Structural characterization and dynamics of AdhE ultrastructures fromClostridium thermocellum:A containment strategy for toxic intermediates

Clostridium thermocellum, a cellulolytic thermophilic anaerobe, is considered by many to be a prime candidate for the realization of consolidated bioprocessing (CBP) and is known as an industry standard for biofuel production.C. thermocellumis among the best biomass degraders identified to date in nature and produces ethanol as one of its main products. Many studies have helped increase ethanol titers in this microbe, however ethanol production usingC. thermocellumis still not economically viable. Therefore, a better understanding of its ethanol synthesis pathway is required. The main pathway for ethanol production inC. thermocelluminvolves the bifunctional aldehyde-alcohol dehydrogenase (AdhE). To better understand the function of theC. thermocellumAdhE, we used cryo-electron microscopy (cryo-EM) to obtain a 3.28 Å structure of the AdhE complex. This high-resolution structure, in combination with molecular dynamics simulations, provides insight into the substrate channeling of the toxic intermediate acetaldehyde, indicates the potential role ofC. thermocellumAdhE to regulate activity and cofactor pools, and establishes a basis for future engineering studies. The containment strategy found in this enzyme offers a template that could be replicated in other systems where toxic intermediates need to be sequestered to increase the production of valuable biochemicals.