Cofactor engineering in Thermoanaerobacterium aotearoense SCUT27 for maximizing ethanol yield and revealing an enzyme complex with high ferredoxin-NAD+ reductase activity

[Display omitted] •BCD complex has high ferredoxin-NAD+ reductase activity under low redox state.•Bifunctional BCD complex regulates C2/C4 alcohols ratio through redox state.•Ethanol yield of strain Δldh/Δrex/ΔhfsB::BCD was close to the theoretical yield.•Ethanol productivity from lignocellulosic hydrolysates was up to 0.45–0.51 g/L/h. Thermoanaerobacterium aotearoense SCUT27 is a prominent producer of biofuels from lignocellulosic materials. To provide sufficient NAD(P)H for ethanol production, redox-related genes, including lactate dehydrogenase (ldh), redox-sensing transcriptional repressor (rex), and hydrogenase (hfsB), were knocked out. However, the growth of strain PRH (Δldh/Δrex/ΔhfsB) was suppressed due to the intracellular redox state imbalance with the increased NADH concentration. Coincidentally, when the Bcd–EtfAB (BCD) complex was overexpressed, the resulting strain PRH–B3 (Δldh/Δrex/ΔhfsB::BCD) grew rapidly and produced ethanol with a high yield. With lignocellulosic hydrolysates, PRH-BA (Δldh/Δrex/ΔhfsB::BCD::adhE) demonstrated high ethanol productivity and yield, reaching levels of 0.45–0.51 g/L/h and 0.46–0.53 g/g sugars, respectively. The study results shed light on the cofactor balance for cell stability and the high ferredoxin–NAD+ reductase activity of the BCD complex under an intracellular low redox state. They also provide an essential reference for developing strains for improved biofuel production.