Dataset Information

Redox controls metabolic robustness in the gas-fermenting acetogen Clostridium autoethanogenum

ABSTRACT: Living biological systems display a fascinating ability to self-organise their metabolism. This ability ultimately determines metabolic robustness that is fundamental to control cellular behaviour. However, fluctuations in metabolism can affect cellular homeostasis through transient oscillations. For example, yeast cultures exhibit rhythmic oscillatory behaviour in high-cell density continuous cultures. Oscillatory behaviour provides a unique opportunity for quantitating the robustness of metabolism, as cells respond to changes by inherently compromising metabolic efficiency. Here, we quantify the limits of metabolic robustness in self-oscillating autotrophic continuous cultures of the gas-fermenting acetogen Clostridium autoethanogenum. On-line gas analysis and high-resolution temporal metabolomics showed oscillations in gas uptake rates and extracellular by-products synchronised with biomass levels. Loss of H2 uptake makes CO the sole carbon and energy source until cells recover uptake of H2 in synchrony with increasing biomass levels. Intriguingly, oscillations are not linked to translational control as no differences were observed in protein expression during oscillations. However, intracellular metabolomics analysis revealed decreasing levels of redox ratios in perfect synchrony with the cycles. Therefore, we developed a thermodynamic metabolic flux analysis (tMFA) model to investigate if regulation in acetogens is controlled at the thermodynamic level. The data shows that the feasible range for the thermodynamic driving force of the Nfn transhydrogenase complex (i.e. NADH/NAD+×NADP+/NADPH) closely matched the experimentally observed range. The data indicate that metabolic oscillations in gas fermentation acetogens are controlled at the thermodynamic level. Our work suggests thermodynamic control of metabolism, potentially contributing to metabolic efficiency and working as a mean of energy conservation.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Clostridium Autoethanogenum

TISSUE(S): Prokaryotic Cell, Cell Culture

SUBMITTER: Kaspar Valgepea

LAB HEAD: Esteban Marcellin

PROVIDER: PXD016381 | Pride | 2020-05-18

REPOSITORIES: Pride

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Redox controls metabolic robustness in the gas-fermenting acetogen <i>Clostridium autoethanogenum</i>.

Mahamkali Vishnuvardhan V Valgepea Kaspar K de Souza Pinto Lemgruber Renato R Plan Manuel M Tappel Ryan R Köpke Michael M Simpson Séan Dennis SD Nielsen Lars Keld LK Marcellin Esteban E

Proceedings of the National Academy of Sciences of the United States of America 20200529 23

Living biological systems display a fascinating ability to self-organize their metabolism. This ability ultimately determines the metabolic robustness that is fundamental to controlling cellular behavior. However, fluctuations in metabolism can affect cellular homeostasis through transient oscillations. For example, yeast cultures exhibit rhythmic oscillatory behavior in high cell-density continuous cultures. Oscillatory behavior provides a unique opportunity for quantitating the robustness of m ...[more]

PMID: 32471945

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Project description:Background: The global demand for affordable carbon has never been stronger, and there is an imperative in many industrial processes to use waste streams to make products. Gas-fermenting acetogens offer a potential solution and several commercial gas fermentation plants are currently under construction. As energy limits acetogen metabolism, supply of H2 should diminish substrate loss to CO2 and facilitate production of reduced and energy-intensive products. However, the effects of H2 supply on CO-grown acetogens have yet to be experimentally quantified under controlled growth conditions. Results: Here, we quantify the effects of H2 supplementation by comparing growth on CO, syngas, and a high-H2 CO gas mix using chemostat cultures of Clostridium autoethanogenum. Cultures were characterised at the molecular level using metabolomics, proteomics, gas analysis, and a genome-scale metabolic model (GEM). CO-limited chemostats operated at two steady-state biomass concentrations facilitated co-utilisation of CO and H2. We show that H2 supply strongly impacts carbon distribution with a four-fold reduction in substrate loss as CO2 (61% vs. 17%) and a proportional increase of flux to ethanol (15% vs. 61%). Notably, H2 supplementation lowers the molar acetate/ethanol ratio by five-fold. At the molecular level, quantitative proteome analysis showed no obvious changes leading to these metabolic rearrangements suggesting the involvement of post-translational regulation. Metabolic modelling showed that H2 availability provided reducing power via H2 oxidation and saved redox as cells reduced all the CO2 to formate directly using H2 in the Wood-Ljungdahl pathway. Modelling further indicated that the methylene-THF reductase reaction was ferredoxin-reducing under all conditions. In combination with proteomics, modelling also showed that ethanol was synthesised through the acetaldehyde:ferredoxin oxidoreductase (AOR) activity. Conclusions: Our quantitative molecular analysis revealed that H2 drives rearrangements at several layers of metabolism and provides novel links between carbon, energy, and redox metabolism advancing our understanding of energy conservation in acetogens. We conclude that H2 supply can substantially increase the efficiency of gas fermentation and thus the feed gas composition can be considered an important factor in developing gas fermentation-based bioprocesses.

Project description:Oscillatory phenomena are ubiquitous across different biological scales, from individual cells to tissues and whole organisms. In physical systems, oscillatory behaviour can emerge from weak coupling of independent stochastic elements. Once established, oscillations can drive coordinated group behaviour and encode robust information through oscillation frequency or amplitude. Here we use experimentation and modelling to understand the emergence and information coding properties of oscillatory cyclic adenosine monophosphate (cAMP) signaling in D. discoideum. Oscillatory cAMP signaling drives aggregation of individual amoebae and coordinated temporal shifts in gene expression. However, it is unknown how the collective behaviour emerges and attenuates in an auto-regulatory manner where temporal transcriptional regulation must decode variation in cAMP pulse count, frequency, or amplitude. To address these questions, we identified a transcription factor, Hbx5, that is essential for the initiation of collective behaviour and its activity provides a single-cell readout of the earliest sign of cAMP signaling. This reveals stochastic pulses in cAMP signaling precede collective oscillations, with Hbx5 dependent transcriptional feedback enhancing signal sensitivity necessary for the emergence of collective oscillations. We demonstrate that once collective oscillations have been established, another cAMP-regulated transcription factor, GtaC, required for subsequent developmental progression becomes activated. Modelling and experimentation suggest that temporal differences in transcription factor activation arise from differences in the threshold at which the protein kinase, ErkB, influences their activity. This leads us to propose that changes in the amplitude of cAMP oscillations during development encode temporal information, driving the orderly progression of development. These studies thus provide crucial insights into the principles driving collective behaviour and the evolution of multicellular systems.

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