Fig. 1

Engineering the mitochondrial isobutanol biosynthetic pathway in a xylose-utilizing strain of S. cerevisiae. Two different heterologous xylose utilization pathways have been used in yeast to convert xylose to xylulose: the isomerase pathway (used in this study), which uses xylose isomerase (XI); and the oxidoreductase pathway, consisting of xylose reductase (XR) and xylitol dehydrogenase (XHD). In both pathways, xylulose is subsequently phosphorylated to xylulose-5-phosphate (X5P) by xylulokinase (XK), and then channeled to glycolysis through the non-oxidative pentose phosphate pathway (PPP). a Mitochondrial isobutanol biosynthesis involves an upstream pathway that consists of the ILV genes including acetolactate synthase (ILV2), ketol-acid reductoisomerase (ILV5), and dihydroxyacid dehydratase (ILV3), as well as a downstream pathway that consists of mitochondrially targeted α-ketoacid decarboxylases (KDC) and alcohol dehydrogenases (ADH). b There is a considerable overlap between the upstream pathways for isobutanol and 2-methyl-1-butanol production, except the isoleucine precursor α-keto-β-methylvalerate (α-KMV) is synthesized by Ilv2p from one pyruvate and one α-ketobutyrate produced from threonine deamination catalyzed by threonine deaminase (ILV1); from there, the downstream Ehrlich degradation pathway for both branched-chain alcohols is identical. Genes overexpressed in our strains are shown in blue, while genes deleted are shown in red. ALD6: cytosolic aldehyde dehydrogenase, BAT1: mitochondrial branched-chain amino acid aminotransferase, BAT2: cytosolic branched-chain amino acid aminotransferase, PDCs: pyruvate decarboxylases, PHO13: alkaline phosphatase, α-KIV: α-ketoisovalerate, IBAL: isobutyraldehyde, IBU: Isobutyrate, α-KMV: α-keto-β-methylvalerate, 2MBAL: 2-methyl-1-butyraldehyde, 2MBU: 2-methyl-1-butyrate