Guterl et al. developed an advanced cell-free system that converted pyruvate to isobutanol using four additional enzymes. First, two pyruvate molecules were joined by acetolactate synthase (ALS) to yield acetolactate, which was further converted by ketolacid reductoisomerase (KARI) into the natural dihydroxyisovalerate (DHAD) substrate. DHAD then catalyzed the conversion of dihydroxyisovalerate into 2-ketoisovalerate. Then, 2-ketoacid decarboxylase (KDC) and alcohol dehydrogenase (ADH) produced isobutanol via isobutyraldehyde. The efficiency of this system was quite high as it converted 19.1 mM KU-60019 into 10.3 mM isobutanol within 23 h corresponding to a molar yield of 53% . In a more recent study, Krutsakorn et al. reported in vitro production of bio-butanol from glucose. They developed an artificial, cofactor balanced, oxygen-sensitive metabolic pathway that consisted of 16 thermotolerant enzymes. The pathway was divided into seven parts, in which each of NAD(H)-dependent enzymes was assigned to be the last step and the fluxes through each part were spectrophotometrically determined. Under optimized conditions, that metabolic pathway successfully produced bio-butanol at a rate of 8.2 μmol L−1 min−1 with a molar yield of 82% .