Adaptive evolution of electron transfer pathways in <i>Thermoanaerobacterium saccharolyticum</i>

ABSTRACT Thermoanaerobacterium saccharolyticum is an anaerobic, thermophilic bacterium that has been proposed for use in consolidated bioprocessing in coculture with cellulolytic bacteria such as Clostridium thermocellum for ethanol production. Although the mixed acid fermentation of both species has been engineered to produce ethanol as the major fermentation product, the maximum titer produced thus far by C. thermocellum is half that produced by T. saccharolyticum . This has motivated us to understand the mechanistic basis of the robust T. saccharolyticum ethanol pathway, so that key features can be recapitulated in C. thermocellum . Previously, we characterized the individual role of the main genes responsible for electron transfer in the ethanol production of T. saccharolyticum . However, the consequences of the combined loss of function of these genes has not been investigated, nor has the way in which fermentative metabolism adapts to such constraints. Here, we combined knockouts of ferredoxin nicotinamide oxidoreductases ( nfnA and nfnB ) and hydrogenases ( hydA and hfsD ). We showed that these genetic modifications together impair growth and decrease electron transfer from reduced ferredoxin, thereby redirecting flux from the pyruvate ferredoxin oxidoreductase enzyme to the pyruvate formate lyase enzyme. We also performed adaptive evolution of these mutants to rescue their growth and observed a mutation in the alcohol dehydrogenase adhA gene. We determined that this point mutation causes a structural change that impairs the AdhA specificity for the NADPH cofactor and increases NADH-linked activity to restore redox balance. These findings consolidate our understanding of electron transfer pathways in this organism. IMPORTANCE Thermoanaerobacterium saccharolyticum has the potential to be used for the conversion of lignocellulose-derived sugars into bioethanol via consolidated bioprocessing in cocultures with Clostridium thermocellum or via transfer of its ethanol production pathway to the same bacterium. However, attempts to transfer the pathway to increase ethanol titer have not yet been successful. A deeper understanding of how electron transfer pathways operate in this thermophile and how they adapt to metabolic disturbances (such as the absence of metabolic pathways for ferredoxin oxidation, for example) will improve our ability to engineer this organism for increased product formation and improve our ability to transfer its remarkable ethanol production phenotype to other microbes. These, in turn, have potential benefits for sustainable production of fuels and chemicals.