Adenosine Triphosphate and Carbon Efficient Route to Second Generation Biofuel Isopentanol

Climate change necessitates the development of CO₂ neutral or negative routes to chemicals currently produced from fossil carbon. In this paper we demonstrate a pathway from the renewable resource glucose to next generation biofuel isopentanol by pairing the isovaleryl-CoA biosynthesis pathway from <i>Myxococcus xanthus</i> and a butyryl-CoA reductase from <i>Clostridium acetobutylicum</i>. The best plasmid and <i>Escherichia coli</i> strain combination makes 80.50 ± 8.08 (SD) mg/L of isopentanol after 36 h under microaerobic conditions with an oleyl alcohol overlay. In addition, the system also shows a strong preference for isopentanol production over prenol in microaerobic conditions. Finally, the pathway requires zero adenosine triphosphate and can be paired theoretically with nonoxidative glycolysis, the combination being redox balanced from glucose thus avoiding unnecessary carbon loss as CO₂. These pathway properties make the isovaleryl-CoA pathway an attractive isopentanol production route for further optimization.