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Expression of a library of fungal β-glucosidases in Saccharomyces cerevisiae for the development of a biomass fermenting strain

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Abstract

Converting cellulosic biomass to ethanol involves the enzymatic hydrolysis of cellulose and the fermentation of the resulting glucose. The yeast Saccharomyces cerevisiae is naturally ethanologenic, but lacks the enzymes necessary to degrade cellulose to glucose. Towards the goal of engineering S. cerevisiae for hydrolysis of and ethanol production from cellulose, 35 fungal β-glucosidases (BGL) from the BGL1 and BGL5 families were screened for their ability to be functionally expressed and displayed on the cell surface. Activity assays revealed that the BGL families had different substrate specificities, with only the BGL1s displaying activity on their natural substrate, cellobiose. However, growth on cellobiose showed no correlation between the specific growth rates, the final cell titer, and the level of BGL1 activity that was expressed. One of the BGLs that expressed the highest levels of cellobiase activity, Aspergillus niger BGL1 (Anig-Bgl101), was then used for further studies directed at developing an efficient cellobiose-fermenting strain. Expressing Anig-Bgl101 from a plasmid yielded higher ethanol levels when secreted into the medium rather than anchored to the cell surface. In contrast, ethanol yields from anchored and secreted Anig-Bgl101 were comparable when integrated on the chromosome. Flow cytometry analysis revealed that chromosomal integration of Anig-Bgl101 resulted in a higher percentage of the cell population that displayed the enzyme but with overall lower expression levels.

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Acknowledgements

We would like to thank Kane LaRue for his useful insights into the manuscript. We are grateful to Danièle Gagné from the Institute for Research in Immunology and Cancer of the Université de Montréal for helping us with the flow cytometry experiments. This work was supported by research grants to V.J.J.M. and R.S. from the Natural Sciences and Engineering Research Council of Canada grant number NETGP 350246-07, the Fonds Québécois de la Recherche sur la Nature et les Technologies grant number 125961, Agriculture and Agri-Food Canada, grant number ABIP000159, and a Canada Research Chair to V.J.J.M.

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  1. Caroline Wilde

    Present address: Lallemand Inc, 6100 Royalmount Avenue, Montréal, Québec, H4P 2R2, Canada

Authors and Affiliations

  1. Department of Biology, Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke West, Montréal, Québec, H4B 1R6, Canada

    Caroline Wilde, Nicholas D. Gold, Nancy Bawa, José Humberto M. Tambor, Lina Mougharbel, Reginald Storms & Vincent J. J. Martin

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Correspondence to Vincent J. J. Martin.

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Caroline Wilde and Nicholas D. Gold shared first authorship.

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Wilde, C., Gold, N.D., Bawa, N. et al. Expression of a library of fungal β-glucosidases in Saccharomyces cerevisiae for the development of a biomass fermenting strain. Appl Microbiol Biotechnol 95, 647–659 (2012). https://doi.org/10.1007/s00253-011-3788-z

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