Structural characterization and dynamics of AdhE ultrastructures from <i>Clostridium thermocellum</i> show a containment strategy for toxic intermediates.

Abstract

<i>Clostridium thermocellum</i>, 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. <i>C. thermocellum</i> is 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 using <i>C. thermocellum</i> is still not economically viable. Therefore, a better understanding of its ethanol synthesis pathway is required. The main pathway for ethanol production in <i>C. thermocellum</i> involves the bifunctional aldehyde-alcohol dehydrogenase (AdhE). To better understand the function of the <i>C. thermocellum</i> AdhE, 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 of <i>C. thermocellum</i> AdhE 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.