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

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 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 Online gas analysis and high-resolution temporal metabolomics showed oscillations in gas uptake rates and extracellular byproducts synchronized with biomass levels. The data show initial growth on CO, followed by growth on CO and H2 Growth on CO and H2 results in an accelerated growth phase, after which a downcycle is observed in synchrony with a loss in H2 uptake. Intriguingly, oscillations are not linked to translational control, as no differences were observed in protein expression during oscillations. Intracellular metabolomics analysis revealed decreasing levels of redox ratios in synchrony with the cycles. We then developed a thermodynamic metabolic flux analysis model to investigate whether regulation in acetogens is controlled at the thermodynamic level. We used endo- and exo-metabolomics data to show that the thermodynamic driving force of critical reactions collapsed as H2 uptake is lost. The oscillations are coordinated with redox. The data indicate that metabolic oscillations in acetogen gas fermentation are controlled at the thermodynamic level.

Systems-level engineering and characterisation of Clostridium autoethanogenum through heterologous production of poly-3-hydroxybutyrate (PHB).

Gas fermentation is emerging as an economically attractive option for the sustainable production of fuels and chemicals from gaseous waste feedstocks. Clostridium autoethanogenum can use CO and/or CO2 + H2 as its sole carbon and energy sources. Fermentation of C. autoethanogenum is currently being deployed on a commercial scale for ethanol production. Expanding the product spectrum of acetogens will enhance the economics of gas fermentation. To achieve efficient heterologous product synthesis, limitations in redox and energy metabolism must be overcome. Here, we engineered and characterised at a systems-level, a recombinant poly-3-hydroxybutyrate (PHB)-producing strain of C. autoethanogenum. Cells were grown in CO-limited steady-state chemostats on two gas mixtures, one resembling syngas (20% H2) and the other steel mill off-gas (2% H2). Results were characterised using metabolomics and transcriptomics, and then integrated using a genome-scale metabolic model reconstruction. PHB-producing cells had an increased expression of the Rnf complex, suggesting energy limitations for heterologous production. Subsequent optimisation of the bioprocess led to a 12-fold increase in the cellular PHB content. The data suggest that the cellular redox state, rather than the acetyl-CoA pool, was limiting PHB production. Integration of the data into the genome-scale metabolic model showed that ATP availability limits PHB production. Altogether, the data presented here advances the fundamental understanding of heterologous product synthesis in gas-fermenting acetogens.

Quantitative analysis of tetrahydrofolate metabolites from clostridium autoethanogenum.

INTRODUCTION: Quantification of tetrahydrofolates (THFs), important metabolites in the Wood-Ljungdahl pathway (WLP) of acetogens, is challenging given their sensitivity to oxygen. OBJECTIVE: To develop a simple anaerobic protocol to enable reliable THFs quantification from bioreactors. METHODS: Anaerobic cultures were mixed with anaerobic acetonitrile for extraction. Targeted LC-MS/MS was used for quantification. RESULTS: Tetrahydrofolates can only be quantified if sampled anaerobically. THF levels showed a strong correlation to acetyl-CoA, the end product of the WLP. CONCLUSION: Our method is useful for relative quantification of THFs across different growth conditions. Absolute quantification of THFs requires the use of labelled standards.

Development of a new strategy for production of medium-chain-length polyhydroxyalkanoates by recombinant Escherichia coli via inexpensive non-fatty acid feedstocks.

Pseudomonas putida KT2440 is capable of producing medium-chain-length polyhydroxyalkanoates (MCL-PHAs) when grown on unrelated carbon sources during nutrient limitation. Transcription levels of genes putatively involved in PHA biosynthesis were assessed by quantitative real-time PCR (qRT-PCR) in P. putida grown on glycerol as a sole carbon source. The results showed that two genes, phaG and the PP0763 gene, were highly upregulated among genes potentially involved in the biosynthesis of MCL-PHAs from unrelated carbon sources. Previous studies have described phaG as a 3-hydroxyacyl-acyl carrier protein (ACP)-coenzyme A (CoA) transferase, and based on homology, the PP0763 gene was predicted to encode a medium-chain-fatty-acid CoA ligase. High expression levels of these genes during PHA production in P. putida led to the hypothesis that these two genes are involved in PHA biosynthesis from non-fatty acid carbon sources, such as glucose and glycerol. The phaG(pp) and PP0763 genes from P. putida were cloned and coexpressed with the engineered Pseudomonas sp. 61-3 PHA synthase gene phaCl (STQK)(ps) in recombinant Escherichia coli. Up to 400 mg liter(-1) MCL-PHAs was successfully produced from glucose. This study has produced the largest amount of MCL-PHAs reported from non-fatty acid carbon sources in recombinant E. coli to date and opens up the possibility of using inexpensive feedstocks to produce MCL-PHA polymers.

Biosynthesis of poly[(R)-3-hydroxyalkanoate] copolymers with controlled repeating unit compositions and physical properties.

As applications for biodegradable and biologically produced poly[(R)-3-hydroxyalkanoates] (PHAs) grow into more specialized areas, the need to precisely control the repeating unit composition and consequently the physical properties of these polymers has become essential. A previous study reported our development of Escherichia coli LSBJ in order to produce PHA polymers composed of single repeating units ranging from 4 to 12 carbon atoms. This investigation expands the scope of our effort toward controlling the repeating unit composition of a variety of PHA copolymers. The sizes for the repeating units within the copolymers were modulated by feeding specific ratios of fatty acids with defined carbon lengths to E. coli LSBJ, which resulted in defined mole ratios for the repeating units. Various physical properties of the copolymers (including the Young's modulus, elongation to break, and glass-transition temperature) were shown to be strongly dependent upon the mole ratios of repeating units. This work demonstrates that copolymers of PHAs with repeating units from 4 to 12 carbons can be incorporated accurately to obtain any desired mole ratio within the PHA copolymers. Our methodology may thus be extended to generate tailor-made PHA copolymers with prescribed values for key sets of physical properties.

Absolute Proteome Quantification in the Gas-Fermenting Acetogen Clostridium autoethanogenum.

Microbes that can recycle one-carbon (C1) greenhouse gases into fuels and chemicals are vital for the biosustainability of future industries. Acetogens are the most efficient known microbes for fixing carbon oxides CO2 and CO. Understanding proteome allocation is important for metabolic engineering as it dictates metabolic fitness. Here, we use absolute proteomics to quantify intracellular concentrations for >1,000 proteins in the model acetogen Clostridium autoethanogenum grown autotrophically on three gas mixtures (CO, CO+H2, or CO+CO2+H2). We detect the prioritization of proteome allocation for C1 fixation and the significant expression of proteins involved in the production of acetate and ethanol as well as proteins with unclear functions. The data also revealed which isoenzymes are likely relevant in vivo for CO oxidation, H2 metabolism, and ethanol production. The integration of proteomic and metabolic flux data demonstrated that enzymes catalyze high fluxes with high concentrations and high in vivo catalytic rates. We show that flux adjustments were dominantly accompanied by changing enzyme catalytic rates rather than concentrations. IMPORTANCE Acetogen bacteria are important for maintaining biosustainability as they can recycle gaseous C1 waste feedstocks (e.g., industrial waste gases and syngas from gasified biomass or municipal solid waste) into fuels and chemicals. Notably, the acetogen Clostridium autoethanogenum is being used as a cell factory in industrial-scale gas fermentation. Here, we perform reliable absolute proteome quantification for the first time in an acetogen. This is important as our work advances both rational metabolic engineering of acetogen cell factories and accurate in silico reconstruction of their phenotypes. Furthermore, this absolute proteomics data set serves as a reference toward a better systems-level understanding of the ancient metabolism of acetogens.

Enhancing CO2-Valorization Using Clostridium autoethanogenum for Sustainable Fuel and Chemicals Production.

Acetogenic bacteria can convert waste gases into fuels and chemicals. Design of bioprocesses for waste carbon valorization requires quantification of steady-state carbon flows. Here, steady-state quantification of autotrophic chemostats containing Clostridium autoethanogenum grown on CO2 and H2 revealed that captured carbon (460 ± 80 mmol/gDCW/day) had a significant distribution to ethanol (54 ± 3 C-mol% with a 2.4 ± 0.3 g/L titer). We were impressed with this initial result, but also observed limitations to biomass concentration and growth rate. Metabolic modeling predicted culture performance and indicated significant metabolic adjustments when compared to fermentation with CO as the carbon source. Moreover, modeling highlighted flux to pyruvate, and subsequently reduced ferredoxin, as a target for improving CO2 and H2 fermentation. Supplementation with a small amount of CO enabled co-utilization with CO2, and enhanced CO2 fermentation performance significantly, while maintaining an industrially relevant product profile. Additionally, the highest specific flux through the Wood-Ljungdahl pathway was observed during co-utilization of CO2 and CO. Furthermore, the addition of CO led to superior CO2-valorizing characteristics (9.7 ± 0.4 g/L ethanol with a 66 ± 2 C-mol% distribution, and 540 ± 20 mmol CO2/gDCW/day). Similar industrial processes are commercial or currently being scaled up, indicating CO-supplemented CO2 and H2 fermentation has high potential for sustainable fuel and chemical production. This work also provides a reference dataset to advance our understanding of CO2 gas fermentation, which can contribute to mitigating climate change.

A TetR-Family Protein (CAETHG_0459) Activates Transcription From a New Promoter Motif Associated With Essential Genes for Autotrophic Growth in Acetogens.

Acetogens can fix carbon (CO or CO2) into acetyl-CoA via the Wood-Ljungdahl pathway (WLP) that also makes them attractive cell factories for the production of fuels and chemicals from waste feedstocks. Although most biochemical details of the WLP are well understood and systems-level characterization of acetogen metabolism has recently improved, key transcriptional features such as promoter motifs and transcriptional regulators are still unknown in acetogens. Here, we use differential RNA-sequencing to identify a previously undescribed promoter motif associated with essential genes for autotrophic growth of the model-acetogen Clostridium autoethanogenum. RNA polymerase was shown to bind to the new promoter motif using a DNA-binding protein assay and proteomics enabled the discovery of four candidates to potentially function directly in control of transcription of the WLP and other key genes of C1 fixation metabolism. Next, in vivo experiments showed that a TetR-family transcriptional regulator (CAETHG_0459) and the housekeeping sigma factor (σA) activate expression of a reporter protein (GFP) in-frame with the new promoter motif from a fusion vector in Escherichia coli. Lastly, a protein-protein interaction assay with the RNA polymerase (RNAP) shows that CAETHG_0459 directly binds to the RNAP. Together, the data presented here advance the fundamental understanding of transcriptional regulation of C1 fixation in acetogens and provide a strategy for improving the performance of gas-fermenting bacteria by genetic engineering.

H2 drives metabolic rearrangements in gas-fermenting Clostridium autoethanogenum.

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. 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 fourfold 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 fivefold. 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.

Maintenance of ATP Homeostasis Triggers Metabolic Shifts in Gas-Fermenting Acetogens.

Acetogens are promising cell factories for producing fuels and chemicals from waste feedstocks via gas fermentation, but quantitative characterization of carbon, energy, and redox metabolism is required to guide their rational metabolic engineering. Here, we explore acetogen gas fermentation using physiological, metabolomics, and transcriptomics data for Clostridium autoethanogenum steady-state chemostat cultures grown on syngas at various gas-liquid mass transfer rates. We observe that C. autoethanogenum shifts from acetate to ethanol production to maintain ATP homeostasis at higher biomass concentrations but reaches a limit at a molar acetate/ethanol ratio of ∼1. This regulatory mechanism eventually leads to depletion of the intracellular acetyl-CoA pool and collapse of metabolism. We accurately predict growth phenotypes using a genome-scale metabolic model. Modeling revealed that the methylene-THF reductase reaction was ferredoxin reducing. This work provides a reference dataset to advance the understanding and engineering of arguably the first carbon fixation pathway on Earth.

Gas Fermentation-A Flexible Platform for Commercial Scale Production of Low-Carbon-Fuels and Chemicals from Waste and Renewable Feedstocks.

There is an immediate need to drastically reduce the emissions associated with global fossil fuel consumption in order to limit climate change. However, carbon-based materials, chemicals, and transportation fuels are predominantly made from fossil sources and currently there is no alternative source available to adequately displace them. Gas-fermenting microorganisms that fix carbon dioxide (CO2) and carbon monoxide (CO) can break this dependence as they are capable of converting gaseous carbon to fuels and chemicals. As such, the technology can utilize a wide range of feedstocks including gasified organic matter of any sort (e.g., municipal solid waste, industrial waste, biomass, and agricultural waste residues) or industrial off-gases (e.g., from steel mills or processing plants). Gas fermentation has matured to the point that large-scale production of ethanol from gas has been demonstrated by two companies. This review gives an overview of the gas fermentation process, focusing specifically on anaerobic acetogens. Applications of synthetic biology and coupling gas fermentation to additional processes are discussed in detail. Both of these strategies, demonstrated at bench-scale, have abundant potential to rapidly expand the commercial product spectrum of gas fermentation and further improve efficiencies and yields.

Precise control of repeating unit composition in biodegradable poly(3-hydroxyalkanoate) polymers synthesized by Escherichia coli.

The composition of medium-chain-length (MCL) poly(3-hydroxyalkanoate) (PHA) biopolymers is normally an uncontrollable random mixture of repeating units with differing side chain lengths. Attempts to generate MCL PHA homopolymers and control repeating unit composition have been published in native PHA-producing organisms but have limited ranges for the different sizes of repeating units that can be synthesized. In this study, a new Escherichia coli-based system that exhibits control over repeating unit composition for both MCL PHAs and short-chain-length (SCL) PHAs has been developed, covering an unprecedented range of repeating units. The fadB and fadJ genes from the ß-oxidation pathway were eliminated from the chromosome of E. coli LS5218. The subsequent blockage in ß-oxidation caused a buildup of enoyl-CoA intermediates, which were converted to PHAs by an (R)-specific enoyl-CoA hydratase (PhaJ4) and PHA synthase [PhaC1(STQK)] expressed from a plasmid DNA construct. Fatty acid substrates were converted to PHAs with repeating units equal in the number of carbon atoms to the fatty acid substrate. The broad substrate specificities of the PhaJ4 and PhaC1(STQK) enzymes allowed for the production of homopolymers with strict control over the repeating unit composition from substrates of four to twelve carbons in length. Polymers were purified and analyzed by GC, GC-MS, and NMR for structural composition and by DSC, TGA, and GPC for thermal and physical characteristics. This study marks the development of the first single biological system to achieve consistent repeating unit control over such a broad range of repeating units in PHAs.

Glycerine and levulinic acid: renewable co-substrates for the fermentative synthesis of short-chain poly(hydroxyalkanoate) biopolymers.

Glycerine (a biodiesel co-product) and levulinic acid (a pulp and paper co-product) were used as co-substrates for the fermentative synthesis of short-chain polyhydroxyalkanoate (sc-PHA) biopolymers with tunable monomer and molecular weight characteristics. Pseudomonas oleovorans NRRL B-14682 utilized glycerine alone to produce poly(3-hydroxybutyrate) (PHB). When levulinic acid was added to the media at shake-flask scale in concentrations ≤0.6 wt.%, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/V) copolymers were produced with 3-HV contents ranging from 37 to 97 mol%; a glycerine:levulinic acid ratio of 0.2%:0.8% (w/v) resulted in poly(3-hydroxyvalerate) (PHV). Ten-liter batch fermentations using glycerine:levulinic acid ratios of 1%:0, 0.75%:0.25%, 0.5%:0.5% and 0.25%:0.75% (w/v) resulted in PHB, P(73%-3HB-co-27%-3HV), P(30%-3HB-co-70%-3HV) and PHV with increasing number average molecular weights (×10(3) g/mol) of 328, 511, 728 and 1330, respectively, owing to glycerine-based chain termination. These results provide a novel means by which glycerine and levulinic acid can be used collectively to produce an array of distinct sc-PHA biopolymers.