The development of biofuels has received much attention due to the global warming and limited resources associated with fossil fuels. Butanol has been identified as a potential option due to its advantages over ethanol, for example higher energy density, compatibility with current infrastructure and its possibility to be blended with gasoline at any ratio. Yeast Saccharomyces cerevisiae can be used as a producer of butanol. However, butanol toxicity to the host limits the yield produced. In this study, four strains of yeast isolated from the habitats of lager beer, ale, wine and baker ́s yeast were grown in YPD media containing isobutanol concentrations of 1.5 %, 2 %, 3 % and 4 %. Growth was measured to determine the most tolerant strain. Gene expression for the genes RPN4, RTG1 and ILV2 was also measured, to determine its involvement in butanol stress. The genes have in previous studies seen to be involved in butanol tolerance or production, and the hypothesis was that they all should be upregulated in response to butanol exposure. It was found that the yeast strain isolated from lager beer was most tolerant to isobutanol concentrations of 2 % and 3 %. It was also found that the gene RPN4 was upregulated in response to isobutanol stress. There was no upregulation of RTG1 or ILV2, which was unexpected. The yeast strain isolated from lager beer and the gene RPN4 is proposed to be investigated further, to be able to engineer a suitable producer of the biofuel butanol.

Today, most transportation fuels are derived from crude oil. However, fossil fuels are limited resources and contribute to climate change, and are therefore not considered as sustainable. Biofuels are highly relevant candidates for replacing fossil fuels and research has gone into butanol as a biofuel. It has a high energy density, is less hygroscopic and can be blended up to 85% with gasoline. The yeast Saccharomyces cerevisiae is considered a good host for bio- butanol production; it produces small amounts of isobutanol naturally through the Ehrlich pathway, is easy to manipulate genetically and can therefore be engineered to produce higher titres of butanol. End-product toxicity, however, is a problem that needs to be solved to make butanol production in S. cerevisiae more effective, since the organism cannot tolerate higher concentrations of butanol than 2%. Four different S. cerevisiae strains were cultivated in 1.5%, 2%, 3% and 4% isobutanol by spot tests and in liquid media to evaluate their tolerance. Gene expression was measured for genes RPN4, RTG1 and ILV2 to examine their up-regulation and relevance in butanol tolerance. S. cerevisiae strain Saflager 34/70 was determined as the most tolerant strain and was able to grow in 2% liquid isobutanol and 3% isobutanol on agar plates. A three-fold up-regulation of RPN4, a transcription factor involved in the regulation of proteasome gene expression, was observed. These results contribute to the progress of genetic engineering of butanol host organisms, which is needed to create a more effective production of butanol as a biofuel.

The global warming on earth has been obvious since the 1950’s. Fossil fuels have a big impact on the observed warming and it is time to replace them with more environmentally friendly fuels. Biobutanol has been proven to be a preferred substitute to fossil fuels. The yeast Saccharomyces cerevisiae is a potential butanol producer. A problem in the biobutanol production is that the product, butanol, is toxic to the producer. In this study four S. cerevisiae strains were investigated for 1- and 2-butanol tolerance with spot tests and growth measurements with different concentrations of 1- and 2-butanol.  One of the four strains, an ale yeast, showed a higher tolerance for 1- and 2-butanol. 2-butanol was overall more tolerated by the yeast. The gene expression for the genes TMC1, LPL1, FLR1 and RPN4 was also investigated at exposure of 3 % 2-butanol. RPN4 is important in the proteasome protein degradation, which is associated with butanol tolerance. TMC1, LPL1 and FLR1 are associated to RPN4, which make them potential genes coupled to butanol tolerance. The genes TMC1 and RPN4 showed an up-regulation when exposed to 3 % 2-butanol. In conclusion, 2-butanol is preferred as a biofuel produced by ale yeast and the ideal genes to use in genetic engineering to achieve a higher butanol tolerance is TMC1 and RPN4. These results contribute to the development of an effective production of biobutanol by S. cerevisiae.