Biobutanol represents a second generation biofuel, which can be producedfrom renewable resources by microorganisms. A Saccharomyces cerevisiae strainbearing the five butanol synthetic genes (hbd, adhe2, crt, ccr and ERG10) wasconstructed, where the hbd, adhe2, crt and ccr genes are derived from Clostridiumbeijerinckii, while ERG10 is a yeast gene. The genes were transformed individually onsingle cassettes, which integrated into specific chromosomal sites. The single integrantstrains were back‐crossed to create a strain bearing all five butanol synthetic genes. The butanol synthetic enzymes appeared to be highly expressed in the cytosol,however, very little butanol was obtained (< 10 ppm). Therefore, additional geneticmanipulations were made with a view to restoring any redox imbalance channellingthe carbon flux toward the butanol pathway. Deletion of the ADH1 gene in strains withthe butanol pathway improved production to ~250 ppm (203 mg/L) butanol. Furtherimprovement to 360 ppm (292 mg/L) was gained by overexpressing the ALD6 and ACS2genes, that are involved in synthesis of acetyl‐CoA; the precursor for butanolbiosynthesis. However, the replacement of ALD6 with ALD2, which produces NADHinstead of NADPH, didn’t improve butanol yields. In addition, no significantimprovement of butanol yield was obtained when dehydrogenase enzymes from theglycerol biosynthetic pathway were deleted. An initial assessment of the bestconditions for butanol production were semi‐anaerobic growth at 30°C in 2% glucosewith a starting OD600 of 0.1.In this project, another key question was addressed: does the sensitivity of cellsto short chain alcohols like butanol affect butanol production? Previous work in theAshe lab has identified specific point mutations in the translation initiation factor,eIF2B, which generate resistance or sensitive phenotypes to exogenously addedbutanol. Here a comparison of butanol production in sensitive and resistantbackgrounds showed that the butanol yield was 1.5‐2 fold higher in a butanol resistantstrain compared to the sensitive mutant. Generating a ‘super’ butanol resistant strainbearing a GCD2‐S131A mutation in eIF2B promoted a higher butanol yield per cell. However, another consequence of this mutation was reduced growth. So thecombination of these effects meant that the overall butanol concentration in mediawas similar to the control. Overall this work highlights that S. cerevisiae can producebutanol but that further optimisation both at the level of the strain and processengineering would be necessary before this would be of interest to the commercialsector.