AUTHOR=Hermann Maria , Teleki Attila , Weitz Sandra , Niess Alexander , Freund Andreas , Bengelsdorf Frank Robert , Dürre Peter , Takors Ralf 

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

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=9

YEAR=2021

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2021.647853

DOI=10.3389/fbioe.2021.647853

ISSN=2296-4185

ABSTRACT=<p><italic>Clostridium ljungdahlii</italic> (<italic>C. ljungdahlii</italic>, CLJU) is natively endowed producing acetic acid, 2,3-butandiol, and ethanol consuming gas mixtures of CO<sub>2</sub>, CO, and H<sub>2</sub> (syngas). Here, we present the syngas-based isobutanol formation using <italic>C. ljungdahlii</italic> 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<sup>–1</sup>. Additional blocking of valine synthesis in CLJU[KAIA]:<italic>ilvE</italic> increased isobutanol production by factor 6.5 finally reaching 0.13 g L<sup>–1</sup>. Future metabolic engineering should focus on debottlenecking NADPH availability, whereas NADH supply is already equilibrated in the current generation of strains.</p>