Thermodynamic metabolic modeling of growth and bioproduction potential of the acetogen Acetobacterium woodii with and without redox cofactor swaps

Acetogenic bacteria such as Acetobacterium woodii use the Wood-Ljungdahl pathway to convert H 2 /CO 2 and other C1 substrates into acetate and couple it via chemiosmotic energy conservation with ATP synthesis. To coordinate the associated electron flows under tight thermodynamic constraints, acetogens use not only NAD(H) and NAD(P)H but also ferredoxin as a third major redox cofactor. In this work, we systematically explore how cofactor specificity in redox reactions, including potential cofactor swaps, may influence growth and bioproduction. We initially reconstructed and validated a large-scale constraint-based metabolic model of A. woodii equipped with standard Gibbs-free-energy values and metabolite concentration bounds. We then analyzed the effects of swapping redox cofactors in the model. This analysis revealed that, in theory, suitable cofactor swaps could increase the growth rate by a factor of 5 compared to the wild type, but only under very high H 2 and CO 2 concentrations and with tight ranges for the redox states of the cofactors. More realistic solutions with higher driving forces and broader concentration ranges would still enable an up to 2.5-fold increase in growth rate and are based on two key swaps: (a) replacement of the hydrogen-dependent CO 2 reductase (HDCR) by a NADPH-dependent formate dehydrogenase and (b) substitution of NAD + by NADP + in the bifurcating hydrogenase. These variants, which were frequently favored by the algorithm in different scenarios and are used by other acetogens, increase the amount of reduced ferredoxin available for subsequent ATP generation. Unexpectedly, our analysis further revealed that the use of ferredoxin and NADH alone (in combination with suitable cofactor swaps) could lead to similar growth rates and driving forces as for the wild type. We discuss possible reasons why these solutions may not have been selected by evolution. In particular, we show that the native redox cofactor specificities of A. woodii facilitate near-maximal driving forces under a wide range of H 2 and CO 2 concentrations. Finally, we evaluated production of 15 native and heterologous chemicals from four C1-substrate regimes. This analysis reveals that targeted cofactor engineering can in many (but not all) cases (i) enable growth-coupled synthesis of a target chemical if it is infeasible in the native A. woodii strain or (ii) enhance the thermodynamic driving force of product synthesis. Overall, this work provides a valuable resource and a generalizable thermodynamics-based framework for evaluating redox engineering strategies in acetogens and other energy-limited microorganisms.