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Background  Butanol isomers are regarded as more suitable fuel substitutes than bioethanol. n-Butanol is naturally produced by some  Clostridia  species, but due to inherent problems with clostridial fermentations, industrially more relevant organisms have been genetically engineered for n-butanol production. Although the yeast  Saccharomyces cerevisiae  holds significant advantages in terms of scalable industrial fermentation, n-butanol yields and titers obtained so far are only low.    Results  Here we report a thorough analysis and significant improvements of n-butanol production from glucose with yeast via the acetoacetyl-CoA-derived pathway. First, we established an improved n-butanol pathway by testing various isoenzymes of different pathway reactions. This resulted in n-butanol titers around 15 mg/L in synthetic medium after 74 h. As the initial substrate of the n-butanol pathway is acetyl-coenzyme A (acetyl-CoA) and most intermediates are bound to coenzyme A (CoA), we increased CoA synthesis by overexpression of the pantothenate kinase  coaA  gene from  Escherichia coli . Supplementation with pantothenate increased n-butanol production up to 34 mg/L. Additional reduction of ethanol formation by deletion of alcohol dehydrogenase genes  ADH1 - 5  led to n-butanol titers of 71 mg/L. Further expression of a mutant form of an ATP independent acetylating acetaldehyde dehydrogenase, adhE A267T/E568K , converting acetaldehyde into acetyl-CoA, resulted in 95 mg/L n-butanol. In the final strain, the n-butanol pathway genes,  coaA  and  adhE  A267T/E568K , were stably integrated into the yeast genome, thereby deleting another alcohol dehydrogenase gene,  ADH6 , and  GPD2 -encoding glycerol-3-phosphate dehydrogenase. This led to a further decrease in ethanol and glycerol by-product formation and elevated redox power in the form of NADH. With the addition of pantothenate, this strain produced n-butanol up to a titer of 130 ± 20 mg/L and a yield of 0.012 g/g glucose. These are the highest values reported so far for  S. cerevisiae  in synthetic medium via an acetoacetyl-CoA-derived n-butanol pathway.    Conclusions  By gradually increasing substrate supply and redox power in the form of CoA, acetyl-CoA, and NADH, and decreasing ethanol and glycerol formation, we could stepwise increase n-butanol production in  S. cerevisiae . However, still further bottlenecks in the n-butanol pathway must be deciphered and improved for industrially relevant n-butanol production levels.    Electronic supplementary material  The online version of this article (doi:10.1186/s13068-016-0456-7) contains supplementary material, which is available to authorized users.

n-Butanol production in Saccharomyces cerevisiae is limited by the availability of coenzyme A and cytosolic acetyl-CoA