Abstract
BackgroundButanol is a chemical with potential uses as biofuel and solvent, which can be produced by microbial fermentation. However, the end product toxicity is one of the main obstacles for developing the production process irrespective of the choice of production organism. The long-term goal of the present project is to produce 2-butanol in Saccharomyces cerevisiae. Therefore, unraveling the toxicity mechanisms of solvents such as butanol and understanding the mechanisms by which tolerant strains of S. cerevisiae adapt to them would be an important contribution to the development of a bio-based butanol production process.ResultsA butanol tolerant S. cerevisiae was achieved through a series of sequential batch cultures with gradual increase of 2-butanol concentration. The final mutant (JBA-mut) tolerates all different alcohols tested at higher concentrations compared to the wild type (JBA-wt). Proteomics analysis of the two strains grown under mild butanol-stress revealed 46 proteins changing their expression by more than 1.5-fold in JBA-mut, 34 of which were upregulated. Strikingly, 21 out of the 34 upregulated proteins were predicted constituents of mitochondria. Among the non-mitochondrial up-regulated proteins, the minor isoform of Glycerol-3-phosphatase (Gpp2) was the most notable, since it was the only tested protein whose overexpression was found to confer butanol tolerance.ConclusionThe study demonstrates several differences between the butanol tolerant mutant and the wild type. Upregulation of proteins involved in the mitochondrial ATP synthesizing machinery constituents and glycerol biosynthesis seem to be beneficial for a successful adaptation of yeast cells to butanol stress.
Highlights
Butanol is a chemical with potential uses as biofuel and solvent, which can be produced by microbial fermentation
We conclude that the JBA mut strain displays an increased tolerance especially to inhibitory concentrations of higher alcohols and might serve as a suitable model to elucidate the cellular adaptation mechanisms leading to alcohol tolerance
Based on the proteomics data, which are consistent with the physiological characterization, higher mitochondrial activity of the Mutated strain (JBA-mut), together with an increased expression of the glycerol 3-phosphatase isoform glycerol 3phosphatase 2 (Gpp2) and increased expression of some of the stress metabolic enzymes (e.g. Glo1 and Hsp42), seem to be the main characteristics providing the JBA-mut with the observed increase in butanol tolerance
Summary
Butanol is a chemical with potential uses as biofuel and solvent, which can be produced by microbial fermentation. Unraveling the toxicity mechanisms of solvents such as butanol and understanding the mechanisms by which tolerant strains of S. cerevisiae adapt to them would be an important contribution to the development of a bio-based butanol production process. [1] and it has several advantages in comparison with ethanol as a biofuel, such as higher energy density, lower water content and vapor pressure [1,2,3]. It has mostly been produced as a petrochemical but there are biological alternatives available [4,5]. Different alcohol toxicity mechanisms have been described such as accumulation of toxic metabolites [27], changes in membrane fluidity and transport disturbance [19,20,22,23,24,25,26,28,29,30] as well as disorders in translation initiation [31]
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