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Genome-scale analyses of butanol tolerance in Saccharomyces cerevisiae reveal an essential role of protein degradation

Daniel González-Ramos12, Marcel van den Broek12, Antonius JA van Maris12, Jack T Pronk123 and Jean-Marc G Daran123*

Author Affiliations

1 Department of Biotechnology, Delft University of Technology, Julianalaan 67, Delft 2628 BC, The Netherlands

2 Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 5057, Delft 2600 GA, The Netherlands

3 Platform for Green Synthetic Biology, P.O. Box 5057, Delft 2600 GA, The Netherlands

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Biotechnology for Biofuels 2013, 6:48  doi:10.1186/1754-6834-6-48

Published: 3 April 2013



n-Butanol and isobutanol produced from biomass-derived sugars are promising renewable transport fuels and solvents. Saccharomyces cerevisiae has been engineered for butanol production, but its high butanol sensitivity poses an upper limit to product titers that can be reached by further pathway engineering. A better understanding of the molecular basis of butanol stress and tolerance of S. cerevisiae is important for achieving improved tolerance.


By combining a screening of the haploid S. cerevisiae knock-out library, gene overexpression, and genome analysis of evolutionary engineered n-butanol-tolerant strains, we established that protein degradation plays an essential role in tolerance. Strains deleted in genes involved in the ubiquitin-proteasome system and in vacuolar degradation of damaged proteins showed hypersensitivity to n-butanol. Overexpression of YLR224W, encoding the subunit responsible for the recognition of damaged proteins of an ubiquitin ligase complex, resulted in a strain with a higher n-butanol tolerance. Two independently evolved n-butanol-tolerant strains carried different mutations in both RPN4 and RTG1, which encode transcription factors involved in the expression of proteasome and peroxisomal genes, respectively. Introduction of these mutated alleles in the reference strain increased butanol tolerance, confirming their relevance in the higher tolerance phenotype. The evolved strains, in addition to n-butanol, were also more tolerant to 2-butanol, isobutanol and 1-propanol, indicating a common molecular basis for sensitivity and tolerance to C3 and C4 alcohols.


This study shows that maintenance of protein integrity plays an essential role in butanol tolerance and demonstrates new promising targets to engineer S. cerevisiae for improved tolerance.

Saccharomyces cerevisiae; Butanol tolerance; Evolutionary engineering; Deletion collection screening; Whole genome sequencing; Proteasome; Multivesicular bodies