Excess Reducing Equivalents Research Articles

Elucidation of triacylglycerol catabolism in Yarrowia lipolytica: How cells balance acetyl-CoA and excess reducing equivalents

Yarrowia lipolytica is an industrial yeast that can convert waste oil to value-added products. However, it is unclear how this yeast metabolizes lipid feedstocks, specifically triacylglycerol (TAG) substrates. This study used 13C-metabolic flux analysis (13C-MFA), genome-scale modeling, and transcriptomics analyses to investigate Y. lipolytica W29 growth with oleic acid, glycerol, and glucose. Transcriptomics data were used to guide 13C-MFA model construction and to validate the 13C-MFA results. The 13C-MFA data were then used to constrain a genome-scale model (GSM), which predicted Y. lipolytica fluxes, cofactor balance, and theoretical yields of terpene products. The three data sources provided new insights into cellular regulation during catabolism of glycerol and fatty acid components of TAG substrates, and how their consumption routes differ from glucose catabolism. We found that (1) over 80% of acetyl-CoA from oleic acid is processed through the glyoxylate shunt, a pathway that generates less CO2 compared to the TCA cycle, (2) the carnitine shuttle is a key regulator of the cytosolic acetyl-CoA pool in oleic acid and glycerol cultures, (3) the oxidative pentose phosphate pathway and mannitol cycle are key routes for NADPH generation, (4) the mannitol cycle and alternative oxidase activity help balance excess NADH generated from β-oxidation of oleic acid, and (5) asymmetrical gene expressions and GSM simulations of enzyme usage suggest an increased metabolic burden for oleic acid catabolism.

Acclimation of anaerobic fermentation microbiome with acetate and ethanol for chain elongation and the biochemical response

Medium chain fatty acids (MCFAs) production is a promising method for resource recovery from organic wastes. In this study, the microbial community structure shift along the long-term acclimation experiment and the concomitant effect of H2 level on chain elongation performance was investigated. Chain elongation microbiome could be rapidly acclimated from traditional anaerobic fermentation consortia. Genera Caproiciproducens, Clostridium sensu stricto 12, Rummeliibacillus and Oscillibacter was found to be dominant during the operation. The H2 was accumulated in the headspace by increasing the ethanol input, which inhibited oxidation of caproate and butyrate immediately, while its inhibition effect on chain elongation was delayed. H2 level in the headspace was positively correlated to the MCFAs production related bacteria. However, too much H2 accumulated might be suppressive for MCFAs production in the long term. It might result from the thermodynamic barrier for discarding excess reducing equivalents under high H2 level, which further gave rise to ethanol accumulation in this system.

Alternative Pathway Is Involved in Hydrogen Peroxide-Enhanced Cadmium Tolerance in Hulless Barley Roots.

Hulless barley, grown in the Qinghai Tibet Plateau, has a wide range of environmental stress tolerance. Alternative pathway (AP) and hydrogen peroxide (H2O2) are involved in enhancing plant tolerance to environmental stresses. However, the relationship between H2O2 and AP in hulless barley tolerance to cadmium (Cd) stress remains unclear. In the study, the role and relationship of AP and H2O2 under Cd stress were investigated in hulless barley (Kunlun14) and common barley (Ganpi6). Results showed that the expression level of alternative oxidase (AOX) genes (mainly AOX1a), AP capacity (Valt), and AOX protein were clearly induced more in Kunlun14 than in Ganpi 6 under Cd stress; moreover, these parameters were further enhanced by applying H2O2. Malondialdehyde (MDA) content, electrolyte leakage (EL) and NAD(P)H to NAD(P) ratio also increased in Cd-treated roots, especially in Kunlun 14, which can be markedly alleviated by exogenous H2O2. However, this mitigating effect was aggravated by salicylhydroxamic acid (SHAM, an AOX inhibitor), suggesting AP contributes to the H2O2-enhanced Cd tolerance. Further study demonstrated that the effect of SHAM on the antioxidant enzymes and antioxidants was minimal. Taken together, hulless barley has higher tolerance to Cd than common barley; and in the process, AP exerts an indispensable function in the H2O2-enhanced Cd tolerance. AP is mainly responsible for the decrease of ROS levels by dissipating excess reducing equivalents.

Respiration responses of wheat seedlings to treatment with trehalose under heat stress

Heat stress limits wheat production and trehalose can improve stress tolerance. How trehalose affects wheat respiration is unclear. In this study, we investigated the effects of exogenous trehalose on the respiration of wheat seedlings during heat stress and the subsequent recovery period. Trehalose pretreatment significantly increased the expression of the alternative oxidase genes AOX1a and AOX1c under heat stress, indicating that trehalose pretreatment increased the capacity of the alternative respiration pathway (AP) in response to heat stress. Trehalose pretreatment also enhanced the activity of the malate-oxaloacetate (Mal-OAA) shuttle and ameliorated the decrease in photosynthetic activity caused by heat stress. However, when the AP was inhibited by salicylhydroxamic acid under heat stress, both Mal-OAA shuttle activity and photosynthetic efficiency were substantially reduced in the control and trehalose pretreatment groups. In addition, trehalose pretreatment helped to maintain inner mitochondrial respiratory activity and the activity of Complex II during heat stress, particularly the coupling of oxidative phosphorylation with the Complex II electron transport chain, thereby mitigating heat-related damage to the cytochrome pathway (CP). Taken together, these results suggest that exogenous trehalose enhanced the AP and reduced damage to the CP under heat stress in wheat seedlings, thus maintaining cellular energy metabolism. Up-regulation of the AP by trehalose pretreatment may improve the heat tolerance of wheat seedlings by dissipating excess reducing equivalents transported through the Mal-OAA shuttle, thereby protecting photosynthetic performance.

A Balance between the Activities of Chloroplasts and Mitochondria Is Crucial for Optimal Plant Growth.

Energy metabolism in plant cells requires a balance between the activities of chloroplasts and mitochondria, as they are the producers and consumers of carbohydrates and reducing equivalents, respectively. Recently, we showed that the overexpression of Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2), a phosphatase dually anchored on the outer membranes of chloroplasts and mitochondria, can boost the plant growth and seed yield of Arabidopsis thaliana by coordinating the activities of both organelles. However, when AtPAP2 is solely overexpressed in chloroplasts, the growth-promoting effects are less optimal, indicating that active mitochondria are required for dissipating excess reducing equivalents from chloroplasts to maintain the optimal growth of plants. It is even more detrimental to plant productivity when AtPAP2 is solely overexpressed in mitochondria. Although these lines contain high level of adenosine triphosphate (ATP), they exhibit low leaf sucrose, low seed yield, and early senescence. These transgenic lines can be useful tools for studying how hyperactive chloroplasts or mitochondria affect the physiology of their counterparts and how they modify cellular metabolism and plant physiology.

Upregulation of mitochondrial alternative oxidase pathway protects photosynthetic apparatus against photodamage under chilling stress in Rumex K-1 leaves

This study aimed to clarify the role of mitochondrial alternative oxidase (AOX) pathway upregulation in photoprotection under chilling stress in Rumex K-1 leaves. Malate-oxaloacetate shuttleꞌs activity and AOX pathwayꞌs capacity were synchronously enhanced with light intensity increase both in control and chilling-treated leaves. Besides, the AOX pathway was upregulated more significantly when irradiated by light in chilling-treated leaves than that of control. These results suggested that excess reducing equivalents generated by photosynthesis were exported from chloroplasts and oxidized by the AOX pathway at a potentially higher rate in chilling-treated leaves than in control, which avoided the overaccumulation of excess reducing equivalents under light conditions. Also, inhibition of the AOX pathway led to more severe accumulation of hydrogen peroxide in chilling-treated leaves than that in control. Therefore, the mitochondrial AOX pathway was specifically upregulated and employed for sinking excess reducing equivalents produced by photosynthesis, which protected photosynthetic apparatus from photodamage under chilling stress in Rumex K-1 leaves.

Antioxidant enzymes and the mitochondrial alternative oxidase pathway play important roles in chilling tolerance of Haematococcus pluvialis at the green motile stage

During outdoor large-scale cultivation, Haematococcus pluvialis cells, especially at the green motile stage, are frequently exposed to chilling stress in winter. However, the physiological responses and adaptation mechanisms to chilling in H. pluvialis have not been characterized. In order to better understand the chilling tolerance mechanisms of H. pluvialis in the green motile stage, the responses of photosynthetic characteristics and photoprotective mechanisms to chilling were investigated. Chilling stress significantly decreased the activities of key enzymes (ribulose-1,5-bisphosphate carboxylase/oxygenase and NADP glyceraldehyde-3-phosphate dehydrogenase) in photosynthetic carbon assimilation, which would cause the accumulation of excess reducing equivalents and an imbalance of light absorption and energy utilization, leading to more severe photoinhibition. After chilling treatment, there was no induction of cyclic electron flow, non-photochemical quenching, fluorescence emission, or catalase, indicating these factors do not protect H. pluvialis from photoinhibition. However, chilling significantly enhanced the activities of antioxidant enzymes (superoxide dismutase, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase), which efficiently dissipated excess electrons generated by photosynthetic linear electron flow (LEF). The malate-oxaloacetate shuttle was activated and the mitochondrial alternative oxidase (AOX) pathway was significantly up-regulated after chilling, indicating the transport of excess reducing equivalents generated by photosynthetic LEF to cytosol and mitochondria and oxidization by the AOX pathway, the mitochondrial non-phosphorylating pathway. As a result, the LEF was not inhibited by negative feedback and the H. pluvialis cells were well protected under chilling stress in the light, with no change in the effective quantum yield of PSII (ΦPSII) and the yield of non-regulated energy dissipation of PSII (ΦNO). The results indicate that antioxidant enzymes and the mitochondrial AOX pathway play important roles in protecting H. pluvialis at the green motile stage against chilling stress.

Impact of iron reduction on the metabolism of Clostridium acetobutylicum.

SummaryIron is essential for most living organisms. In addition, its biogeochemical cycling influences important processes in the geosphere (e.g., the mobilization or immobilization of trace elements and contaminants). The reduction of Fe(III) to Fe(II) can be catalysed microbially, particularly by metal‐respiring bacteria utilizing Fe(III) as a terminal electron acceptor. Furthermore, Gram‐positive fermentative iron reducers are known to reduce Fe(III) by using it as a sink for excess reducing equivalents, as a form of enhanced fermentation. Here, we use the Gram‐positive fermentative bacterium Clostridium acetobutylicum as a model system due to its ability to reduce heavy metals. We investigated the reduction of soluble and solid iron during fermentation. We found that exogenous (resazurin, resorufin, anthraquinone‐2,6‐disulfonate) as well as endogenous (riboflavin) electron mediators enhance solid iron reduction. In addition, iron reduction buffers the pH, and elicits a shift in the carbon and electron flow to less reduced products relative to fermentation. This study underscores the role fermentative bacteria can play in iron cycling and provides insights into the metabolic profile of coupled fermentation and iron reduction with laboratory experiments and metabolic network modelling.

Microarray analysis of high light intensity stress on hydrogen production metabolism of Rhodobacter capsulatus

Biohydrogen obtained from purple non sulfur bacteria (PNSB) is an environmentally friendly alternative for hydrogen production. PNSB can be employed in large scale outdoor photobioreactors to produce hydrogen by photofermentation with sunlight as the light source. In external environmental conditions, however, bacteria can experience stress due to high light intensities, which can inhibit or slow down hydrogen production. Previous studies with other PNSB showed varying responses to light intensities (above 4000 lux), in some cases improving, and in others adversely affecting hydrogen production.In this study, Rhodobacter capsulatus, a PNSB species that produce hydrogen efficiently from dark fermenter effluents containing acetate, was used to investigate the effects of high light intensity stress on the hydrogen production metabolism at the gene expression level. A microarray analysis was carried out using a custom-design Affymetrix GeneChip TR_RCH2a520699F. R. capsulatus DSM1710 was grown under a cyclic illumination of 2000 and 7000 lux (12 h light/12 h dark) in a hydrogen production medium having 30 mM acetate and 2 mM glutamate, and was exposed to a high light intensity (10,000 lux) for 1 h in the middle of a light period. The results reveal that photosynthetic reaction center genes were down-regulated in order to protect the photosynthetic membrane from damage. On the other hand, the expression of nitrogenase and electron transport system genes were enhanced by high light intensity. These results show that a high light intensity stress drives R. capsulatus to direct gene expression towards hydrogen production, which supports the hypothesis that hydrogen production is a way for the disposal of excess reducing equivalents to maintain the internal redox balance.

Constraints-based analysis identifies NAD+ recycling through metabolic reprogramming in antibiotic resistant Chromobacterium violaceum.

In the post genomic era, high throughput data augment stoichiometric flux balance models to compute accurate metabolic flux states, growth and energy phenotypes. Investigating altered metabolism in the context of evolved resistant genotypes potentially provide simple strategies to overcome drug resistance and induce susceptibility to existing antibiotics. A genome-scale metabolic model (GSMM) for Chromobacterium violaceum, an opportunistic human pathogen, was reconstructed using legacy data. Experimental constraints were used to represent antibiotic susceptible and resistant populations. Model predictions were validated using growth and respiration data successfully. Differential flux distribution and metabolic reprogramming were identified as a response to antibiotics, chloramphenicol and streptomycin. Streptomycin resistant populations (StrpR) redirected tricarboxylic acid (TCA) cycle flux through the glyoxylate shunt. Chloramphenicol resistant populations (ChlR) resorted to overflow metabolism producing acetate and formate. This switch to fermentative metabolism is potentially through excess reducing equivalents and increased NADH/NAD ratios. Reduced proton gradients and changed Proton Motive Force (PMF) induced by antibiotics were also predicted and verified experimentally using flow cytometry based membrane potential measurements. Pareto analysis of NADH and ATP maintenance showed the decoupling of electron transfer and ATP synthesis in StrpR. Redox homeostasis and NAD+ cycling through rewiring metabolic flux was implicated in re-sensitizing antibiotic resistant C. violaceum. These approaches can be used to probe metabolic vulnerabilities of resistant pathogens. On the verge of a post-antibiotic era, we foresee a critical need for systems level understanding of pathogens and host interaction to extend shelf life of antibiotics and strategize novel therapies.

24-epibrassinolide alleviate drought-induced photoinhibition in Capsicum annuum via up-regulation of AOX pathway

The aim of this study was to explore whether application of BRs could alleviate drought-induced photoinhibition in pepper leaves by improving the photosynthetic electron transport via up-regulation of alternative oxidase (AOX) pathway, we examined the effects of 24-epibrassinolide (EBR, 0.1 μM) and salicylhydroxamic acid (SHAM, 1 mM) on the AOX pathway capacity, matale/oxaloacetate (OAA) shuttle, and chlorophyll fluorescence in pepper (Capsicum annuum L.) leaves under drought condition (treated by 15% polyethylene glycol, PEG). In this study, the leaf relative water content (LRWC) decreased from 92.0% to 47.0% by drought treatment for 4 d. Drought induced the up-regulation of AOX pathway and increased the activities of NADP-malate dehydrogenase (MDH) and NAD-MDH. Inhibition of AOX pathway decreased NADP-MDH and NAD-MDH activities and enhanced the increases of NADPH and NADPH/NADP+ ratio in leaves under drought condition. Meanwhile, inhibition of AOX pathway aggravated the decreases of maximal quantum efficiency of PSII (Fv/Fm), quantum efficiency of PSII (ФPSII), photochemical quenching coefficient (qP), and photosynthetic electron transport rate (ETR) in leaves under drought condition. These results indicated that up-regulation of AOX pathway could activate malate/OAA shuttle and dissipate the excess reducing equivalents in chloroplast, resulting in an optimization of PSII efficiency and photosynthetic electron transport. EBR treatment up-regulated the activity of AOX pathway in leaves under well-watered and drought conditions. EBR treatment also significantly alleviated the decreases of Fv/Fm, ФPSII, qP, and ETR in leaves with/without SHAM treatment under drought condition, however, the improvement degree was more efficiently in leaves with SHAM treatment. Furthermore, EBR increased the activities of NADP-MDH and NAD-MDH, and significantly decreased NADPH/NADP+ ratio in leaves with/without SHAM treatment under drought condition. These results suggested that EBR-induced protection against photoinhibition was partly related to the up-regulation of AOX pathway by dissipating excess reducing equivalents.

Reductive Stress in Inflammation-Associated Diseases and the Pro-Oxidant Effect of Antioxidant Agents.

Reductive stress (RS) is the counterpart oxidative stress (OS), and can occur in response to conditions that shift the redox balance of important biological redox couples, such as the NAD+/NADH, NADP+/NADPH, and GSH/GSSG, to a more reducing state. Overexpression of antioxidant enzymatic systems leads to excess reducing equivalents that can deplete reactive oxidative species, driving the cells to RS. A feedback regulation is established in which chronic RS induces OS, which in turn, stimulates again RS. Excess reducing equivalents may regulate cellular signaling pathways, modify transcriptional activity, induce alterations in the formation of disulfide bonds in proteins, reduce mitochondrial function, decrease cellular metabolism, and thus, contribute to the development of some diseases in which NF-κB, a redox-sensitive transcription factor, participates. Here, we described the diseases in which an inflammatory condition is associated to RS, and where delayed folding, disordered transport, failed oxidation, and aggregation are found. Some of these diseases are aggregation protein cardiomyopathy, hypertrophic cardiomyopathy, muscular dystrophy, pulmonary hypertension, rheumatoid arthritis, Alzheimer’s disease, and metabolic syndrome, among others. Moreover, chronic consumption of antioxidant supplements, such as vitamins and/or flavonoids, may have pro-oxidant effects that may alter the redox cellular equilibrium and contribute to RS, even diminishing life expectancy.

The Physiological Functions and Structural Determinants of Catalytic Bias in the [FeFe]-Hydrogenases CpI and CpII of Clostridium pasteurianum Strain W5.

The first generation of biochemical studies of complex, iron-sulfur-cluster-containing [FeFe]-hydrogenases and Mo-nitrogenase were carried out on enzymes purified from Clostridium pasteurianum (strain W5). Previous studies suggested that two distinct [FeFe]-hydrogenases are expressed differentially under nitrogen-fixing and non-nitrogen-fixing conditions. As a result, the first characterized [FeFe]-hydrogenase (CpI) is presumed to have a primary role in central metabolism, recycling reduced electron carriers that accumulate during fermentation via proton reduction. A role for capturing reducing equivalents released as hydrogen during nitrogen fixation has been proposed for the second hydrogenase, CpII. Biochemical characterization of CpI and CpII indicated CpI has extremely high hydrogen production activity in comparison to CpII, while CpII has elevated hydrogen oxidation activity in comparison to CpI when assayed under the same conditions. This suggests that these enzymes have evolved a catalytic bias to support their respective physiological functions. Using the published genome of C. pasteurianum (strain W5) hydrogenase sequences were identified, including the already known [NiFe]-hydrogenase, CpI, and CpII sequences, and a third hydrogenase, CpIII was identified in the genome as well. Quantitative real-time PCR experiments were performed in order to analyze transcript abundance of the hydrogenases under diazotrophic and non-diazotrophic growth conditions. There is a markedly reduced level of CpI gene expression together with concomitant increases in CpII gene expression under nitrogen-fixing conditions. Structure-based analyses of the CpI and CpII sequences reveal variations in their catalytic sites that may contribute to their alternative physiological roles. This work demonstrates that the physiological roles of CpI and CpII are to evolve and to consume hydrogen, respectively, in concurrence with their catalytic activities in vitro, with CpII capturing excess reducing equivalents under nitrogen fixation conditions. Comparison of the primary sequences of CpI and CpII and their homologs provides an initial basis for identifying key structural determinants that modulate hydrogen production and hydrogen oxidation activities.

Metabolic engineering of Rhodopseudomonas palustris for the obligate reduction of n-butyrate to n-butanol

BackgroundRhodopseudomonas palustris is a versatile microbe that encounters an innate redox imbalance while growing photoheterotrophically with reduced substrates. The resulting excess in reducing equivalents, together with ATP from photosynthesis, could be utilized to drive a wide range of bioconversions. The objective of this study was to genetically modify R. palustris to provide a pathway to reduce n-butyrate into n-butanol for maintaining redox balance.ResultsHere, we constructed and expressed a plasmid-based pathway for n-butanol production from Clostridium acetobutylicum ATCC 824 in R. palustris. We maintained the environmental conditions in such a way that this pathway functioned as the obligate route to re-oxidize excess reducing equivalents, resulting in an innate selection pressure. The engineered strain of R. palustris grew under otherwise restrictive redox conditions and achieved concentrations of 1.5 mM n-butanol at a production rate of 0.03 g L−1 day−1 and a selectivity (i.e., products compared to the consumed substrate) of close to 40%. Since the theoretical maximum selectivity is 45%, the engineered strain converted close to its maximum selectivity.ConclusionsThe innate redox imbalance of R. palustris can be used to drive the reduction of n-butyrate into n-butanol after expression of a plasmid-based enzyme from a butanol-producing Clostridium strain.

Contribution of the Alternative Respiratory Pathway to PSII Photoprotection in C3 and C4 Plants

The mechanism by which the mitochondrial alternative oxidase (AOX) pathway contributes to photosystem II (PSII) photoprotection is in dispute. It was generally thought that the AOX pathway protects photosystems by dissipating excess reducing equivalents exported from chloroplasts through the malate/oxaloacetate (Mal/OAA) shuttle and thus preventing the over-reduction of chloroplasts. In this study, using the aox1a Arabidopsis mutant and nine other C3 and C4 plant species, we revealed an additional action model of the AOX pathway in PSII photoprotection. Although the AOX pathway contributes to PSII photoprotection in C3 leaves treated with high light, this contribution was observed to disappear when photorespiration was suppressed. Disruption or inhibition of the AOX pathway significantly decreased the photorespiration in C3 leaves. Moreover, the AOX pathway did not respond to high light and contributed little to PSII photoprotection in C4 leaves possessing a highly active Mal/OAA shuttle but with little photorespiration. These results demonstrate that the AOX pathway contributes to PSII photoprotection in C3 plants by maintaining photorespiration to detoxify glycolate and via the indirect export of excess reducing equivalents from chloroplasts by the Mal/OAA shuttle. This new action model explains why the AOX pathway does not contribute to PSII photoprotection in C4 plants.

Elucidation of the co-metabolism of glycerol and glucose in Escherichia coli by genetic engineering, transcription profiling, and (13)C metabolic flux analysis.

BackgroundGlycerol, a byproduct of biodiesel, has become a readily available and inexpensive carbon source for the production of high-value products. However, the main drawback of glycerol utilization is the low consumption rate and shortage of NADPH formation, which may limit the production of NADPH-requiring products. To overcome these problems, we constructed a carbon catabolite repression-negative ΔptsGglpK* mutant by both blocking a key glucose PTS transporter and enhancing the glycerol conversion. The mutant can recover normal growth by co-utilization of glycerol and glucose after loss of glucose PTS transporter. To reveal the metabolic potential of the ΔptsGglpK* mutant, this study examined the flux distributions and regulation of the co-metabolism of glycerol and glucose in the mutant.ResultsBy labeling experiments using [1,3-13C]glycerol and [1-13C]glucose, 13C metabolic flux analysis was employed to decipher the metabolisms of both the wild-type strain and the ΔptsGglpK* mutant in chemostat cultures. When cells were maintained at a low dilution rate (0.1 h−1), the two strains showed similar fluxome profiles. When the dilution rate was increased, both strains upgraded their pentose phosphate pathway, glycolysis and anaplerotic reactions, while the ΔptsGglpK* mutant was able to catabolize much more glycerol than glucose (more than tenfold higher). Compared with the wild-type strain, the mutant repressed its flux through the TCA cycle, resulting in higher acetate overflow. The regulation of fluxomes was consistent with transcriptional profiling of several key genes relevant to the TCA cycle and transhydrogenase, namely gltA, icdA, sdhA and pntA. In addition, cofactor fluxes and their pool sizes were determined. The ΔptsGglpK* mutant affected the redox NADPH/NADH state and reduced the ATP level. Redox signaling activated the ArcA regulatory system, which was responsible for TCA cycle repression.ConclusionsThis work employs both 13C-MFA and transcription/metabolite analysis for quantitative investigation of the co-metabolism of glycerol and glucose in the ΔptsGglpK* mutant. The ArcA regulatory system dominates the control of flux redistribution. The ΔptsGglpK* mutant can be used as a platform for microbial cell factories for the production of biofuels and biochemicals, since most of fuel molecule (e.g., alcohols) synthesis requires excess reducing equivalents.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0591-1) contains supplementary material, which is available to authorized users.

Using directed evolution to improve hydrogen production in chimeric hydrogenases from Clostridia species.

Hydrogenases are enzymes that play a key role in controlling excess reducing equivalents in both photosynthetic and anaerobic organisms. This enzyme is viewed as potentially important for the industrial generation of hydrogen gas; however, insufficient hydrogen production has impeded its use in a commercial process. Here, we explore the potential to circumvent this problem by directly evolving the Fe-Fe hydrogenase genes from two species of Clostridia bacteria. In addition, a computational model based on these mutant sequences was developed and used as a predictive aid for the isolation of enzymes with even greater efficiency in hydrogen production. Two of the improved mutants have a logarithmic increase in hydrogen production in our in vitro assay. Furthermore, the model predicts hydrogenase sequences with hydrogen productions as high as 540-fold over the positive control. Taken together, these results demonstrate the potential of directed evolution to improve the native bacterial hydrogenases as a first step for improvement of hydrogenase activity, further in silico prediction, and finally, construction and demonstration of an improved algal hydrogenase in an in vivo assay of C. reinhardtii hydrogen production.

Single-Genotype Syntrophy by Rhodopseudomonas palustris Is Not a Strategy to Aid Redox Balance during Anaerobic Degradation of Lignin Monomers.

Rhodopseudomonas palustris has emerged as a model microbe for the anaerobic metabolism of p-coumarate, which is an aromatic compound and a primary component of lignin. However, under anaerobic conditions, R. palustris must actively eliminate excess reducing equivalents through a number of known strategies (e.g., CO2 fixation, H2 evolution) to avoid lethal redox imbalance. Others had hypothesized that to ease the burden of this redox imbalance, a clonal population of R. palustris could functionally differentiate into a pseudo-consortium. Within this pseudo-consortium, one sub-population would perform the aromatic moiety degradation into acetate, while the other sub-population would oxidize acetate, resulting in a single-genotype syntrophy through acetate sharing. Here, the objective was to test this hypothesis by utilizing microbial electrochemistry as a research tool with the extracellular-electron-transferring bacterium Geobacter sulfurreducens as a reporter strain replacing the hypothesized acetate-oxidizing sub-population. We used a 2 × 4 experimental design with pure cultures of R. palustris in serum bottles and co-cultures of R. palustris and G. sulfurreducens in bioelectrochemical systems. This experimental design included growth medium with and without bicarbonate to induce non-lethal and lethal redox imbalance conditions, respectively, in R. palustris. Finally, the design also included a mutant strain (NifA*) of R. palustris, which constitutively produces H2, to serve both as a positive control for metabolite secretion (H2) to G. sulfurreducens, and as a non-lethal redox control for without bicarbonate conditions. Our results demonstrate that acetate sharing between different sub-populations of R. palustris does not occur while degrading p-coumarate under either non-lethal or lethal redox imbalance conditions. This work highlights the strength of microbial electrochemistry as a tool for studying microbial syntrophy.

Direct bioconversion of brown algae into ethanol by thermophilic bacterium Defluviitalea phaphyphila.

BackgroundBrown algae are promising feedstocks for biofuel production with inherent advantages of no structural lignin, high growth rate, and no competition for land and fresh water. However, it is difficult for one microorganism to convert all components of brown algae with different oxidoreduction potentials to ethanol. Defluviitalea phaphyphila Alg1 is the first characterized thermophilic bacterium capable of direct utilization of brown algae.ResultsDefluviitalea phaphyphila Alg1 can simultaneously utilize mannitol, glucose, and alginate to produce ethanol, and high ethanol yields of 0.47 g/g-mannitol, 0.44 g/g-glucose, and 0.3 g/g-alginate were obtained. A rational redox balance system under obligate anaerobic condition in fermenting brown algae was revealed in D. phaphyphila Alg1 through genome and redox analysis. The excess reducing equivalents produced from mannitol metabolism were equilibrated by oxidizing forces from alginate assimilation. Furthermore, D. phaphyphila Alg1 can directly utilize unpretreated kelp powder, and 10 g/L of ethanol was accumulated within 72 h with an ethanol yield of 0.25 g/g-kelp. Microscopic observation further demonstrated the deconstruction process of brown algae cell by D. phaphyphila Alg1.ConclusionsThe integrated biomass deconstruction system of D. phaphyphila Alg1, as well as its high ethanol yield, provided us an excellent alternative for brown algae bioconversion at elevated temperature.

Persistent Mycobacterium bovis-BCG is resistant to glutathione induced reductive stress killing

This study focuses on the redox stress response in mycobacteria elicited by a host-derived, thiol-based detoxification molecule, glutathione (GSH). Although the growth and viability of Mycobacterium bovis-BCG (BCG) was hampered by exposure to 8 mM GSH, oxygen depleted, persistent BCG (NRP BCG) resisted GSH-mediated killing. Fast growing mycobacteria also resisted GSH-mediated killing. To determine the mechanisms behind these observations, we evaluated the levels of intracellular ATP in both BCG and NRP BCG exposed to 8 mM GSH. Intracellular ATP levels increased from 0.13 to 2.3 μM in BCG upon exposure to GSH. The levels of ATP remained low and unchanged when NRP BCG was exposed to GSH. Using both HPLC and a cell-based thiol detection assay, it was determined that GSH stimulates the production of mycothiol (MSH) by BCG approximately 5.7 fold. The levels of MSH did not change upon exposure of NRP BCG to GSH. MSH is an alternative, thiol-based detoxification molecule employed by mycobacteria. Changes in the cytoplasmic concentrations of this molecule are suggestive of redox imbalances. Together, GSH and MSH may introduce excess reducing equivalents into the mycobacterial cytoplasm; leading to reductive stress. The modulation of NAD+ levels through alterations in ATP metabolism can enhance the cells ability to bind excess reducing equivalents and serve as a mechanism to restore the cellular redox balance when cells experience reductive stress. These data suggest that killing of BCG by GSH may result from reductive stress that cannot be controlled. NRP BCG appears to be resistant to GSH-induced reductive stress.