Abstract
Backgroundn-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.ResultsBy 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.ConclusionsThis 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.
Highlights
Four-carbon alcohols, including n-butanol, produced from renewable biomass, are promising alternatives to ethanol as biofuels: they are less volatile, less hygroscopic and less corrosive than ethanol, and have higher energy content
The aim of the present study was to identify the metabolic functions associated with n-butanol tolerance in S. cerevisiae
Experimental design and evaluation of n-butanol tolerance of S. cerevisiae strains BY4741 and CEN.PK113-7D To assess the impact of n-butanol on growth rate and biomass yield, S. cerevisiae strains BY4741 and CEN
Summary
Four-carbon alcohols, including n-butanol, produced from renewable biomass, are promising alternatives to ethanol as biofuels: they are less volatile, less hygroscopic and less corrosive than ethanol, and have higher energy content. They can be added to gasoline as a fuel additive or even replace it completely without n-Butanol can be naturally produced by some Clostridium species through the so called ABE fermentation, a process that yields acetone, butanol and ethanol [3]. ‘Biobutanol’ has been historically produced through this process [4], it exhibits several drawbacks that limit its current application for large scale production. Attempts have been made to engineer Clostridium in order to overcome these limitations [3,5,6], its complex physiology and lack of efficient genetic tools make it difficult to engineer [5]
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