AUTHOR=Weitz Sandra , Hermann Maria , Linder Sonja , Bengelsdorf Frank R. , Takors Ralf , Dürre Peter 

TITLE=Isobutanol Production by Autotrophic Acetogenic Bacteria

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=9

YEAR=2021

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2021.657253

DOI=10.3389/fbioe.2021.657253

ISSN=2296-4185

ABSTRACT=<p>Two different isobutanol synthesis pathways were cloned into and expressed in the two model acetogenic bacteria <italic>Acetobacterium woodii</italic> and <italic>Clostridium ljungdahlii</italic>. <italic>A. woodii</italic> is specialized on using CO<sub>2</sub> + H<sub>2</sub> gas mixtures for growth and depends on sodium ions for ATP generation by a respective ATPase and Rnf system. On the other hand, <italic>C. ljungdahlii</italic> grows well on syngas (CO + H<sub>2</sub> + CO<sub>2</sub> mixture) and depends on protons for energy conservation. The first pathway consisted of ketoisovalerate ferredoxin oxidoreductase (Kor) from <italic>Clostridium thermocellum</italic> and bifunctional aldehyde/alcohol dehydrogenase (AdhE2) from <italic>C. acetobutylicum</italic>. Three different <italic>kor</italic> gene clusters are annotated in <italic>C. thermocellum</italic> and were all tested. Only in recombinant <italic>A. woodii</italic> strains, traces of isobutanol could be detected. Additional feeding of ketoisovalerate increased isobutanol production to 2.9 mM under heterotrophic conditions using <italic>kor</italic>3 and to 1.8 mM under autotrophic conditions using <italic>kor</italic>2. In <italic>C. ljungdahlii</italic>, isobutanol could only be detected upon additional ketoisovalerate feeding under autotrophic conditions. <italic>kor</italic>3 proved to be the best suited gene cluster. The second pathway consisted of ketoisovalerate decarboxylase from <italic>Lactococcus lactis</italic> and alcohol dehydrogenase from <italic>Corynebacterium glutamicum</italic>. For increasing the carbon flux to ketoisovalerate, genes encoding ketol-acid reductoisomerase, dihydroxy-acid dehydratase, and acetolactate synthase from <italic>C. ljungdahlii</italic> were subcloned downstream of <italic>adhA</italic>. Under heterotrophic conditions, <italic>A. woodii</italic> produced 0.2 mM isobutanol and 0.4 mM upon additional ketoisovalerate feeding. Under autotrophic conditions, no isobutanol formation could be detected. Only upon additional ketoisovalerate feeding, recombinant <italic>A. woodii</italic> produced 1.5 mM isobutanol. With <italic>C. ljungdahlii</italic>, no isobutanol was formed under heterotrophic conditions and only 0.1 mM under autotrophic conditions. Additional feeding of ketoisovalerate increased these values to 1.5 mM and 0.6 mM, respectively. A further increase to 2.4 mM and 1 mM, respectively, could be achieved upon inactivation of the <italic>ilvE</italic> gene in the recombinant <italic>C. ljungdahlii</italic> strain. Engineering the coenzyme specificity of IlvC of <italic>C. ljungdahlii</italic> from NADPH to NADH did not result in improved isobutanol production.</p>