Date Published: May 20, 2012
Author(s): Siyavuya Ishmael Bulani, Lucy Moleleki, Jacobus Albertyn, Ntsane Moleleki.
In this study, a novel rDNA based plasmid was developed for display of heterologous proteins on the cell surface of Yarrowia lipolytica using the C-terminal end of the glycosylphosphatidylinositol (GPI) anchored Y. lipolytica cell wall protein 1 (YlCWP1). mCherry was used as a model protein to assess the efficiency of the constructed plasmid. Y. lipolytica transformants harbouring the expression cassettes showed a purple colour phenotype on selective YNB-casamino plates as compared to control cells indicating that mCherry was displayed on the cells. Expression of mCherry on cells of Y. lipolytica was confirmed by both fluorescent microscopy and flow cytometry. Furthermore, SDS-PAGE analysis and matrix-assisted laser desorption/ionization (MALDI)-time-of (TOF)-mass spectrometry (MS) peptide mass fingerprinting (PMF) confirmed that the protein cleaved from the yeast cells using enterokinase was mCherry. Efficient cleavage of mCherry reported in this work offers an alternative purification method for displayed heterologous proteins on Y. lipolytica cells using the plasmid constructed in this study. The developed displaying system offers great potential for industrial production and purification of heterologous proteins at low cost.
Since the first development of a cell surface display system on bacteriophage by ([Smith 1985]), various yeast cell surface displaying systems have been developed for expression of heterologous proteins ([Sergeeva et al. 2006]). Yeast cell surface display has been used as a method of choice for expression of heterologous proteins. This is because yeast cell surface display is convenient, shows ease of handling of displayed heterologous proteins and has been found to be comparatively stable against environmental changes ([Inaba et al. 2010]). In addition, the advantage of yeast cell surface display over bacterial display is that yeast has a post-translational modification system that resembles the mammalian system for efficient processing and folding of proteins ([Kondo and Ueda,2004]). Cell surface displaying systems in yeast, in particular Saccharomyces cerevisiae, have been studied extensively ([Kondo and Ueda, 2004];[ Furukawa et al. 2006]). Although S. cerevisiae emerged as the most favourable microorganism for displaying heterologous proteins ([Kondo and Ueda, 2004]), hyperglycosylation of expressed proteins has remained a major drawback ([Gemmill and Trimble, 1999]). Hyperglycosylation of heterologous proteins has a potential to affect protein activity ([Wang et al. 2007]). Other yeasts, such as Pichia pastoris have been reported to express heterologous proteins with reduced glycosylation ([Choi et al. 2003]). More recently, cell surface display of active heterologous proteins in both Y. lipolytica and P. pastoris has gained momentum.
Cell surface display has shown great potential for various applications, such as whole-cell biocatalysis and combinatorial library construction [(Furukawa et al. 2006]). Its application in Y. lipolytica, which has been reported to secrete a wide range of proteins ([Beckerich et al. 1998]), could be essential for immobilization of active heterologous proteins. In this study, we constructed a novel rDNA based plasmid for surface display of heterologous proteins on Y. lipolytica. The plasmid of [Yue et al. (2007]) and ([Yang et al. 2009]) employ ura3d1 and LEU2 selection markers for single copy integration into the zeta targeting sequence and the pBR322 docking platform, respectively [(Madzak et al. 2000]; [Nicaud et al. 2002]). In addition, vectors carrying both a zeta sequence and ura3d4 are integrated into Y. lipolytica genome with low transformation frequencies [(Juretzek et al. 2001]). The plasmid developed in this study uses ura3d4 allele for homologous multiple integration into the rDNA cluster [(Juretzek et al. 2001]). Similar to the zeta-based plasmid constructed by ([Yue et al. 2007]) for cell surface display of heterologous proteins on Y. lipolytica, the constructed rDNA based displaying plasmid in this study uses a strong recombinant growth phase dependent hp4d promoter [(Madzak et al. 2004]). The hp4d promoter has traits optimal for heterologous protein expression as it operates almost unaffected by environmental conditions such as pH, carbon and nitrogen sources and presence of peptones [(Madzak et al. 1995,2000]). In addition, the expression cassette used to transform Y. lipolytica is devoid of a bacterial moiety including antibiotic resistance genes as a result retaining its GRAS (generally regarded as safe) status ([Nicaud et al. 1998]; [Pignede et al. 2000a]). As a result of these characteristics, the plasmid pKOV410 was used to construct a novel plasmid for enhanced cell surface display on Y. lipolytica using the GPI-anchored YlCWP1. The efficiency of the displaying plasmid for enhanced display of heterologous proteins was demonstrated using mCherry as a model protein. When Y. lipolytica was transformed with the expression cassettes, purple transformants were observed on the YNB-casamino selective plates compared to the Y. lipolytica negative control transformants (Figure 2). This colour change on the transformants served as a quick visual indication that mCherry was displayed on the yeast cell surface. The purple colour was observed on cells grown on both solid agar plates and liquid YPD medium, respectively (Figure 2A & C). Similar results were first reported by ([Keppler-Ross et al. 2008]) in S. cerevisiae overexpressing codon optimised enhanced monomeric red fluorescent protein (EmRFP). In addition, [(Gerami-Nejad et al. 2009]) constructed a synthetic codon optimised monomeric red fluorescent protein from Discosoma sp. (DsRed) that produced transformants detectable at colony level based on colour. Recent studies by [(Wu et al. 2011]) demonstrated a change in colour of E. coli cells displaying enhanced green fluorescent protein (EGFP) after induction with IPTG. However, the fluorescence was insufficient to change the colour of the growth medium to green. Studies by [(Kuroda et al. 2009]) did not report transformants colour changes when the DsRed-monomer was displayed on S. cerevisiae cell surface. To our knowledge, the purple colour change observation in the Yl-mch1 engineered in this study constitutes the first report of such an effect of displayed mCherry on both solid and liquid medium.
The authors declare that they have no competing interests.