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Over-expression of native Saccharomyces cerevisiae exocytic SNARE genes increased heterologous cellulase secretion

Applied Microbiology and Biotechnology volume 98, pages 5567–5578 (2014)Cite this article

Abstract

SNAREs (soluble NSF [N-ethylmaleimide-sensitive factor] attachment receptor proteins) are required at the majority of fusion events during intracellular membrane transport and play crucial roles in facilitating protein trafficking between the various membrane-enclosed organelles and the plasma membrane. We demonstrate increases in the secretion of the Talaromyces emersonii Cel7A (a cellobiohydrolase) and the Saccharomycopsis fibuligera Cel3A (a β-glucosidase), through the separate and simultaneous over-expression of different components of the exocytic SNARE complex in Saccharomyces cerevisiae. Over-expression of SNC1 yielded the biggest improvement in Te-Cel7A secretion (71 %), whilst SSO1 over-expression lead to the highest increases in Sf-Cel3A secretion (43.8 %). Simultaneous over-expression of differential combinations of these SNARE components yielded maximal increases of ~52 % and ~49 % for the secretion of Te-Cel7A and Sf-Cel3A, respectively. These increases generally did not cause deleterious growth effects, whilst differential improvement patterns were observed for the two reporter proteins (Sf-Cel3A and Te-Cel7A). Simultaneous over-expression of up to three of these components, in strains secreting the more efficiently expressed Sf-Cel3A, illustrated a slight decrease in osmotic tolerance at elevated NaCl concentrations, as well as a detectable decrease in ethanol tolerance at increased concentrations. This work illustrates the potential of engineering components of the anterograde secretory pathway, particularly its SNARE components, for the improvement of heterologous cellulase secretion.

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References

  • Burri L, Lithgow T (2004) A complete set of SNAREs in yeast. Traffic 5:45–52

    CAS  PubMed  Article  Google Scholar 

  • Byrne KP, Wolfe KH (2005) The yeast gene order browser: combining curated homology and syntenic context reveals gene fate in polyploid species. Gen Res 15:1456–1461

    CAS  Article  Google Scholar 

  • Çelik E, Çalık P (2012) Production of recombinant proteins by yeast cells. Biotechnol Adv 30:1108–1118

    PubMed  Article  Google Scholar 

  • Demain A, Vaishnav P (2009) Production of recombinant proteins by microbes and higher organisms. Biotechnol Adv 27:297–306

    CAS  PubMed  Article  Google Scholar 

  • Den Haan R, Rose S, Lynd L, Van Zyl WH (2007) Hydrolysis and fermentation of amorphous cellulose by recombinant Saccharomyces cerevisiae. Metab Eng 9:87–94

    Article  Google Scholar 

  • Den Haan R, Kroukamp H, Van Zyl JHDV, Van Zyl WH (2013) Cellobiohydrolase secretion by yeast: current state and prospects for improvement. Process Biochem 48:1–12

    Article  Google Scholar 

  • Di Sansebastiano G (2013) Defining new SNARE functions: the i-SNARE. Front Plant Sci 4:99

    PubMed Central  PubMed  Article  Google Scholar 

  • Galdieri L, Mehrotra S, Yu S, Vancura A (2010) Transcriptional regulation in yeast during diauxic shift and stationary phase. OMICS 14:629–638

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Gasser B, Sauer M, Maurer M, Stadlmayr G, Mattanovich D (2007) Transcriptomics-based identification of novel factors enhancing heterologous protein secretion in yeasts. Appl Environ Microbiol 73:6499–6507

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Harford N, Cabezon T, Colau B, Delisse AM, Rutgers T, De Wilde M (1987) Construction and characterization of a Saccharomyces cerevisiae strain (RIT4376) expressing hepatitis B surface antigen. Postgrad Med J 63(2):65–70

    CAS  PubMed  Google Scholar 

  • Hill J, Donald KA, Griffiths DE, Donald G (1991) DMSO-enhanced whole cell yeast transformation. Nucleic Acids Res 19(20):5791

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Hoffman CS, Winston F (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267–272

    CAS  PubMed  Article  Google Scholar 

  • Hou J, Tyo K, Liu Z, Petranovic D, Nielsen J (2012) Engineering of vesicle trafficking improves heterologous protein secretion in Saccharomyces cerevisiae. Metab Eng 14:120–127

    CAS  PubMed  Article  Google Scholar 

  • Idiris A, Tohda H, Kumagai H, Takegawa K (2010) Engineering of protein secretion in yeast: strategies and impact on protein production. Appl Microbiol Biotechnol 86:403–417

    CAS  PubMed  Article  Google Scholar 

  • Ilmén M, Den Haan R, Brevnova E, McBride J, Wiswall E, Froehlich A, Koivula A, Voutilainen SP, Siika-aho M, La Grange DC, Thorngren N, Ahlgren S, Mellon M, Deleault K, Rajgarhia V, Van Zyl WH, Pentillä M (2011) High level secretion of cellobiohydrolases by Saccharomyces cerevisiae. Biotechnol Biofuels 4:30

    PubMed Central  PubMed  Article  Google Scholar 

  • Jahn R, Scheller RH (2006) SNAREs — engines for membrane fusion. Nat Rev Mol Cell Bio 7:631–643

    CAS  Article  Google Scholar 

  • Jäntti J, Aalto M, Oyen M, Sundqvist L, Keränen S, Ronne H (2002) Characterization of temperature-sensitive mutations in the yeast syntaxin 1 homologues Sso1p and Sso2p, and evidence of a distinct function for Sso1p in sporulation. J Cell Sci 115:409–420

    PubMed  Google Scholar 

  • Jüschke C, Wächter A, Schwappach B, Seedorf M (2005) SEC18/NSF-independent, protein sorting pathway from the yeast cortical ER to the plasma membrane. J Cell Biol 169(4):613–622

    PubMed Central  PubMed  Article  Google Scholar 

  • Kloepper TH, Kienle CN, Fasshauer D (2008) SNAREing the basis of multicellularity: consequences of protein family expansion during evolution. Mol Biol Evol 25(9):2055–2068

    CAS  PubMed  Article  Google Scholar 

  • Krantz M, Ahmadpour D, Ottosson L, Warringer J, Waltermann C, Nordlander B, Klipp E, Blomberg A, Hohmann S, Kitano H (2009) Robustness and fragility in the yeast high osmolarity glycerol (HOG) signal transduction pathway. Mol Syst Biol 5:281

    PubMed Central  PubMed  Article  Google Scholar 

  • Kroukamp H, Den Haan R, Van Wyk N, Van Zyl WH (2013) Over-expression of native PSE1 and SOD1 in Saccharomyces cerevisiae improved heterologous cellulase secretion. Appl Energy 102:150–156

    CAS  Article  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685

    CAS  PubMed  Article  Google Scholar 

  • La Grange DC, Pretorius IS, Claeyssens M, Van Zyl WH (2001) Degradation of xylan to d-xylose by recombinant Saccharomyces cerevisiae coexpressing the Aspergillus niger β-xylosidase (xlnD) and the Trichoderma reesei xylanase II (Xyn2) genes. Appl Environ Microbiol 67(12), 5512v5519

    Article  Google Scholar 

  • Lee FW, Da Silva NA (1997) Sequential delta integration for the regulated insertion of cloned genes in Saccharomyces cerevisiae. Biotechnol Progr 13(4):368–373

    CAS  Article  Google Scholar 

  • Lynd L, Van Zyl W, McBride J, Laser M (2005) Consolidated bioprocessing of cellulosic biomass: an update. Curr Opin Biotechnol 16:577–583

    CAS  PubMed  Article  Google Scholar 

  • Malsam J, Kreye S, Söllner T (2008) Membrane fusion: SNAREs and regulation. CMLS Cell Mol Life S 65:2814–2832

    CAS  Article  Google Scholar 

  • McBride JE, Zietsman JJ, Van Zyl WH, Lynd LR (2005) Utilization of cellobiose by recombinant β-glucosidase-expressing strains of Saccharomyces cerevisiae: characterization and evaluation of the sufficiency of expression. Enzyme Microb Technol 37:93–101

    CAS  Article  Google Scholar 

  • McBride JEE, Deleault KM, Lynd LR, Pronk JT (2008) Recombinant yeast strains expressing tethered cellulase enzymes. Patent PCT/US2007/085390

  • Nigam JM, Ireland RS, Margaritus A, Lachance MA (1985) Isolation and screening of yeasts that ferment d-xylose directly to ethanol. Appl Environ Microbiol 50(6):1486–1489

    CAS  PubMed Central  PubMed  Google Scholar 

  • Ostergaard S, Olsson L, Nielsen J (2000) Metabolic engineering of Saccharomyces cerevisiae. Microbiol Mol Biol R 64(1):34–50

    CAS  Article  Google Scholar 

  • Protopopov V, Govindan B, Novick P, Gerst JE (1993) Homologues of the synaptobrevin/VAMP family of synaptic vesicle proteins function on the late secretory pathway in S. cerevisiae. Cell 74(5):855–861

    CAS  PubMed  Article  Google Scholar 

  • Rader RA (2007) Biopharmaceutical products in the US and European markets, 6th edn. BioPlan Associates, Rockville

    Google Scholar 

  • Romanos MA, Scorer CA, Clarke JJ (1992) Foreign gene expression in yeast: a review. Yeast 8:423–488

    CAS  PubMed  Article  Google Scholar 

  • Romanos M (1995) Advances in the use of Pichia pastoris for high-level gene expression. Curr Opin Biotechnol 6:527–533

    CAS  Article  Google Scholar 

  • Ruohonen L, Toikkanen J, Tieaho V, Outola M, Soderlund H, Keranen S (1997) Enhancement of protein secretion in Saccharomyces cerevisiae by overproduction of Sso protein, a late-acting component of the secretory pathway. Yeast 13(4):337–351

    CAS  PubMed  Article  Google Scholar 

  • Sambrook J, Russel DB (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Schmidt FR (2004) Recombinant expression systems in the pharmaceutical industry. Appl Microbiol Biotechnol 65:363–372

    CAS  PubMed  Article  Google Scholar 

  • Schröder M (2008) Engineering eukaryotic protein factories. Biotechnol Lett 30:187–196

    PubMed Central  PubMed  Article  Google Scholar 

  • Shen D, Yuan H, Hutagalung A, Verma A, Kümmel D, Wu X, Reinisch K, McNew JA, Novick P (2013) The synaptobrevin homologue Snc2p recruits the exocyst to the secretory vesicle by binding to Sec6p. J Cell Biol 202(3):509–526

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Stephen JD, Mabee WE, Saddler JN (2012) Will second-generation ethanol be able to compete with first-generation ethanol? Opportunities for cost reduction. Biofuels Bioprod Bioref 6:159–176

    CAS  Article  Google Scholar 

  • Teste M, Duquenne M, François JM, Parrou J (2009) Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae. BMC Mol Biol 10:99

    PubMed Central  PubMed  Article  Google Scholar 

  • Van Rensburg E, Den Haan R, Smith J, Van Zyl WH, Görgens JF (2012) The metabolic burden of cellulase expression by recombinant Saccharomyces cerevisiae Y294 in aerobic batch culture. Appl Microbiol Biotechnol 96(1):197–209

    CAS  PubMed  Article  Google Scholar 

  • Van Rooyen R, Hahn-Hägerdal B, La Grange DC, Van Zyl WH (2005) Construction of cellobiose-growing Saccharomyces cerevisiae strains. J Biotechnol 120:284–295

    PubMed  Article  Google Scholar 

  • Weinberger A, Kamena F, Kama R, Spang A, Gerst J (2005) Control of Golgi morphology and function by Sed5 t-SNARE phosphorylation. Mol Biol Cell 16:4918–4930

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Xu L, Shen Y, Hou J, Peng B, Tang H, Bao X (2013) Secretory pathway engineering enhances secretion of cellobiohydrolase I from Trichoderma reesei in Saccharomyces cerevisiae. J Biosci Bioeng. doi:10.1016/j.jbiosc.2013.06.017

    Google Scholar 

  • Yoshikawa K, Tanaka T, Ida Y, Furusawa C, Hirasawa T, Shimizu H (2011) Comprehensive phenotypic analysis of single-gene deletion and overexpression strains of Saccharomyces cerevisiae. Yeast 28:349–361

    CAS  PubMed  Article  Google Scholar 

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Acknowledgements

Funding for this project was provided by the National Research Foundation (South Africa).

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The authors declare that there are no conflicts of interest associated with the submission of this study.

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  1. Department of Microbiology, Stellenbosch University, Stellenbosch, 7602, South Africa

    John Henry D. Van Zyl, Riaan Den Haan & Willem H. Van Zyl

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  1. John Henry D. Van Zyl
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  2. Riaan Den Haan
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  3. Willem H. Van Zyl
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Correspondence to Willem H. Van Zyl.

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Van Zyl, J.H.D., Den Haan, R. & Van Zyl, W.H. Over-expression of native Saccharomyces cerevisiae exocytic SNARE genes increased heterologous cellulase secretion. Appl Microbiol Biotechnol 98, 5567–5578 (2014). https://doi.org/10.1007/s00253-014-5647-1

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  • DOI: https://doi.org/10.1007/s00253-014-5647-1

Keywords

  • SNAREs
  • Secretion
  • Improvement
  • Cellulases
  • Yeast