Pyruvate Ferredoxin Oxidoreductase Research Articles

Bioinformatic Analysis of Oxygen Sensitivity in Arcobacter bivalviorum's Pyruvate:Ferredoxin Oxidoreductase (PFOR)

Background: Arcobacter bivalviorum is an emerging foodborne pathogen particularly found in shellfish and other bivalves. This bacterium's metabolic versatility is highlighted by its possession of both the Pyruvate Dehydrogenase Complex (PDC) and Pyruvate:Ferredoxin Oxidoreductase (PFOR). The PFOR enzyme is crucial for linking glycolysis to the citric acid cycle. Previous studies of PFOR enzyme reveal its susceptibility to oxygen damage due to its reliance on Fe-S clusters for electron transfer.Methods: The current study employs bioinformatic approaches to explore the oxygen sensitivity of A. bivalviorum's PFOR, comparing it with Desulfovibrio africanus and Moorella thermoacetica. The UniProt database was used to obtain the sequences of PFOR from D. africanus (CAA70873), A. bivalviorum (AXH11209), and M. thermoacetica (Q2RMD6). This comparative analysis sheds light on the structural similarities and differences between the enzymes, providing a deeper understanding of their functional mechanisms.Result: PFOR enzyme is a heterodimer, with its functional subunits containing three [4Fe-4S]²⁺ clusters. Its exposed Fe-S clusters are vulnerable to oxygen and ROS, leading to enzyme inactivation. A comparison of PFOR sequences from D. africanus, M. thermoacetica, and A. bivalviorum reveals a crucial difference: the final 43 residues at the C-terminal of D. africanus are missing in the other two enzymes, depriving the M. thermoacetica and A. bivalviorum enzymes of a self-protective mechanism.Conclusion: Pyruvate:ferredoxin oxidoreductase (PFOR) in A. bivalviorum lacks protection from oxidative damage due to the absence of the final 43 amino acids at the C-terminal.Keywords: PFOR; Homology modeling; oxygen sensitivity; Foodborne pathogen

In silico Investigation of Caged Xanthone Compounds Isolated from Cratoxylum sumatranum Stem Bark against Entamoeba histolytica Enzymes

Background: Amoebiasis is caused by Entamoeba histolytica, a pathogenic species living on human colon tissues. Metronidazole is currently used for the treatment of amoebiasis, but resistance of E. histolytica to the use of such treatment has been reported. Therefore, the development of new anti-amoebic drugs is still very much needed for clinical treatment. Preliminary research on extract and fractions from Cratoxylum sumatranum stem bark has shown their anti-amoebic activity. Two compounds from the cage xanthone groups, cochinchinoxanthone and cochinchinone D, have been isolated from the active fraction of C. sumatranum stem bark. Objective: This study aimed to investigate the anti-amoebic activity of the two known compounds against E. histolytica. Methods: The in silico method used was molecular docking with several receptors, including thioredoxin reductase, triose phosphate isomerase, pyruvate ferredoxin oxidoreductase, Giardia fructose-1,6-bisphosphate aldolase, serine acetyltransferase, and phosphoserine phosphatase. The prediction of ADMET properties was also carried out for both the compounds. Results: The results showed cochinchinone D to have a higher binding affinity to thioredoxin reductase, pyruvate ferredoxin oxidoreductase, and Giardia fructose-1,6-bisphosphate aldolase receptors than cochinchinoxanthone. In contrast, cochinchinoxanthone bound better to the triose phosphate isomerase and phosphoserine phosphatase receptors, while both exhibited the same affinity for serine acetyltransferase. In general, the two compounds were also found to have similar ADMET profiles. Conclusion: In conclusion, caged xanthone compounds from C. sumatranum have the potential to be developed as anti-amoebic agents against E. histolytica through the mechanism of inhibition of these enzymes.

Structural organization of pyruvate: ferredoxin oxidoreductase from the methanogenic archaeon Methanosarcina acetivorans.

Enzymes of the 2-oxoacid:ferredoxin oxidoreductase (OFOR) superfamily catalyze the reversible oxidation of 2-oxoacids to acyl-coenzyme A esters and carbon dioxide (CO2)using ferredoxin or flavodoxin as the redox partner. Although members of the family share primary sequence identity, a variety of domain and subunit arrangements are known. Here, we characterize the structure of a four-subunit family member: the pyruvate:ferredoxin oxidoreductase (PFOR) from the methane producing archaeon Methanosarcina acetivorans (MaPFOR). The 1.92Å resolution crystal structure of MaPFOR shows a protein fold like those of single- or two-subunit PFORs that function in 2-oxoacid oxidation, including the location of the requisite thiamine pyrophosphate (TPP), and three [4Fe-4S] clusters. Of note, MaPFOR typically functions in the CO2 reductive direction, and structural comparisons to the pyruvate oxidizing PFORs show subtle differences in several regions of catalytical relevance. These studies provide a framework that may shed light on the biochemical mechanisms used to facilitate reductive pyruvate synthesis.

Beyond traditional pathways: Uncovering ultrasonication’s novel routes to enhance hydrogen yields in Clostridium pasteurianum

This study presents a comparative analysis of metabolic end-products and proteomic profiles in Clostridium (C.) pasteurianum during dark fermentation, in the presence and absence of ultrasonication. The study revealed significant changes in the abundance of six proteins associated with dark fermentation pathways, particularly those involved in hydrogen generation pyruvate:ferredoxin oxidoreductase (PFOR) and pyruvate formate lyase (PFL)), glycolysis, butyrate pathway, and adenosine triphosphate (ATP) synthesis. Ultrasonication was efficient at degassing with dissolved hydrogen concentrations reduced from 2.63 ± 0.09 mM to 0.65 ± 0.04 mM. This led to an 11 % increase in produced hydrogen concentration, and a 37 % increase in hydrogen yield. Contrary to expectations, ultrasonication resulted in a deceleration of both glycolytic and hydrogen generation pathways, as evidenced by a decrease in the proteomic expression of key enzymes like pyruvate:ferredoxin oxidoreductase and pyruvate formate lyase. The study also discovered an increased ATP demand and an altered carbon flux through metabolic pathways in C. pasteurianum. This flux initially favoured the acetate pathway due to the sudden ATP demand. The long-term maintenance of this ATP demand required balancing the carbon flux between lactate, acetate, and butyrate pathways, to achieve both a stable redox balance and ATP demand. For the first time, this study revealed opportunities to enhance hydrogen yields using strategies other than enhancing the hydrogen generation pathways, specifically by reducing dissolved hydrogen concentrations.

Comparison of anaerobic co-digestion of vacuum toilet blackwater and kitchen waste under mesophilic and thermophilic conditions: Reactor performance, microbial response and metabolic pathway

Co-digestion of kitchen waste (KW) and black water (BW) can be considered as an attractive method to efficiently achieve the clean energy from waste. To find the optimal operation parameters for the co-digestion, the effects of different temperatures (35 and 55 °C) and BW:KW ratios on the reactor performances, microbial communities and metabolic pathways were studied. The results showed that the optimum BW:KW ratio was 1:3.6 and 1:4.5 for mesophilic and thermophilic optimal reactors, with methane production of 449.04 mL/g VS and 411.90 mL/g VS, respectively. Microbial communities showed significant differences between the reactors under different temperatures. For bacteria, increasing BW:KW ratio significantly promoted Defluviitoga enrichment (1.1%–9.5%) under thermophilic condition. For Archaea, the increase in BW:KW ratio promoted the enrichment of Methanosaeta (8.6%–56.4%) in the mesophilic reactor and Methanothermobacter (62.0%–89.2%) in the thermophilic reactor. The analysis of the key enzymes showed that, acetoclastic methanogenic pathway performed as the dominant under mesophilic condition, with high abundance of Acetate-CoA ligase (EC:6.2.1.1) and Pyruvate synthase (EC:1.2.7.1). Hydrogenotrophic methanogenic pathway was the main pathway in the thermophilic reactors, with high abundance of Formylmethanofuran dehydrogenase (EC:1.2.99.5).

Phylogenetic Analysis of Pyruvate-Ferredoxin Oxidoreductase, a Redox Enzyme Involved in the Pharmacological Activation of Nitro-Based Prodrugs in Bacteria and Protozoa.

The present frontrunners in the chemotherapy of infections caused by protozoa are nitro-based prodrugs that are selectively activated by PFOR-mediated redox reactions. This study seeks to analyze the distribution of PFOR in selected protozoa and bacteria by applying comparative genomics to test the hypothesis that PFOR in eukaryotes was acquired through horizontal gene transfer (HGT) from bacteria. Furthermore, to identify other putatively acquired genes, proteome-wide and gene enrichment analyses were used. A plausible explanation for the patchy occurrence of PFOR in protozoa is based on the hypothesis that bacteria are potential sources of genes that enhance the adaptation of protozoa in hostile environments. Comparative genomics of Entamoeba histolytica and the putative gene donor, Desulfovibrio vulgaris, identified eleven candidate genes for HGT involved in intermediary metabolism. If these results can be reproduced in other PFOR-possessing protozoa, it would provide more validated evidence to support the horizontal transfer of pfor from bacteria.

Flocculation-enhanced photobiological hydrogen production by microalgae: Flocculant composition, hydrogenase activity and response mechanism

The practical application of microalgal photobiological hydrogen was limited by its low yield and short production time. In this study, the promotion of inorganic flocculants (polymeric aluminum chloride, PAC) and organic flocculants (cationic polyacrylamide, CPAM) to the aggregate formation and photobiological hydrogen production of prokaryotic microalgae (Synechocystis sp.) and eukaryotic microalgae (Chlamydomonas reinhardtii) were investigated. The effectiveness of PAC-CPAM composite flocculants in promoting aggregation was compared to single PAC or CPAM, with flocculation efficiencies exceeding 92.2 % for both microalgal strains. After aggregate formation, hydrogen production in Synechocystis and C. reinhardtii increased by 65.9 % and 80.2 %, respectively, compared with the control (without flocculation). Their hydrogenase activity rose to 1.14 and 28.2 μmol H2/(mg chlorophyll·h), respectively. Aggregation sustained the photosynthetic activity of both Synechocystis and C. reinhardtii, with the photosynthetic system electron transfer rate (ETR) 1.54–1.72 times higher than that of the control. The formation of aggregates enhanced microalgal respiration, accelerating the reduction of oxygen content, and increasing hydrogenase activity. Notably, the increased respiration in Synechocystis and C. reinhardtii aggregates supplied more electrons, enhancing hydrogen production by consuming intracellular organic matter. Aggregate formation upregulated the expression of Synechocystis and C. reinhardtii hydrogenase genes by average 2.1-fold and 2.5-fold, respectively. Simultaneously, the expression of genes related to photosynthetic system Ⅱ, ferredoxin, pyruvate ferredoxin-oxidoreductase, and oxidative phosphorylation were upregulated, with a significant effect observed in C. reinhardtii aggregates. The results of this study advanced the utilization and industrialization of microalgae-based green hydrogen production, contributing to carbon neutrality.

Identification and characterization of the low molecular mass ferredoxins involved in central metabolism in Heliomicrobium modesticaldum.

The homodimeric Type I reaction center (RC) from Heliomicrobium modesticaldum lacks the PsaC subunit found in Photosystem I and instead uses the interpolypeptide [4Fe-4S] cluster FX as the terminal electron acceptor. Our goal was to identify which of the small mobile dicluster ferredoxins encoded by the H. modesticaldum genome are capable of accepting electrons from the heliobacterial RC (HbRC) and pyruvate:ferredoxin oxidoreductase (PFOR), a key metabolic enzyme. Analysis of the genome revealed seven candidates: HM1_1462 (PshB1), HM1_1461 (PshB2), HM1_2505 (Fdx3), HM1_0869 (FdxB), HM1_1043, HM1_0357, and HM1_2767. Heterologous expression in Escherichia coli and studies using time-resolved optical spectroscopy revealed that only PshB1, PshB2, and Fdx3 are capable of accepting electrons from the HbRC and PFOR. Modeling studies using AlphaFold show that only PshB1, PshB2, and Fdx3 should be capable of docking on PFOR at a positively charged patch that overlays a surface-proximal [4Fe-4S] cluster. Proteomic analysis of wild-type and gene deletion strains ΔpshB1, ΔpshB2, ΔpshB1pshB2, and Δfdx3 grown under nitrogen-replete conditions revealed that Fdx3 is undetectable in the wild-type, ΔpshB1, and Δfdx3 strains, but it is present in the ΔpshB2 and ΔpshB1pshB2 strains, implying that Fdx3 may substitute for PshB2. When grown under nitrogen-deplete conditions, Fdx3 is present in the wild-type and all deletion strains except for Δfdx3. None of the knockout strains demonstrated significant impairment during chemotrophic dark growth on pyruvate, photoheterotrophic light growth on pyruvate, or phototrophic growth on acetate+CO2, indicating a high degree of redundancy among these three electron transfer proteins. Loss of both PshB1 and PshB2, but not FdxB, resulted in poor growth under N2-fixing conditions.

Metronidazole Induces the Metazoan Death of Giardia Trophozoites with the Aid of Pyruvate

Background: Metronidazole is the most common drug for the treatment of infectious agent Giardia. The trophozoites need to fight against the oxidative stress generated by metronidazole for their survival. It has been reported that trophozoites possesses several enzymes involved in response to oxidative stress like pyruvate-ferredoxin oxidoreductase, NADH oxidase, peroxiredoxin to combat the harsh condition. These enzyme systems generally act on the amitochondriate trophozoites to attenuate the reactive oxygen species generation which causes cytotoxicity but the actual mechanism of trophozoites death due to metronidazole treatment was still not clear. Methods: The present study aims to establish the effects of pyruvate in Giardia trophozoites exposed to metronidazole treatment. Intracellular reactive oxygen species (ROS) production by Giardia trophozoite suspension was monitored in the presence and absence of pyruvate with the help of a dichlorodihydrofluoresceine diacetate (H2DCFDA) based assay. In the present study, we have investigated the effects of pyruvate on DNA damage in the trophozoites during metronidazole stress. We have also looked into the expression levels of some genes to show their relevance to metronidazole stress. Results: The exogenously addition of physiologically relevant concentration of pyruvate was shown to elevate the rate of ROS generation in Giardia suspension under metronidazole stress. Our results provide evidence that exogenously added pyruvate have induced lipid peroxidation of stressed Giardia. Several known genes are modulated due to the exposure of metronidazole in trophozoites. Conclusion: These results suggest that pyruvate is the key regulatory metabolite that helps generation of different radicals to initiate apoptotic like death in Giardia trophozoites during metronidazole exposure.

Phenolic chalcones as agents against Trichomonas vaginalis

Trichomonas vaginalis, a flagellated and anaerobic protozoan, is a causative agent of trichomoniasis. This disease is among the world’s most common non-viral sexually transmitted infection. A single class drug, nitroimidazoles, is currently available for the trichomoniasis treatment. However, resistant isolates have been identified from unsuccessfully treated patients. Thus, there is a great challenge for a discovery of innovative anti-T. vaginalis agents. As part of our ongoing search for antiprotozoal chalcones, we designed and synthesized a series of 21 phenolic chalcones, which were evaluated against T. vaginalis trophozoites. Structure-activity relationship indicated hydroxyl group plays a role key in antiprotozoal activity. 4′-Hydroxychalcone (4HC) was the most active compound (IC50 = 27.5 µM) and selected for detailed bioassays. In vitro and in vivo evaluations demonstrated 4HC was not toxic against human erythrocytes and Galleria mellonella larvae. Trophozoites of T. vaginalis were treated with 4HC and did not present significant reactive oxygen species (ROS) accumulation. However, compound 4HC was able to increase ROS accumulation in neutrophils coincubated with T. vaginalis. qRT-PCR Experiments indicated that 4HC did not affect the expression of pyruvate:ferredoxin oxidoreductase (PFOR) and β-tubulin genes. In silico simulations, using purine nucleoside phosphorylase of T. vaginalis (TvPNP), corroborated 4HC as a promising ligand. Compound 4HC was able to establish interactions with residues D21, G20, M180, R28, R87 and T90 through hydrophobic interactions, π-donor hydrogen bond and hydrogen bonds. Altogether, these results open new avenues for phenolic chalcones to combat trichomoniasis, a parasitic neglected infection.

Synthesis of isobutanol using acetate as sole carbon source in Escherichia coli

BackgroundWith concerns about depletion of fossil fuel and environmental pollution, synthesis of biofuels such as isobutanol from low-cost substrate by microbial cell factories has attracted more and more attention. As one of the most promising carbon sources instead of food resources, acetate can be utilized by versatile microbes and converted into numerous valuable chemicals.ResultsAn isobutanol synthetic pathway using acetate as sole carbon source was constructed in E. coli. Pyruvate was designed to be generated via acetyl-CoA by pyruvate-ferredoxin oxidoreductase YdbK or anaplerotic pathway. Overexpression of transhydrogenase and NAD kinase increased the isobutanol titer of recombinant E. coli from 121.21 mg/L to 131.5 mg/L under batch cultivation. Further optimization of acetate supplement concentration achieved 157.05 mg/L isobutanol accumulation in WY002, representing the highest isobutanol titer by using acetate as sole carbon source.ConclusionsThe utilization of acetate as carbon source for microbial production of valuable chemicals such as isobutanol could reduce the consumption of food-based substrates and save production cost. Engineering strategies applied in this study will provide a useful reference for microbial production of pyruvate derived chemical compounds from acetate.

New insight into effects of waste scrap iron on sludge anaerobic digestion: Performances, microbial community, and potential metabolic functions

The low sludge degradation efficiency and hydrogen sulfide (H2S) generation impair the development of sludge anaerobic digestion. This study explored the effects of waste scrap iron (WSI) addition on methane (CH4) production and H2S control in sludge anaerobic digestion. The maximum cumulative CH4 yield of 205.02 mL/g VSS was obtained at the WSI dosage of 0.7 g/g VSS, which increased by 21.34 % compared to that of control without WSI addition. The increase in CH4 yield was attributed to the enhancement of sludge degradation and soluble organic conversion. Meanwhile, the cumulative H2S yield reduced by 62.00 % compared to that of control. The pH value and content of insoluble sulfide precipitate increased with the increase in WSI dosage, which were two main reasons for H2S control. Microbial community results indicated that relative abundance of unclassified_f_Clostridiaceae_1, Bacteroides, vadinBC27_wastewater-sludge_group, Clostridium_sensu-stricto-1, Macellibacteroides, and hydrogenotrophic methanogens increased with WSI addition, meaning that WSI addition promoted syntrophic interaction between key bacteria and hydrogenotrophic methanogens. Gene prediction analysis demonstrated that WSI addition promoted microbial proliferation and respiration, carbohydrate and amino acid metabolism, and activity of butyrate kinase, acetate kinase, pyruvate ferredoxin oxidoreductase, and coenzyme F420 hydrogenase. In view of the enhancement of ABC transporters, lipopolysaccharide biosynthesis, quorum sensing, two-component systems, and ribosome metabolic pathway, it could be considered that WSI addition promoted information recognition and substance exchange among microorganisms, increasing microbial metabolic activity of anaerobic system.

The effect of single dose albendazole (400 mg) treatment on the human gut microbiome of hookworm-infected Ghanaian individuals

Microbes play a key role in human gut homeostasis, metabolic, immunologic and physiopathology of the body. A longitudinal study conducted during 2018–2021 in the Kintampo North Municipality in Ghana demonstrated low hookworm infection cure rates following treatment with a single dose of 400 mg albendazole in some communities. To investigate associations between hookworm infection and the gut microbiome, we examined stool samples from consented participants who were either cured or remained infected after treatment. At each time point, stool was collected prior to and 10–14 days after albendazole treatment. We used 16S rRNA amplicon sequencing of DNA extracted from stool samples to investigate the composition and diversity of the gut microbiota and to identify potential microbial biomarkers associated with treatment outcomes. Hookworm infection was associated with increased species richness (p = 0.0093). Among treated individuals, there was also a significant variation in microbiota composition at 10–14 days following single-dose albendazole treatment. Individuals cured of hookworm infection after treatment showed a significant reduction in microbiota composition when compared to their pre-treatment state (ANOSIM; p = 0.02), whilst individuals who failed to clear the infection showed no change in microbiota composition (ANOSIM; p = 0.35). Uninfected individuals and those who were successfully treated were similar in their microbial composition and structure. We also found that the abundance of Clostridia spp. was increased in infected individuals pre- or post-treatment. Predictive functional profiling revealed the enrichment of two pyruvate ferredoxin oxidoreductase subunit pathways in individuals who remained infected after treatment (p < 0.05), alluding to an upturn of strictly anaerobic commensal bacteria such as Clostridia spp. This study suggests a relationship between human gut microbiome dysbiosis and albendazole therapy outcomes of hookworm infection. Future studies will further characterize specific biomarkers identified within this study to establish their potential for assessment of pharmacological responses to anthelminthic therapies, as well as explore the possibility of using probiotic supplementation as an adjunct treatment to increase albendazole effectiveness against hookworm.

In-situ production of lactate driving the biotransformation of waste activated sludge to medium-chain fatty acid

Medium-chain fatty acids (MCFAs) have drawn great attention due to their high energy density and superior hydrophobicity. Waste activated sludge (WAS) has been documented as a renewable feedstock for MCFAs production via anaerobic fermentation. However, MCFAs production from WAS depends on exogenous addition of electron donor (ED, e.g., lactate) for chain elongation (CE) bioprocess, which results in increased economic cost and limited practical application. In this study, a novel biotechnology was proposed to produce MCFAs from WAS with in-situ self-formed lactate by inoculating Yoghurt starter powder containing with Lactobacillales cultures. The batch experimental results revealed that the lactate was in-situ generated from WAS and the maximum production of MCFAs increased from 1.17 to 3.99 g COD/L with the increased addition of Lactobacillales cultures from 6✕107 to 2.3✕108 CFU/mL WAS. In continuous long-term test over 97 days, average MCFA production reached up to 3.94 g COD/L with a caproate yield of 82.74% at sludge retention time (SRT) 12 days, and the average MCFA production increased to 5.87 g COD/L with 69.28% caproate and 25.18% caprylate at SRT 15 days. A comprehensive analysis of the metagenome and metatranscriptome demonstrated that the genus of Lactobacillus and Streptococcus were capable of producing lactate from WAS and upgrading to MCFAs. Moreover, another genus, i.e., Candidatus Promineofilum, was firstly revealed that it might be responsible for lactate and MCFAs production. Further investigation of related microbial pathways and enzyme expression suggested that D-lactate dehydrogenase and pyruvate ferredoxin oxidoreductase contributed to lactate and acetyl-CoA production, which were the crucial steps for MCFAs generation and were most actively expressed. This study provides a conceptual framework of MCFAs from WAS with endogenous ED, potentially enhancing the energy recovery from WAS treatment.

Transcriptional profile of Trichomonas vaginalis in response to metronidazole

BackgroundTrichomoniasis caused by Trichomonas vaginalis, combined with its complications, has long frequently damaged millions of human health. Metronidazole (MTZ) is the first choice for therapy. Therefore, a better understanding of its trichomonacidal process to ultimately reveal the global mechanism of action is indispensable. To take a step toward this goal, electron microscopy and RNA sequencing were performed to fully reveal the early changes in T. vaginalis at the cellular and transcriptome levels after treatment with MTZ in vitro.ResultsThe results showed that the morphology and subcellular structures of T. vaginalis underwent prominent alterations, characterized by a rough surface with bubbly protrusions, broken holes and deformed nuclei with decreased nuclear membranes, chromatin and organelles. The RNA-seq data revealed a total of 10,937 differentially expressed genes (DEGs), consisting of 4,978 upregulated and 5,959 downregulated genes. Most DEGs for the known MTZ activators, such as pyruvate:ferredoxin oxidoreductase (PFOR) and iron-sulfur binding domain, were significantly downregulated. However, genes for other possible alternative MTZ activators such as thioredoxin reductase, nitroreductase family proteins and flavodoxin-like fold family proteins, were dramatically stimulated. GO and KEGG analyses revealed that genes for basic vital activities, proteostasis, replication and repair were stimulated under MTZ stress, but those for DNA synthesis, more complicated life activities such as the cell cycle, motility, signaling and even virulence were significantly inhibited in T. vaginalis. Meanwhile, increased single nucleotide polymorphism (SNP) and insertions - deletions (indels) were stimulated by MTZ.ConclusionsThe current study reveals evident nuclear and cytomembrane damage and multiple variations in T. vaginalis at the transcriptional level. These data will offer a meaningful foundation for a deeper understanding of the MTZ trichomonacidal process and the transcriptional response of T. vaginalis to MTZ-induced stress or even cell death.

Phenanthrene regulates metabolic pathways for hydrogen accumulation in sludge alkaline dark fermentation

The influence of phenanthrene (PHE), a general polycyclic aromatic hydrocarbon in waste activated sludge, on sludge alkaline dark fermentation for hydrogen accumulation was investigated prospectively. The yield of hydrogen was 16.2 mL/g TSS with 50 mg/kg TSS PHE, which was 1.3-fold greater than that of the control. Mechanism research demonstrated that hydrogen production and the abundance of functional microorganisms were facilitated, whereas those of homoacetogenesis were reduced. The activity of pyruvate ferredoxin oxidoreductase in the conversion of pyruvate to reduced ferredoxin for hydrogen production was promoted by 57.2%, and that of carbon monoxide dehydrogenase and formyltetrahydrofolate synthetase, closely associated with hydrogen consumption, was suppressed by 60.5% and 55.9%, respectively. Moreover, the encoding genes involved in pyruvate metabolism were significantly up-regulated, while genes related to consuming hydrogen to reduce carbon dioxide and produce 5-methyltetrahydrofolate were down-regulated. This study notably illustrates the effect of PHE on hydrogen accumulation from metabolic pathways.

Iron zero valent nanoparticles applied in the biorefinery concept: Functional genes in continuous bioreactor fermenting vinasse

Increased production of biogas and volatile acids was evaluated by the addition of Fe0 Nanoparticles (NP) on Anaerobic Fluidized Bed Reactor (AFBR-NP) with expanded clay as support material (45.1 mg Fe0 g−1 expanded clay), fermenting vinasse, and operated at 37 °C and 3h Hydraulic Retention Time (HRT). Higher H2 percentage in the biogas composition was observed in AFBR-NP (50–65%) when compared to AFBR without NP, AFBR-Control (24–43%). A 1.2-fold higher valeric acid production was also observed as a result of NPs supplementation. Genes related to the first steps of vinasse anaerobic degradation in AFBR-NP (beta-glucosidase, glucokinase and pyruvate-ferredoxin oxidoreductase) and the genes related to volatile acids production (L- and d-lactate dehydrogenase, 3-hydroxybutyryl-CoA dehydrogenase, acetate kinase, pyruvate kinase and fumarate reductase flavoprotein) were observed by metagenome sequencing. A 4.2-fold higher relative abundance of Secundilactobacillus genus in AFBR-NP was evidenced. The genetic and microbial community differences observed with the addition of NP resulted in improved biorefinery products of anaerobic digestion.

Manipulating Fermentation Pathways in the Hyperthermophilic Archaeon Pyrococcus furiosus for Ethanol Production up to 95°C Driven by Carbon Monoxide Oxidation.

Genetic engineering of hyperthermophilic organisms for the production of fuels and other useful chemicals is an emerging biotechnological opportunity. In particular, for volatile organic compounds such as ethanol, fermentation at high temperatures could allow for straightforward separation by direct distillation. Currently, the upper growth temperature limit for native ethanol producers is 72°C in the bacterium Thermoanaerobacter ethanolicus JW200, and the highest temperature for heterologously-engineered bioethanol production was recently demonstrated at 85°C in the archaeon Pyrococcus furiosus. Here, we describe an engineered strain of P. furiosus that synthesizes ethanol at 95°C, utilizing a homologously-expressed native alcohol dehydrogenase, termed AdhF. Ethanol biosynthesis was compared at 75°C and 95°C with various engineered strains. At lower temperatures, the acetaldehyde substrate for AdhF is most likely produced from acetate by aldehyde ferredoxin oxidoreductase (AOR). At higher temperatures, the effect of AOR on ethanol production is negligible, suggesting that acetaldehyde is produced by pyruvate ferredoxin oxidoreductase (POR) via oxidative decarboxylation of pyruvate, a reaction known to occur only at higher temperatures. Heterologous expression of a carbon monoxide dehydrogenase complex in the AdhF overexpression strain enabled it to use CO as a source of energy, leading to increased ethanol production. A genome reconstruction model for P. furiosus was developed to guide metabolic engineering strategies and understand outcomes. This work opens the door to the potential for 'bioreactive distillation' since fermentation can be performed well above the normal boiling point of ethanol. IMPORTANCE Previously, the highest temperature for biological ethanol production was 85°C. Here, we have engineered ethanol production at 95°C by the hyperthermophilic archaeon Pyrococcus furiosus. Using mutant strains, we showed that ethanol production occurs by different pathways at 75°C and 95°C. In addition, by heterologous expression of a carbon monoxide dehydrogenase complex, ethanol production by this organism was driven by the oxidation of carbon monoxide. A genome reconstruction model for P. furiosus was developed to guide metabolic engineering strategies and understand outcomes.

Proteomic analysis of metronidazole resistance in the human facultative pathogen Bacteroides fragilis.

The anaerobic gut bacteria and opportunistic pathogen Bacteroides fragilis can cause life-threatening infections when leaving its niche and reaching body sites outside of the gut. The antimicrobial metronidazole is a mainstay in the treatment of anaerobic infections and also highly effective against Bacteroides spp. Although resistance rates have remained low in general, metronidazole resistance does occur in B. fragilis and can favor fatal disease outcomes. Most metronidazole-resistant Bacteroides isolates harbor nim genes, commonly believed to encode for nitroreductases which deactivate metronidazole. Recent research, however, suggests that the mode of resistance mediated by Nim proteins might be more complex than anticipated because they affect the cellular metabolism, e.g., by increasing the activity of pyruvate:ferredoxin oxidoreductase (PFOR). Moreover, although nim genes confer only low-level metronidazole resistance to Bacteroides, high-level resistance can be much easier induced in the laboratory in the presence of a nim gene than without. Due to these observations, we hypothesized that nim genes might induce changes in the B. fragilis proteome and performed comparative mass-spectrometric analyses with B. fragilis 638R, either with or without the nimA gene. Further, we compared protein expression profiles in both strains after induction of high-level metronidazole resistance. Interestingly, only few proteins were repeatedly found to be differentially expressed in strain 638R with the nimA gene, one of them being the flavodiiron protein FprA, an enzyme involved in oxygen scavenging. After induction of metronidazole resistance, a far higher number of proteins were found to be differentially expressed in 638R without nimA than in 638R with nimA. In the former, factors for the import of hemin were strongly downregulated, indicating impaired iron import, whereas in the latter, the observed changes were not only less numerous but also less specific. Both resistant strains, however, displayed a reduced capability of scavenging oxygen. Susceptibility to metronidazole could be widely restored in resistant 638R without nimA by supplementing growth media with ferrous iron sulfate, but not so in resistant 638R with the nimA gene. Finally, based on the results of this study, we present a novel hypothetic model of metronidazole resistance and NimA function.

Carbon Fixation in the Chemolithoautotrophic Bacterium Aquifex aeolicus Involves Two Low-Potential Ferredoxins as Partners of the PFOR and OGOR Enzymes

Aquifex aeolicus is a microaerophilic hydrogen- and sulfur -oxidizing bacterium that assimilates CO2 via the reverse tricarboxylic acid cycle (rTCA). Key enzymes of this pathway are pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxin oxidoreductase (OGOR), which are responsible, respectively, for the reductive carboxylation of acetyl-CoA to pyruvate and of succinyl-CoA to 2-oxoglutarate, two energetically unfavorable reactions that require a strong reduction potential. We have confirmed, by biochemistry and proteomics, that A. aeolicus possesses a pentameric version of these enzyme complexes ((αβγδε)2) and that they are highly abundant in the cell. In addition, we have purified and characterized, from the soluble fraction of A. aeolicus, two low redox potential and oxygen-stable [4Fe-4S] ferredoxins (Fd6 and Fd7, E0 = -440 and -460 mV, respectively) and shown that they can physically interact and exchange electrons with both PFOR and OGOR, suggesting that they could be the physiological electron donors of the system in vivo. Shotgun proteomics indicated that all the enzymes assumed to be involved in the rTCA cycle are produced in the A. aeolicus cells. A number of additional enzymes, previously suggested to be part of a putative partial Wood-Ljungdahl pathway used for the synthesis of serine and glycine from CO2 were identified by mass spectrometry, but their abundance in the cell seems to be much lower than that of the rTCA cycle. Their possible involvement in carbon assimilation is discussed.