title: Increased isobutanol production in Saccharomyces cerevisiae by overexpression of genes in valine metabolism.
abstract: 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.
authors.affiliation: Center for Microbial Biotechnology, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 223, DK-2800 Kgs, Lyngby, Denmark. xc@bio.dtu.dk.
authors.forename: Xiao
authors.initials: X
authors.lastname: Chen
authors.forename: Kristian F
authors.initials: KF
authors.lastname: Nielsen
authors.forename: Irina
authors.initials: I
authors.lastname: Borodina
authors.forename: Morten C
authors.initials: MC
authors.lastname: Kielland-Brandt
authors.forename: Kaisa
authors.initials: K
authors.lastname: Karhumaa