Metabolic engineering of Clostridium acetobutylicum ATCC 824 for the high-yield production of a biofuel composed of an isopropanol/butanol/ethanol mixture

• Metabolic engineering of C. acetobutylicum for stable biofuel production with low amount of by-products.
• Construction of synthetic isopropanol operon for early isopropanol production to minimize acetate accumulation.
• Final [biofuel] of 21 g/l, a yield of 0.34 g/g glucose, a productivity of 0.8 g/l h after culture condition optimization.
• The isopropanol flux was identified to be limited by the [acetate]int and the high Km of CoA-transferase for acetate.

Clostridium acetobutylicum was metabolically engineered to produce a biofuel consisting of an isopropanol/butanol/ethanol mixture. For this purpose, different synthetic isopropanol operons were constructed and introduced on plasmids in a butyrate minus mutant strain (C. acetobutylicum ATCC 824 Δcac15ΔuppΔbuk). The best strain expressing the isopropanol operon from the thl promoter was selected from batch experiments at pH 5. By further optimizing the pH of the culture, a biofuel mixture with almost no by-products was produced at a titer, a yield and productivity never reached before, opening the opportunities to develop an industrial process for alternative biofuels with Clostridial species. Furthermore, by performing in vivo and in vitro flux analysis of the synthetic isopropanol pathway, this flux was identified to be limited by the [acetate]int and the high Km of CoA-transferase for acetate. Decreasing the Km of this enzyme using a protein engineering approach would be a good target for improving isopropanol production and avoiding acetate accumulation in the culture medium.