13C-Metabolic Flux Analysis of Substrate Co-Metabolism in the Methylotrophic Acetogen Eubacterium limosum.
- 1Northeastern University
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 13C metabolic flux analysis (13C-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 CO2-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.
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BibTeX
@misc{hoyt202613cmetabolic,
title = {{13C-Metabolic Flux Analysis of Substrate Co-Metabolism in the Methylotrophic Acetogen Eubacterium limosum.}},
author = {K. O. Hoyt and W. Gasparrini and J. A. Hayes and B. M. Woolston},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.12.758492},
url = {https://www.biorxiv.org/content/10.64898/2026.10.12.758492v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - 13C-Metabolic Flux Analysis of Substrate Co-Metabolism in the Methylotrophic Acetogen Eubacterium limosum.
AU - K. O. Hoyt
AU - W. Gasparrini
AU - J. A. Hayes
AU - B. M. Woolston
PY - 2026
DA - 2026/10/12
PB - bioRxiv
DO - 10.64898/2026.10.12.758492
UR - https://www.biorxiv.org/content/10.64898/2026.10.12.758492v1
AB - 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 13C metabolic flux analysis (13C-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 CO2-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.
ER -