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Background  Isobutanol can be a better biofuel than ethanol due to its higher energy density and lower hygroscopicity. Furthermore, the branched-chain structure of isobutanol gives a higher octane number than the isomeric  n -butanol.  Saccharomyces cerevisiae  was chosen as the production host because of its relative tolerance to alcohols, robustness in industrial fermentations, and the possibility for future combination of isobutanol production with fermentation of lignocellulosic materials.    Results  The yield of isobutanol was improved from 0.16 to 0.97 mg per g glucose by simultaneous overexpression of biosynthetic genes  ILV2, ILV3 , and  ILV5  in valine metabolism in anaerobic fermentation of glucose in mineral medium in  S. cerevisiae . Isobutanol yield was further improved by twofold by the additional overexpression of  BAT2 , encoding the cytoplasmic branched-chain amino-acid aminotransferase. Overexpression of  ILV6 , encoding the regulatory subunit of Ilv2, in the  ILV2 ILV3 ILV5  overexpression strain decreased isobutanol production yield by threefold. In aerobic cultivations in shake flasks in mineral medium, the isobutanol yield of the  ILV2 ILV3 ILV5  overexpression strain and the reference strain were 3.86 and 0.28 mg per g glucose, respectively. They increased to 4.12 and 2.4 mg per g glucose in yeast extract/peptone/dextrose (YPD) complex medium under aerobic conditions, respectively.    Conclusions  Overexpression of genes  ILV2, ILV3, ILV5 , and  BAT2  in valine metabolism led to an increase in isobutanol production in  S. cerevisiae . Additional overexpression of  ILV6  in the  ILV2 ILV3 ILV5  overexpression strain had a negative effect, presumably by increasing the sensitivity of Ilv2 to valine inhibition, thus weakening the positive impact of overexpression of  ILV2, ILV3 , and  ILV5  on isobutanol production. Aerobic cultivations of the  ILV2 ILV3 ILV5  overexpression strain and the reference strain showed that supplying amino acids in cultivation media gave a substantial improvement in isobutanol production for the reference strain, but not for the  ILV2 ILV3 ILV5  overexpression strain. This result implies that other constraints besides the enzyme activities for the supply of 2-ketoisovalerate may become bottlenecks for isobutanol production after  ILV2, ILV3 , and  ILV5  have been overexpressed, which most probably includes the valine inhibition to Ilv2.

Increased isobutanol production in Saccharomyces cerevisiae by overexpression of genes in valine metabolism