Abstract
Abstract
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.
Highlights
13
C MFA was conducted for
E. limosum
grown on methanol, glucose, or cofed.
Cultures reached metabolic and isotopic stationarity in the AneVO chemostat.
Flux distributions showed significant contributions of carbon from cysteine reducing agent.
Cofactor analysis reveals cofeeding benefit is not simply additional ATP