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

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