Generation Of NADH Research Articles

Enhanced aerobic denitrification performance with Bacillus licheniformis via secreting lipopeptide biosurfactant lichenysin

Aerobic denitrification efficiency is not always satisfactory and high nitrite accumulation and N2O emission occur frequently. Though NOx reduction during aerobic denitrification is influenced by neighboring microbes, the underlying mechanism has been seldomly studied. Here, we showed markedly improved denitrification performance of a common aerobic denitrifier in its response to co-culturing with an abundant co-existing bacterium Bacillus licheniformis, and NOx removal by B. licheniformis was marginal. Compared with that in solo culture, the growth rate of denitrifiers in co-culture varied slightly, but the cytoplasmic nitrate reductase (NAR) exhibited obvious reduction capability and its encoding genes narGHI increased to 95.5 ∼ 122.2 times. It suggested that B. licheniformis improved nitrate removal by enhancing NAR activity instead of denitrifiers density. Transwell assay demonstrated non-contacting B. licheniformis could also promote nitrate and nitrite removal, and following LC-MS showed noticeable difference in the concentration of lichenysin, a lipopeptides biosurfactant metabolite from B. licheniformis, in its solo-culture and co-culture with denitrifiers. Following observation that exogenous lichenysin, but lichenysin producing mutant strain, enhanced aerobic denitrification performance verified the critical role of lichenysin here. Further investigation illustrated that lichenysin promoted the membrane permeability of aerobic denitrifiers, which facilitated the transmembrane transport of nitrate and glucose, and subsequently made more nitrate accessible to NAR and more glucose participate into oxidation for NADH generation respectively. By these means, increased nitrate reduction with less nitrite accumulation and N2O emission was realized in the presence of B. licheniformis. Finally, enhancement of B. licheniformis on performance of aerobic denitrifying community from wastewater treatment plant was also observed in laboratory, which suggested the feasibility of its practical application in the future.

Fast Determination of Mitochondrial Metabolism and Respiratory Complex Activity in Permeabilized and Intact Cells.

Assessment of mitochondrial metabolism is multidimensional and time consuming, usually requiring specific training. Respiration, NADH generation, and mitochondrial membrane potential (ΔΨm) are dynamic readouts of the metabolism and bioenergetics of mitochondria. Methodologies available to determine functional parameters in isolated mitochondria and permeabilized cells are sometimes of limited use or inapplicable to studies in live cells. In particular, the sequential assessment of the activity of each complex in the electron transport chain has not been reported in intact cells. Here, we describe a novel approach to sequentially assess electron flow through all respiratory complexes in permeabilized and intact cells by respirometry. We also describe a highly sensitive and fast method to assess ΔΨm and NADH generation in live cells using plate reader assays. Thus, our combined method allows a relatively inexpensive and fast determination of three major readouts of mitochondrial function in a few hours, using equipment that is frequently available in many laboratories worldwide.

High methanol-to-formate ratios induce butanol production in Eubacterium limosum.

SummaryUnlike gaseous C1 feedstocks for acetogenic bacteria, there has been less attention on liquid C1 feedstocks, despite benefits in terms of energy efficiency, mass transfer and integration within existing fermentation infrastructure. Here, we present growth of Eubacterium limosum ATCC8486 using methanol and formate as substrates, finding evidence for the first time of native butanol production. We varied ratios of methanol‐to‐formate in batch serum bottle fermentations, showing butyrate is the major product (maximum specific rate 220 ± 23 mmol‐C gDCW‐1day‐1). Increasing this ratio showed methanol is the key feedstock driving the product spectrum towards more reduced products, such as butanol (maximum titre 2.0 ± 1.1 mM‐C). However, both substrates are required for a high growth rate (maximum 0.19 ± 0.011 h‐1) and cell density (maximum 1.2 ± 0.043 gDCW l‐1), with formate being the preferred substrate. In fact, formate and methanol are consumed in two distinct growth phases – growth phase 1, on predominately formate and growth phase 2 on methanol, which must balance. Because the second growth varied according to the first growth on formate, this suggests butanol production is due to overflow metabolism, similar to 2,3‐butanediol production in other acetogens. However, further research is required to confirm the butanol production pathway in E. limosum, particularly given, unlike other substrates, methanol likely results in mostly NADH generation, not reduced ferredoxin.

Biological removal of sulfamethoxazole enhanced by S. oneidensis MR-1 via promoting NADH generation and electron transfer and consumption

The bio-removal efficiency of sulfamethoxazole (SMX) from wastewater is usually very poor. In this paper a new efficient method to biodegrade SMX was reported. The SMX biodegradation efficiency by Paracoccus denitrificans was observed to be remarkably enhanced from 48.9% to 94.2% after Shewanella oneidensis MR-1 addition. The mechanisms investigation revealed that P. denitrificans was the dominant microbe for SMX biodegradation. Although SMX biodegradation by S. oneidensis MR-1 alone was negligible, its presence advanced NADH generation. The proteomics assay revealed that the expression of key proteins relevant with complex I and III and cytochrome c in electron transfer chain were increased due to P. denitrificans acquiring iron from periplasm to cytoplasm being improved. In addition, the extracellular electron transfer capability was enhanced as S. oneidensis MR-1 not only produced flavin, but caused P. denitrificans to secret more extracellular polymeric substances. Further investigation indicated that the expression of key enzymes related to electron consumption in SMX biodegradation was up-regulated. Based on these findings, the pathways of S. oneidensis MR-1 promoting SMX biodegradation were proposed. As all nitrate could be removed with almost no nitrite accumulation, this study would also provide an attractive way for simultaneous bio-removal of multiple pollutants from wastewater.

Transcriptomics and Metabolomics Integration Reveals Redox-Dependent Metabolic Rewiring in Breast Cancer Cells.

Simple SummaryMetabolic rewiring fuels cancer proliferation by enhanced glycolysis and the increased NADH/NAD+ ratio. In this study, we highlight the critical role of NADH in the epigenetic landscape mediated by CtBP2 (C-terminal binding protein 2) activation, linking metabolism to epigenetic transcriptional reprogramming. Moreover, using metabolomics and transcriptomics integration, we show that genetic and pharmacological down-regulation of CtBP2 strongly reduces cell proliferation by modulating the redox balance, nucleotide synthesis, reactive oxygen species (ROS) generation, and scavenging. Therefore, we provide evidence that metabolic rewiring plasticity regulates the crosstalk between metabolism and the transcriptional program that sustains energetic and anabolic demands in cancer cells.Rewiring glucose metabolism toward aerobic glycolysis provides cancer cells with a rapid generation of pyruvate, ATP, and NADH, while pyruvate oxidation to lactate guarantees refueling of oxidized NAD+ to sustain glycolysis. CtPB2, an NADH-dependent transcriptional co-regulator, has been proposed to work as an NADH sensor, linking metabolism to epigenetic transcriptional reprogramming. By integrating metabolomics and transcriptomics in a triple-negative human breast cancer cell line, we show that genetic and pharmacological down-regulation of CtBP2 strongly reduces cell proliferation by modulating the redox balance, nucleotide synthesis, ROS generation, and scavenging. Our data highlight the critical role of NADH in controlling the oncogene-dependent crosstalk between metabolism and the epigenetically mediated transcriptional program that sustains energetic and anabolic demands in cancer cells.

Enhanced Bioreduction of Radionuclides by Driving Microbial Extracellular Electron Pumping with an Engineered CRISPR Platform.

Dissimilatory metal-reducing bacteria (DMRB) with extracellular electron transfer (EET) capability show great potential in bioremediating the subsurface environments contaminated by uranium through bioreduction and precipitation of hexavalent uranium [U(VI)]. However, the low EET efficiency of DMRB remains a bottleneck for their applications. Herein, we develop an engineered CRISPR platform to drive the extracellular electron pumping of Shewanella oneidensis, a representative DMRB species widely present in aquatic environments. The CRISPR platform allows for highly efficient and multiplex genome editing and rapid platform elimination post-editing in S. oneidensis. Enabled by such a platform, a genomic promoter engineering strategy (GPS) for genome-widely engineering the EET-encoding gene network was established. The production of electron conductive Mtr complex, synthesis of electron shuttle flavin, and generation of NADH as intracellular electron carrier are globally optimized and promoted, leading to a significantly enhanced EET ability. Applied to U(VI) bioreduction, the edited strains achieve up to 3.62-fold higher reduction capacity over the control. Our work endows DMRB with an enhanced ability to remediate the radionuclides-contaminated environments and provides a gene editing approach to handle the growing environmental challenges of radionuclide contaminations.

TRAP transporter TakP: a key player in the resistance against selenite-induced oxidative stress in Rhodobacter sphaeroides

Almost one-third of all proteins require metal ions as an essential component in key biological processes and approximately half of all enzymes are associated with one or more metal ions. The naturally occurring selenium is very toxic at higher levels, but few bacteria can reduce it into the less toxic insoluble elemental selenium. Selenium is required for the synthesis of selenocysteine, an essential residue involved in the active sites of various enzymes. The purple non-sulphur bacteria, Rhodobacter sphaeroidesis demonstrated for its selenite reduction capacity. The exact mechanism of selenite toxicity is unknown but it reacts with glutathione to form selenodiglutathione, producing the highly toxic compounds namely, H2O2and O2−. A R. sphaeroidesstrain with mutated takP gene, a member of the TRAP (tripartite ATP-independent periplasmic) family of transporter, was reported to be showing more resistance towards selenite in the growth medium but the reason for the resistance is unknown. TRAP transporters are the best-studied family of substrate-binding protein and in our previous study it was confirmed that the gene takP in R. sphaeroides is down-regulated by a small non-coding RNA SorY, providing more resistance to the bacterium against the oxidative stress. By comparative growth analysis and sensitivity assays in the presence of 2 mM selenite, it was observed that the SorY knockout strain is more sensitive to selenite while overexpression of the sRNA conferred more resistance to the bacterium like the takP mutant strain. TakP is involved in the import of malate into the cell, which under oxidative stress needs to be down-regulated to limit malate flux into the cell. Limited malate flux leads to metabolic rearrangements in the cell to avoid excessive generation of prooxidant NADH and facilitate constant generation of antioxidant NADPH. In the presence and absence of selenite, a drastic increase in the NADPH and decrease in the NADH levels are reported respectively. Accumulation of metallic selenium in the cytoplasm was detected via atomic absorption spectrophotometer and our analysis clearly demonstrated the presence of more selenium in the electron micrographs of the SorY knockout strain compared to the takP mutant grown under dark semi-aerobic growth conditions in the presence of selenite. Hence based on our analysis, it is confirmed that lack of TakP transporter led to reduced selenite influx into the cytoplasm, relieving cells with limited generation of ROS, eventually exhibiting more resistance against selenite-induced oxidative stress.

A novel strategy of feeding nitrate for cost-effective production of poly-γ-glutamic acid from crude glycerol by Bacillus licheniformis WX-02

Poly-γ-glutamic acid (γ-PGA) is a natural and multifunctional biopolymer. Glycerol, especially crude glycerol, was used as fermentation raw material for production of a variety of chemical products. Here, glycerol was used with nitrate addition for γ-PGA production by Bacillus licheniformis WX-02 at a 5 L fermenter scale. However, nitrite accumulation was discovered in batch fermentation, which resulted in low γ-PGA yield (19.58 g/L). Furthermore, based on transcription analysis and intracellular NADH measurement, nitrite accumulation was probably due to the low content of intracellular NADH in glycerol-based medium, which might be resulted from low efficiency of NADH generation rate and quick NADH consumption by nitrate reduction. Accordingly, different nitrate feeding strategies were designed and optimized in γ-PGA fermentation with glycerol, then applied in fermentation with crude glycerol. Finally, 35.63 g/L of γ-PGA was achieved with the constant-rate nitrate feeding of 0.38 g/(L h) by using crude glycerol, while 36.83 g/L of γ-PGA was achieved with pH-feedback controlling. Therefore, this study provided a novel strategy for cost-effective production of γ-PGA from crude glycerol.

A system-oriented strategy to enhance electron production of Synechocystis sp. PCC6803 in bio-photovoltaic devices: experimental and modeling insights

Bio-photovoltaic devices (BPVs) harness photosynthetic organisms to produce bioelectricity in an eco-friendly way. However, their low energy efficiency is still a challenge. A comprehension of metabolic constraints can result in finding strategies for efficiency enhancement. This study presents a systemic approach based on metabolic modeling to design a regulatory defined medium, reducing the intracellular constraints in bioelectricity generation of Synechocystis sp. PCC6803 through the cellular metabolism alteration. The approach identified key reactions that played a critical role in improving electricity generation in Synechocystis sp. PCC6803 by comparing multiple optimal solutions of minimal and maximal NADH generation using two criteria. Regulatory compounds, which controlled the enzyme activity of the key reactions, were obtained from the BRENDA database. The selected compounds were subsequently added to the culture media, and their effect on bioelectricity generation was experimentally assessed. The power density curves for different culture media showed the BPV fed by Synechocystis sp. PCC6803 suspension in BG-11 supplemented with NH4Cl achieved the maximum power density of 148.27 mW m−2. This produced power density was more than 40.5-fold of what was obtained for the BPV fed with cyanobacterial suspension in BG-11. The effect of the activators on BPV performance was also evaluated by comparing their overpotential, maximum produced power density, and biofilm morphology under different conditions. These findings demonstrated the crucial role of cellular metabolism in improving bioelectricity generation in BPVs.

NADH/NAD+ Redox Imbalance and Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is a common and severe complication of diabetes mellitus. If left untreated, DKD can advance to end stage renal disease that requires either dialysis or kidney replacement. While numerous mechanisms underlie the pathogenesis of DKD, oxidative stress driven by NADH/NAD+ redox imbalance and mitochondrial dysfunction have been thought to be the major pathophysiological mechanism of DKD. In this review, the pathways that increase NADH generation and those that decrease NAD+ levels are overviewed. This is followed by discussion of the consequences of NADH/NAD+ redox imbalance including disruption of mitochondrial homeostasis and function. Approaches that can be applied to counteract DKD are then discussed, which include mitochondria-targeted antioxidants and mimetics of superoxide dismutase, caloric restriction, plant/herbal extracts or their isolated compounds. Finally, the review ends by pointing out that future studies are needed to dissect the role of each pathway involved in NADH-NAD+ metabolism so that novel strategies to restore NADH/NAD+ redox balance in the diabetic kidney could be designed to combat DKD.

A staged representation electrochemical stimulated strategy to regulate intracellular reducing power for improving succinate production by Escherichia coli AFP111.

Escherichia coli AFP111 was previously engineered for succinate production by eliminating byproducts of synthesis pathways. Still, the succinate yield is limited due to the insufficient NADH supplement, when fed with glucose. Microbial electrolysis cell (MEC) allows microorganisms to perform unbalanced fermentation by establishing polarized cathode interaction. In this study, a cathode electrode was used as an additional electron donor to improve succinate synthesis by E. coli AFP111. In MEC with -0.65V (vs. Ag/AgCl) poised on cathode electrode, 95.72% electrons were transferred into cells via neutral red (NR), and the ratio of NADH/NAD+ increased by 2.5-fold. Meanwhile, compared with the control experiment, the value of oxidation-reduction potential (ORP) changed from -240 to -265mV in MEC, which was beneficial for NADH generation. During two-stage fermentation (no potential growth stage followed by electric stimulation) in MEC, succinate yield was increased by 29.09% (the final yield was 0.71g g-1 ), and glucose consumption rate was enhanced by 36.22%. In addition, the carbon flux was pumped to succinate and pyruvate metabolism was enhanced. Staged representation of electrochemical stimulated strategy is effective for succinate producing in engineered E. coli by regulating intracellular reducing power, which provides a new concept for producing reduced metabolite in unbalanced fermentation.

A Model of Aerobic and Anaerobic Metabolism of Hydrogen in the Extremophile Acidithiobacillus ferrooxidans

Hydrogen can serve as an electron donor for chemolithotrophic acidophiles, especially in the deep terrestrial subsurface and geothermal ecosystems. Nevertheless, the current knowledge of hydrogen utilization by mesophilic acidophiles is minimal. A multi-omics analysis was applied on Acidithiobacillus ferrooxidans growing on hydrogen, and a respiratory model was proposed. In the model, [NiFe] hydrogenases oxidize hydrogen to two protons and two electrons. The electrons are used to reduce membrane-soluble ubiquinone to ubiquinol. Genetically associated iron-sulfur proteins mediate electron relay from the hydrogenases to the ubiquinone pool. Under aerobic conditions, reduced ubiquinol transfers electrons to either cytochrome aa3 oxidase via cytochrome bc1 complex and cytochrome c4 or the alternate directly to cytochrome bd oxidase, resulting in proton efflux and reduction of oxygen. Under anaerobic conditions, reduced ubiquinol transfers electrons to outer membrane cytochrome c (ferrireductase) via cytochrome bc1 complex and a cascade of electron transporters (cytochrome c4, cytochrome c552, rusticyanin, and high potential iron-sulfur protein), resulting in proton efflux and reduction of ferric iron. The proton gradient generated by hydrogen oxidation maintains the membrane potential and allows the generation of ATP and NADH. These results further clarify the role of extremophiles in biogeochemical processes and their impact on the composition of the deep terrestrial subsurface.

Sirt3 is a novel target to treat sepsis induced myocardial dysfunction by acetylated modulation of critical enzymes within cardiac tricarboxylic acid cycle

Sepsis induced myocardial dysfunction (SIMD) results in high morbidity and mortality. However, the effective therapeutic strategies for SIMD treatment remain limited. Sirt3 is the main mitochondrial Sirtuin member and is a key modulator of mitochondrial metabolism and function. In this study, we aimed to investigate the effect and mechanism of Sirt3 on SIMD. SIMD was induced by 20 mg/kg Lipopolysaccharides (LPS) injection for 6 h in mice. Sepsis could induce the reduction of cardiac Sirt3 expression and global deficiency of Sirt3 exacerbated cardiac function. Quantitative acetyl-proteomics and cardiac metabolomics analysis revealed that loss of Sirt3 led to hyper-acetylation of critical enzymes within cardiac tricarboxylic acid (TCA) cycle and generation of lactate and NADH, subsequently promotion of cardiac dysfunction after sepsis. Additionally, to evaluate whether Emodin could be utilized as a potential Sirt3 modulator to treat SIMD, male wild type mice (WT mice) or global Sirt3 deficient mice (Sirt3−/− mice) were intraperitoneally injected with 40 mg/kg Emodin for 5 days followed by 20 mg/kg LPS administration for another 6 h and observed that exogenous administration of Emodin could attenuate myocardial dysfunction in septic WT mice. However, septic Sirt3−/− mice can not gain benefit on cardiac performance from Emodin infusion. In conclusion, this study presented the protective role of Sirt3 targeting SIMD, which may provide a potential novel approach to maintain normal cardiac performance after sepsis.

Effects of Ag NPs on denitrification in suspended sediments via inhibiting microbial electron behaviors

The wide use of silver nanoparticles (Ag NPs) inevitably leads to their increasing emission into aquatic environments. However, before their final deposition into sediments, the ecological effects of Ag NPs in suspended sediment (SPS) systems have not received much attention. Herein, we investigated the influences of Ag NPs on denitrification in SPS systems, and explored the potential toxicity mechanism through microbial metabolism (electron behaviors) and isotope tracing (added 15NO3−). After exposure to 10 mg/L Ag NPs, electron generation, transport and consumption during denitrification were clearly inhibited, which led to a decrease in the SPS denitrification rate. Specifically, the generation of NADH (electron donor) was significantly decreased to 59.92–86.47% with the Ag NPs treatments by affecting the degradation of glucose, one of the major reasons for the decreased denitrification. It also indicated that Ag NPs could affect nitrogen metabolism by influencing carbon metabolism. In addition, ETSA was clearly inhibited by the affected electron transfer and reception during denitrification; that was the most direct way in the microbial electron transport chain to affect the SPS denitrification rate. Furthermore, the particle size and concentration of SPS affected the toxicity of Ag NPs. The denitrification process in SPS systems with a smaller particle size and lower particle concentration was easily affected by Ag NPs, suggesting that SPS systems dominated by clay (particle size < 3.9 μm) or that less turbulence (having low SPS concentration) might be at greater risk factor when exposed to NPs. Thus, it is important to understand the risks of pollutants, such as Ag NPs, to biogeochemical cycles and ecosystem function in SPS systems.

Glutaminase Inhibition Overcomes Acquired Resistance to Mitochondrial Complex I in NOTCH1-Driven T-Cell Acute Lymphoblastic Leukemias (T-ALL) Via Block of Glutamine Driven Reductive Metabolism

Notch1-mutated T-ALL is an aggressive hematologic malignancy lacking targeted therapeutic options. Genomic alterations in Notch1-gene and its activated downstream pathways are associated with metabolic stress response and heightened glutamine (Gln) utilization to fuel oxidative phosphorylation (OxPhos) (Kishton at al., Cell Metabolism 2016, 23:649, Herranz at al., Nat Med, 2015, 21(10): 1182-1189). Hence, targeting NOTCH1-associated OxPhos and/or Gln dependency could constitute a plausible therapeutic strategy for T-ALL. In this study we examined metabolic vulnerabilities of NOTCH1-driven T-ALL and tested pre-clinical efficacy of novel mitochondrial complex I (OxPhosi) IACS-010759 and of glutaminase inhibitor CB-839 (GLSi) in T-ALL models including Notch1-mutated T-ALL cell lines, patient-derived xenograft (PDX) and primary T-ALL cells. We have previously reported and confirmed in this expanded study the anti-leukemia efficacy of IACS-010759 (EC50s 0.1-15 nM) (Molina at al., Nat Med, 2018, 24: 1036; Baran at al., Blood, 2018, 132:4020). Metabolic characterization demonstrated that OxPhosi caused striking dose-dependent decrease in basal and maximal oxygen consumption rate (OCR), ATP and NADH generation in T-ALL cell lines and primary T-ALL samples (p&amp;lt;0.001). OxPhosi, similar to knockout of complex I subunit NDUFS4 using CRISPR-CAS9, induced profound changes in T-ALL mitochondria, with induction of mitochondrial reactive oxygen species (ROS), DNA damage, activation of AMPK and inhibition of mTOR pathway. OxPhosi altered cellular energy homeostasis by reduction of TCA cycle intermediates, glutathione and reduction of intracellular nucleotides ATP, CTP, GTP, and UTP, translating into inhibition of DNA and RNA synthesis (p&amp;lt;0.0001) (by UPLC-MS/MS). IACS-010759 significantly reduced the glucose flux through the TCA cycle, redirected it towards lactate production and triggered increased utilization of Gln for fueling of the TCA cycle and reductive metabolism (Fig.1, Flux metabolic analysis SIRM). In concert with these findings, supplementation with Gln partially rescued growth-inhibitory effects of OxPhos inhibition. These results uncover metabolic gap used by T-ALL to escape OxPhos block, and identifying reliance on glutaminolysis as a critical therapeutic target. To confirm that blockade of Gln entry into TCA cycle with GLSi synergistically reduced viable ALL cell numbers, we studied potential synergy of OxPhosi and GLSi. The key role of Gln in maintaining energy production and cell proliferation via OxPhos in Notch1-mutated T-ALL cells was confirmed by the findings that Gln starvation or pharmacological GLS inhibition by CB-839 reduced ATP production and OCR and decreased cell proliferation by more than 50% in vitro (Fig.2, Fig.3). Dual blockade of OxPhos together with GLS induced DNA damage response via accumulation of ROS upon glutathione deprivation, induced AMPK signaling through profound reduction of all adenosine related intermediates and inhibited mTOR signaling. This translated into significant reduction of leukemia burden and extension of overall survival in vivo (p&amp;lt;0.0001) in Notch1-mutated T-ALL PDX models (n=2) with IACS-010759/CB-839 co-treatment and in the Notch1-mutated GLS fl/fl murine model upon tamoxifen-induced GLS knockout (p&amp;lt;0.0001) (Fig.4, Fig.5). In summary, our findings indicate that dual blockade of metabolic processes by inhibiting complex I of mitochondria and restricting Gln utilization results in metabolic catastrophe in Notch1-mutated T-ALL associated with energy depletion and oxidative stress, which combined severely inhibit T-ALL growth and survival. We postulate that targeting this unique metabolic vulnerability of Notch1-mutated T-ALL cells constitutes a novel therapeutic modality in this aggressive malignancy. Disclosures Kuruvilla: The University of Texas M.D.Anderson Cancer Center: Employment. Jabbour:AbbVie: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Cyclacel LTD: Research Funding; Takeda: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding; Amgen: Consultancy, Research Funding. Konopleva:Agios: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Calithera: Research Funding; Astra Zeneca: Research Funding; Kisoji: Consultancy, Honoraria; Ascentage: Research Funding; Genentech: Honoraria, Research Funding; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Eli Lilly: Research Funding; Cellectis: Research Funding; Forty-Seven: Consultancy, Honoraria; Ablynx: Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents &amp; Royalties; AbbVie: Consultancy, Honoraria, Research Funding.

Pleiotropic regulation of a glucose-specific PTS in Clostridium acetobutylicum for high-efficient butanol production from corn stover without detoxification

BackgroundCorn stover (CS) is evaluated as the most favorable candidate feedstock for butanol production via microbial acetone–butanol–ethanol (ABE) fermentation by Clostridium acetobutylicum. By independent acid pretreatment and enzymatic hydrolysis, fermentable sugars (mainly glucose and xylose) were released, of which glucose was naturally utilized as the most preferred carbon source by C. acetobutylicum. However, the ABE fermentation using corn stover hydrolysate (CSH) without detoxification is typically limited to poor sugars utilization, butanol production and productivity. In the presence of pretreatment-derived inhibitors, the intracellular ATP and NADH, as important factors involved in cell growth, solventogenesis initiation and stress response, are exceedingly challenged owing to disrupted glucose phosphotransferase system (PTS). Therefore, there is a necessity to develop effective engineering approaches to overcome these limitations for high-efficient butanol production from CSH without detoxification.ResultsPTS-engineered C. acetobutylicum strains were constructed via overexpression and knockout of gene glcG encoding glucose-specific PTS IICBA, which pleiotropically regulated glucose utilization, cell growth, solventogenesis and inhibitors tolerance. The PTSGlcG-overexpressing strain exhibited high fermentation efficiency, wherein butanol production and productivity was 11.1 g/L and 0.31 g/L/h, compared to those of 11.0 g/L and 0.15 g/L/h with the PTSGlcG-deficient strain. During CSH culture without detoxification, the PTSGlcG-overexpressing strain exhibited desirable inhibitors tolerance and solventogenesis with butanol production of 10.0 g/L, increased by 300% and 400% compared to those of 2.5 and 2.0 g/L with the control and PTSGlcG-deficient strains, respectively. As a result of extra glucose and 10 g/L CaCO3 addition into CSH, butanol production and productivity were further maximized to 12.5 g/L and 0.39 g/L/h. These validated improvements on the PTSGlcG-overexpressing strain were ascribed to not only efficient glucose transport but also its cascading effects on intracellular ATP and NADH generation, solventogenesis initiation and inhibitors tolerance at the exponential growth phase.ConclusionThe PTSGluG regulation could be an effective engineering approach for high-efficient ABE fermentation from lignocellulosic hydrolysates without detoxification or wastewater generation, providing fundamental information for economically sustainable butanol production with high productivity.

The Human Transient Receptor Potential Melastatin 2 Ion Channel Modulates ROS Through Nrf2

Transient receptor potential melastatin channel subfamily member 2 (TRPM2) has an essential role in protecting cell viability through modulation of oxidative stress. TRPM2 is highly expressed in cancer. When TRPM2 is inhibited, mitochondria are dysfunctional, ROS levels are increased, and cell viability is reduced. Here, the importance of NF-E2-related factor (Nrf2) in TRPM2-mediated suppression of oxidant stress was explored. In TRPM2 depleted cells, antioxidant cofactors glutathione, NADPH, and NADH were significantly reduced. Cytoplasmic and nuclear expression of Nrf2 and of IQGAP1, a modulator of Nrf2 stability regulated by intracellular calcium, were decreased. Antioxidant enzymes transcriptionally regulated by Nrf2 and involved in GSH, NADPH, and NADH generation were significantly lower including PRX1 and PRX3, GPX4, GSTP1, GCLC, and MTHFD2. The glutamine pathway leading to GSH production was suppressed, and ATP and GTP levels were impaired. Reconstitution with wild type TRPM2 or Nrf2, but not TRPM2 pore mutant E960D, rescued expression of enzymes downstream of Nrf2 and restored GSH and GTP. Cell viability, ROS, NADPH, NADH, and ATP levels were fully rescued by TRPM2 and partially by Nrf2. These data show that TRPM2 maintains cell survival following oxidative stress through modulation of antioxidant pathways and cofactors regulated by Nrf2.

Effect of cutting on the reactive oxygen species accumulation and energy change in postharvest melon fruit during storage

The effect of cutting on the accumulation of reactive oxygen species (ROS) and energy change in melon fruit (cv. Xizhoumi-17, stored at 15 °C) during storage was explored. The key metabolites, enzymes, and genes involved in ROS accumulation and energy metabolism were investigated. Cutting melon fruit changed the respiratory metabolism pathway, increased the respiration rate, and maintained higher activities of H+-adenosine triphosphatase and Ca2+-adenosine triphosphatase. This led to the large generation of adenosine triphosphate, adenosine diphosphate, NADPH, and NADH in fresh-cut melon fruit. Furthermore, cutting enhanced the NADPH oxidase activity, which induced a higher ROS level and further resulted in higher malondialdehyde content and electrolyte leakage. However, only the activities of peroxidase (POD) and glutathione peroxidase (GPX) were enhanced in fresh-cut melon during the early stage of storage. In addition, lower antioxidant system activity was observed in fresh-cut melon at the late stage of storage. This was indicated by the lower levels of ascorbic acid, glutathione, POD, GPX, ascorbate peroxidase, and glutathione reductase. These lower levels were related to changes of genes related to ROS accumulation and energy metabolism in fresh-cut melon. These findings demonstrate that cut-wounding stress changed the respiratory metabolism pathway in melon fruit and led to a higher energy status, accompanied by increased ROS accumulation.

Enhancement of acetyl-CoA flux for photosynthetic chemical production by pyruvate dehydrogenase complex overexpression in Synechococcus elongatus PCC 7942

Genetic manipulation in cyanobacteria enables the direct production of valuable chemicals from carbon dioxide. However, there are still very few reports of the production of highly effective photosynthetic chemicals. Several synthetic metabolic pathways (e.g., isopropanol, acetone, isoprene, and fatty acids) have been constructed by branching from acetyl-CoA and malonyl-CoA, which are key intermediates for photosynthetic chemical production downstream of pyruvate decarboxylation. Recent reports of the absolute determination of cellular metabolites in Synechococcus elongatus PCC 7942 have shown that its acetyl-CoA levels corresponded to about one hundredth of the pyruvate levels. In short, one of the reasons for lower photosynthetic chemical production from acetyl-CoA and malonyl-CoA was the smaller flux to acetyl-CoA. Pyruvate decarboxylation is a primary pathway for acetyl-CoA synthesis from pyruvate and is mainly catalyzed by the pyruvate dehydrogenase complex (PDHc). In this study, we tried to enhance the flux toward acetyl-CoA from pyruvate by overexpressing PDH genes and, thus, catalyzing the conversion of pyruvate to acetyl-CoA via NADH generation. The overexpression of PDH genes cloned from S. elongatus PCC 7942 significantly increased PDHc enzymatic activity and intracellular acetyl-CoA levels in the crude cell extract. Although growth defects were observed in overexpressing strains of PDH genes, the combinational overexpression of PDH genes with the synthetic metabolic pathway for acetate or isopropanol resulted in about 7-fold to 9-fold improvement in its production titer, respectively (9.9 mM, 594.5 mg/L acetate, 4.9 mM, 294.5 mg/L isopropanol). PDH genes overexpression would, therefore, be useful not only for the production of these model chemicals, but also for the production of other chemicals that require acetyl-CoA as a key precursor.

Tetrabromobisphenol A (TBBPA) inhibits denitrification via regulating carbon metabolism to decrease electron donation and bacterial population

The potential risks of brominated flame retardants (BFRs) like tetrabromobisphenol A (TBBPA) have attracted much attention. However, the influence of TBBPA on functional microbes remains poorly understood, especially with regards to denitrification, which is closely related with the carbon and nitrogen cycles, eutrophication and greenhouse gas emission. Herein, we found that 1.0 mg/L of TBBPA significantly decreased the total nitrogen removal efficiency by 81.7%, but increased the accumulation of NO2−–N (by 81.5%) and N2O (by 172-fold). This was found to be underlie by the significant decrease in both the denitrifying capability of denitrifiers and total bacterial population. Further investigation revealed that TBBPA inhibited the pathways of glucolysis and pentose phosphate, and promoted glyoxylate bypass via regulating genes expressions of key enzymes (such as glucose-6-phosphate isomerase, pyruvate dehydrogenase, isocitrate lyase, etc.), then decreased the generation of NADH serving as electron donor for denitrification, and inhibited the denitrifying capability of denitrifiers. Moreover, insufficient NADH stimulated the accumulation of denitrifying intermediates (NO2−–N and N2O), which induced the increase of reactive nitrogen species (RNS), whose accumulation decreased proliferation and increased apoptosis of denitrifying bacteria. Finally, the decrease in the denitrifying capability of denitrifier and bacterial population resulted in negative denitrifying performance.