Abstract
The endoglucanase I and II genes (egI or Cel7B and egII or Cel5A) of Trichoderma reesei QM6a were successfully cloned and expressed in Saccharomyces cerevisiae under the transcriptional control of the yeast ENO1 promoter and terminator sequences. Random mutagenesis of the egI-bearing plasmid resulted in a twofold increase in extracellular EGI activity. Both endoglucanase genes were co-expressed with the synthetic, codon-optimised cellobiohydrolase gene (s-cbhI) from T. reesei as well as the β-glucosidase gene (bgl1) from Saccharomycopsis fibuligera in S. cerevisiae. Extracellular endoglucanase activity was lower when co-expressed with s-cbhI or bgl1. Recombinant strains were able to hydrolyse phosphoric acid swollen cellulose, generating mainly cellotriose, cellobiose and glucose. Cellobiose accumulated, suggesting the β-glucosidase activity to be the rate-limiting factor. As a consequence, the recombinant strains were unable to produce enough glucose for growth on amorphous cellulose. The results of this study provide insight into further optimisation of recombinantly expressed cellulase combinations for saccharification and fermentation of cellulose to ethanol.
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Acknowledgements
The authors would like to thank the National Research Foundation (NRF) for financial support. We would also like to express gratitude to Nico de Villiers from Omic Solutions for assistance in the real-time PCR experiments and Anscha Zietsman and Dr Riaan den Haan for supplying plasmids pAZ1 and YEpENO-sCBHI, as well as strain Saccharomyces cerevisiae Y294[BGL1].
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du Plessis, L., Rose, S.H. & van Zyl, W.H. Exploring improved endoglucanase expression in Saccharomyces cerevisiae strains. Appl Microbiol Biotechnol 86, 1503–1511 (2010). https://doi.org/10.1007/s00253-009-2403-z
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DOI: https://doi.org/10.1007/s00253-009-2403-z