Fig. 6.

Gevo’s isobutanol production strategy in the mitochondria [106]. a All enzymes involved in isobutanol synthesis were localized in the mitochondria; KIVD and ADH being in cytosol were expressed in mitochondria with signal sequence. NADPH-dependent enzymes were engineered to an NADH-dependent form, and then NADH was supplied by using an NADH shuttle concept. Acetaldehyde and ethanol produced by fermentation were transported across membranes, and alcohol dehydrogenase in the mitochondria (encoded by ADH3) provided NADH by conversion of ethanol to acetaldehyde. b Isobutyraldehyde was transferred to the cytosol from mitochondria where it is converted to isobutanol under consumption of NADH generated by glycolysis in the cytosol to make more precise cofactor balance