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Overexpression of native Saccharomyces cerevisiae ER-to-Golgi SNARE genes increased heterologous cellulase secretion

Applied Microbiology and Biotechnology volume 100, pages 505–518 (2016)Cite this article

Abstract

Soluble N-ethylmaleimide-sensitive factor attachment receptor proteins (SNAREs) are essential components of the yeast protein-trafficking machinery and are required at the majority of membrane fusion events in the cell, where they facilitate SNARE-mediated fusion between the protein transport vesicles, the various membrane-enclosed organelles and, ultimately, the plasma membrane. We have demonstrated an increase in secretory titers for the Talaromyces emersonii Cel7A (Te-Cel7A, a cellobiohydrolase) and the Saccharomycopsis fibuligera Cel3A (Sf-Cel3A, a β-glucosidase) expressed in Saccharomyces cerevisiae through single and co-overexpression of some of the endoplasmic reticulum (ER)-to-Golgi SNAREs (BOS1, BET1, SEC22 and SED5). Overexpression of SED5 yielded the biggest improvements for both of the cellulolytic reporter proteins tested, with maximum increases in extracellular enzyme activity of 22 % for the Sf-Cel3A and 68 % for the Te-Cel7A. Co-overexpression of the ER-to-Golgi SNAREs yielded proportionately smaller increases for the Te-Cel7A (46 %), with the Sf-Cel3A yielding no improvement. Co-overexpression of the most promising exocytic SNARE components identified in literature for secretory enhancement of the cellulolytic proteins tested (SSO1 for Sf-Cel3A and SNC1 for Te-Cel7A) with the most effective ER-to-Golgi SNARE components identified in this study (SED5 for both Sf-Cel3A and Te-Cel7A) yielded variable results, with Sf-Cel3A improved by 131 % and Te-Cel7A yielding no improvement. Improvements were largely independent of gene dosage as all strains only integrated single additional SNARE gene copies, with episomal variance between the most improved strains shown to be insignificant. This study has added further credence to the notion that SNARE proteins fulfil an essential role within a larger cascade of secretory machinery components that could contribute significantly to future improvements to S. cerevisiae as protein production host.

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Acknowledgments

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

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

    John Henry D. Van Zyl & Willem H. Van Zyl

  2. Department of Biotechnology, University of the Western Cape, Bellville, 7530, South Africa

    Riaan Den Haan

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  1. John Henry D. Van Zyl
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Van Zyl, J.H.D., Den Haan, R. & Van Zyl, W.H. Overexpression of native Saccharomyces cerevisiae ER-to-Golgi SNARE genes increased heterologous cellulase secretion. Appl Microbiol Biotechnol 100, 505–518 (2016). https://doi.org/10.1007/s00253-015-7022-2

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Keywords

  • SNAREs
  • Cellulases
  • Secretion
  • Yeast