¹³ C-Metabolic Flux Analysis of Substrate Co-Metabolism in the Methylotrophic Acetogen Eubacterium limosum

Acetogens are promising microbial catalysts for the sustainable production of fuels and chemicals, metabolizing a range of renewable single-carbon (C1) compounds with high energetic efficiency. The major challenge is their energy-limited anaerobic lifestyle, which results in slow growth and a limited product spectrum on individual C1 substrates. We previously showed in the acetogen Eubacterium limosum that co-metabolism of glucose-methanol or CO-methanol mixtures results in substantial improvements in growth and acetate productivity. To investigate the underlying mechanism(s), here we conducted ¹³ C metabolic flux analysis ( ¹³ C-MFA) of E. limosum grown on a glucose-methanol mixture, as well as in single-substrate controls. The results suggest that the main benefit of glucose cofeeding is not provision of additional ATP, but rather a reduction of flux through the ferredoxin-dependent and kinetically challenging reactions of the Wood-Ljungdahl pathway. Subsequent RNAseq analysis showed no significant differences in the expression level of genes involved in methanol metabolism between cofeeding and methanol-only controls, suggesting this reduction of flux is controlled post-transcriptionally. Most surprisingly, we found that under all growth conditions, flux through pyruvate:ferredoxin oxidoreductase (PFOR) was in the oxidative direction, with cysteine (added as a reducing agent) providing all required gluconeogenic carbon flux under CO ₂ -fixing conditions. This work provides critical insights into the metabolic network of an emerging model acetogen that will facilitate future metabolic engineering efforts to channel flux to value-added products, and highlights the under-appreciated role of cysteine as a carbon source in acetogenic fermentations.