Fig. 1.

Isobutanol biosynthesis pathway in yeast S. cerevisiae. ALAC: 2-acetolactate; DIV: 2,3-dihydroxyisovalerate; KIV: 2-ketoisovalerate. Glucose is converted to two molecules of pyruvate in glycolysis, generating two molecules of NADH. In the native pathway, after transport into mitochondria, two molecules of pyruvate are condensed to one molecule of 2-acetolactate (ALAC) by the Ilv2^Ilv6 complex. Reduction and isomerization of ALAC to 2,3-dihydroxyisovalerate (DIV) by Ilv5 requires redox cofactor NADPH. DIV is dehydrated to 2-ketoisovalerate (KIV) by Ilv3. KIV is either directly released into the cytosol or converted to valine in mitochondria by Bat1. After export into the cytosol, valine is degraded via the Ehrlich pathway to isobutanol, starting with a transamination to KIV by Bat2. KIV is decarboxylated to isobutyraldehyde by Pdc1, 5 and Aro10. Finally, isobutyraldehyde is reduced to isobutanol, generating one molecule of NAD(P)⁺. By transformation of the episomal 2µ-plasmid IsoV100, we overexpressed Ilv2, Ilv5 and Ilv3 in the cytosol. Simultaneous deletion of mitochondrial Ilv2 resulted in a cytosolic isobutanol pathway mixed with part of a mitochondrial pathway