Highly selective ethanol production via the thermophilic bioconversion of sugars and syngas using metabolically engineered Neomoorella thermoacetica

Abstract

Ethanol production from biomass-derived and gaseous substrates (CO, H 2 and CO 2 ) is essential for achieving a carbon-neutral society. The thermophilic acetogen Neomoorella thermoacetica offers notable industrial advantages for converting such diverse feedstocks; however, its physiology and metabolic engineering strategies remain insufficiently understood. Using stoichiometric analysis, we evaluated the metabolic pathways for ethanol production from sugars and gaseous substrates, with particular attention to redox balance and ATP conservation. This analysis suggested that introducing an NADPH-dependent route for reducing acetyl-CoA to ethanol could maintain redox-balanced metabolism under conditions that support sufficient energy generation. Consistent with this prediction, disruption of the major acetate-forming pathway, together with the introduction of heterologous NADPH-dependent reductases, enabled high-selectivity ethanol production (> 90%) from both sugars and syngas. Inactivation of the acetate pathway did not result in growth arrest. In contrast, little to no growth occurred when H 2 was supplied as the sole energy source. However, growth and ethanol production were restored by the addition of an external electron acceptor that supported anaerobic respiration. This result was consistent with the predicted energy deficit. The strain constructed using a redox-guided metabolic design attained > 90% ethanol selectivity from both sugar- and syngas-based substrates. These findings demonstrate that stoichiometric analysis of redox balance and ATP conservation can provide useful guidance for engineering ethanol-producing thermophilic acetogens.