Identifying and Engineering Bottlenecks of Autotrophic Isobutanol Formation in Recombinant C. ljungdahlii by Systemic Analysis

Clostridium ljungdahlii ( C. , CLJU) is natively endowed producing acetic acid, 2,3-butandiol, and ethanol consuming gas mixtures of CO 2 CO, H (syngas). Here, we present the syngas-based isobutanol formation using harboring recombinant amplification “Ehrlich” pathway that converts intracellular KIV to isobutanol. Autotrophic production was studied analyzing two different strains in 3-L gassed stirred bioreactors. Physiological characterization thoroughly applied together with metabolic profiling flux balance analysis. Thereof, pyruvate supply were identified as key “bottlenecking” precursors limiting preliminary CLJU[KAIA] 0.02 g L –1 . Additional blocking valine synthesis CLJU[KAIA]: ilvE increased by factor 6.5 finally reaching 0.13 Future engineering should focus on debottlenecking NADPH availability, whereas NADH already equilibrated current generation strains.