microscopic analyses. Appl Microbiol Biotechnol 55:471–475CrossRefPubMedGoogle Scholar
  44. Shibasaki S, Maeda H, Ueda M (2009) Molecular display technology using yeast—arming technology. Anal Sci 25:41–49CrossRefPubMedGoogle Scholar
  45. Su GD, Zhang X, Lin Y (2010) Surface display of active lipase in Pichia pastoris using Sed1 as an anchor protein. Biotechnol Lett 32:1131–1136CrossRefPubMedGoogle Scholar
  46. Tanaka T, Yamada R, Ogino C, Kondo A (2012) Recent developments in yeast cell surface display toward extended applications in biotechnology. Appl Microbiol Biotechnol 95:577–591CrossRefPubMedGoogle Scholar
  47. The UniProt Consortium (2008) The universal protein resource (UniProt). Nucleic Acids Res 36:D190–D195CrossRefGoogle Scholar
  48. 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 63:615–620PubMedGoogle Scholar
  49. Yamada R, Taniguchi N, Tanaka T, Ogino C, Fukuda H, Kondo A (2010) Cocktail δ-integration: a novel method to construct cellulolytic enzyme expression ratio-optimized yeast strains. Microb Cell Factories 9:32CrossRefGoogle Scholar
  50. Yuen KY, Chan CM, Chan KM, Woo PC, Che XY, Leung AS, Cao L (2001) Characterization of AFMP1: a novel target for serodiagnosis of aspergillosis. J Clin Microbiol 39:3830–3383CrossRefPubMedGoogle Scholar
  51. Zhou Z, Dang Y, Zhou M, Li L, Yu CH, Fu J, Chen S, Liu Y (2016) Codon usage is an important determinant of gene expression levels largely through its effects on transcription. Proc Natl Acad Sci U S A 113:E6117–E6125CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Microbial Biotechnology and Biochemicals Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC)National Science and Technology Development AgencyPathumthaniThailand
  2. 2.Department of Computer Engineering, Faculty of EngineeringChulalongkorn UniversityBangkokThailand

Personalised recommendations