Potential Rate-limiting Step Research Articles

Effect of inoculated sludge concentration on start-up of anammox reactor: Nitrogen removal performance and metabolic pathways

The anammox process is efficient for nitrogen removal but faces challenges due to slow bacterial growth and limited inoculated sludge supply. This study examined the effects of different inoculated sludge concentrations (3.5, 7, and 14 g/L) on start-up and nitrogen metabolism in anammox reactors. Three identical reactors were operated under controlled conditions, with comprehensive analysis of nitrogen removal efficiency, sludge characteristics, and microbial community dynamics through metagenomic and transcriptomic approaches. Results demonstrated that higher inoculated sludge concentrations accelerated reactor start-up, with the 14 g/L reactor achieving stable operation in 13 days compared to 44 days for the 3.5 g/L reactor. However, the improvement in nitrogen removal rate showed a boundary effect, not proportional to the increase in sludge concentration. Notably, reactors with higher inoculated sludge concentrations exhibited lower sludge loads but higher sludge yield coefficients. Metagenomic analysis revealed Candidatus Kuenenia as the dominant anammox bacteria, with decreasing hydrazine dehydrogenase (hdh) gene expression levels observed at higher sludge concentrations, suggesting hydrazine synthesis as a potential rate-limiting step. This study provides novel insights into the optimal range of inoculated sludge concentration for anammox reactor start-up and elucidates the underlying metabolic mechanisms, offering valuable guidance for practical engineering applications.

Inverse metabolic engineering based on metabonomics for efficient production of hydroxytyrosol by Saccharomyces cerevisiae

Metabolic engineering provides a powerful approach to efficiently produce valuable compounds, with the aid of emerging gene editing tools and diverse metabolic regulation strategies. However, apart from the current known biochemical pathway information, a variety of unclear constraints commonly limited the optimization space of cell phenotype. Hydroxytyrosol is an important phenolic compound that serves various industries with prominent health-beneficial properties. In this study, the inverse metabolic engineering based on metabolome analysis was customized and implemented to disclose the hidden rate-limiting steps and thus to improve hydroxytyrosol production in Saccharomyces cerevisiae (S. cerevisiae). The potential rate-limiting steps involved three modules that were eliminated individually via reinforcing and balancing metabolic flow, optimizing cofactor supply, and weakening the competitive pathways. Ultimately, a 118.53 % improvement in hydroxytyrosol production (639.84 mg/L) was achieved by inverse metabolic engineering.

Engineering strategies for enhanced 1′, 4′-trans-ABA diol production by Botrytis cinerea

BackgroundCurrently, industrial fermentation of Botrytis cinerea is a significant source of abscisic acid (ABA). The crucial role of ABA in plants and its wide range of applications in agricultural production have resulted in the constant discovery of new derivatives and analogues. While modifying the ABA synthesis pathway of existing strains to produce ABA derivatives is a viable option, it is hindered by the limited synthesis capacity of these strains, which hinders further development and application.ResultsIn this study, we knocked out the bcaba4 gene of B. cinerea TB-31 to obtain the 1′,4′-trans-ABA-diol producing strain ZX2. We then studied the fermentation broth of the batch-fed fermentation of the ZX2 strain using metabolomic analysis. The results showed significant accumulation of 3-hydroxy-3-methylglutaric acid, mevalonic acid, and mevalonolactone during the fermentation process, indicating potential rate-limiting steps in the 1′,4′-trans-ABA-diol synthesis pathway. This may be hindering the flow of the synthetic pathway. Additionally, analysis of the transcript levels of terpene synthesis pathway genes in this strain revealed a correlation between the bchmgr, bcerg12, and bcaba1-3 genes and 1′,4′-trans-ABA-diol synthesis. To further increase the yield of 1′,4′-trans-ABA-diol, we constructed a pCBg418 plasmid suitable for the Agrobacterium tumefaciens-mediated transformation (ATMT) system and transformed it to obtain a single-gene overexpression strain. We found that overexpression of bchmgr, bcerg12, bcaba1, bcaba2, and bcaba3 genes increased the yield of 1′,4′-trans-ABA-diol. The highest yielding ZX2 A3 strain was eventually screened, which produced a 1′,4′-trans-ABA-diol concentration of 7.96 mg/g DCW (54.4 mg/L) in 144 h of shake flask fermentation. This represents a 2.1-fold increase compared to the ZX2 strain.ConclusionsWe utilized metabolic engineering techniques to alter the ABA-synthesizing strain B. cinerea, resulting in the creation of the mutant strain ZX2, which has the ability to produce 1′,4′-trans-ABA-diol. By overexpressing the crucial genes involved in the 1′,4′-trans-ABA-diol synthesis pathway in ZX2, we observed a substantial increase in the production of 1′,4′-trans-ABA-diol.

Quantum Mechanical/Molecular Mechanical Elucidation of the Catalytic Mechanism of Leukotriene A4 Hydrolase as an Epoxidase.

Leukotriene A4 hydrolase (LTA4H) functions as a mono-zinc bifunctional enzyme with aminopeptidase and epoxidase activities. While the aminopeptidase mechanism is well understood, the epoxidase mechanism remains less clear. In continuation of our prior research, we undertook an in-depth exploration of the LTA4H catalytic role as an epoxidase, employing a combined SCC-DFTB/CHARMM method. In the current work, we found that the conversion of LTA4 to leukotriene B4 (LTB4) involves three successive steps: epoxy ring opening (RO), nucleophilic attack (NA), and proton transfer (PT) reactions at the epoxy oxygen atom. Among these steps, the RO and NA stages constitute the potential rate-limiting step within the entire epoxidase mechanism. Notably, the NA step implicates D375 as the general base catalyst, while the PT step engages protonated E271 as the general acid catalyst. Additionally, we delved into the mechanism behind the formation of the isomer product, Δ6-trans-Δ8-cis-LTB4. Our findings debunked the feasibility of a direct LTB4 to iso-LTB4 conversion. Instead, we highlight the possibility of isomerization from LTA4 to its isomeric conjugate (iso-LTA4), showing comparable energy barriers of 5.1 and 5.5 kcal/mol in aqueous and enzymatic environments, respectively. The ensuing dynamics of iso-LTA4 hydrolysis subsequently yield iso-LTB4 via a mechanism akin to LTA4 hydrolysis, albeit with a heightened barrier. Our computations firmly support the notion that substrate isomerization exclusively takes place prior to or during the initial substrate-binding phase, while LTA4 remains the dominant conformer. Notably, our simulations suggest that irrespective of the active site's constrained L-shape, isomerization from LTA4 to its isomeric conjugate remains plausible. The mechanistic insights garnered from our simulations furnish a valuable understanding of LTA4H's role as an epoxidase, thereby facilitating potential advancements in inhibitor design.

Coordinative Stabilization of Single Bismuth Sites in a Carbon-Nitrogen Matrix to Generate Atom-Efficient Catalysts for Electrochemical Nitrate Reduction to Ammonia.

Electrochemical nitrate reduction to ammonia powered by renewable electricity is not only a promising alternative to the established energy-intense and non-ecofriendly Haber-Bosch reaction for ammonia generation but also a future contributor to the ever-more important denitrification schemes. Nevertheless, this reaction is still impeded by the lack of understanding for the underlying reaction mechanism on the molecular scale which is necessary for the rational design of active, selective, and stable electrocatalysts. Herein, a novel single-site bismuth catalyst (Bi-N-C) for nitrate electroreduction is reported to produce ammonia with maximum Faradaic efficiency of 88.7% and at a high rate of 1.38mg h-1 mgcat -1 at -0.35V versus reversible hydrogen electrode (RHE). The active center(described as BiN2 C2 ) is uncovered by detailed structural analysis. Coupled density functional theory calculations are applied to analyze the reaction mechanism and potential rate-limiting steps for nitrate reduction based on the BiN2 C2 model. The findings highlight the importance of model catalysts to utilize the potential of nitrate reduction as a new-generation nitrogen-management technology based on the construction of efficient active sites.

Selective NOx- Electroreduction to Ammonia on Isolated Ru Sites.

Nitrate and nitrite (NOx-) are widespread contaminants in industrial wastewater and groundwater. Sustainable ammonia (NH3) production via NOx- electroreduction provides a prospective alternative to the energy-intensive industrialized Haber-Bosch process. However, selectively regulating the reaction pathway, which involves complicated electron/proton transfer, toward NH3 generation relies on the robust catalyst. A specific consideration in designing selective NOx--to-NH3 catalysts should meet the criteria to suppress competing hydrogen evolution and avoid the presence of neighboring active sites that are in favor of adverse N-N coupling. Nevertheless, efforts in this regard are still inadequate. Herein, we demonstrate that isolated ruthenium sites can selectively reduce NOx- into NH3, with maximal Faradaic efficiencies of 97.8% (NO2- reduction) and 72.8% (NO3- reduction) at -0.6 and -0.4 V, respectively. Density functional theory calculations simulated the reaction mechanisms and identified the *NO → *NOH as the potential rate-limiting step for NOx--to-NH3 conversion on single-atom Ru sites.

Combining Metabolic Engineering and Multiplexed Screening Methods for 3-Hydroxypropionic Acid Production in Pichia pastoris.

Production of 3-hydroxypropionic acid (3-HP) in Pichia pastoris (syn. Komagataella phaffii) via the malonyl-CoA pathway has been recently demonstrated using glycerol as a carbon source, but the reported metrics were not commercially relevant. The flux through the heterologous pathway from malonyl-CoA to 3-HP was hypothesized as the main bottleneck. In the present study, different metabolic engineering approaches have been combined to improve the productivity of the original 3-HP producing strains. To do so, an additional copy of the gene encoding for the potential rate-limiting step of the pathway, i.e., the C-terminal domain of the malonyl-CoA reductase, was introduced. In addition, a variant of the endogenous acetyl-CoA carboxylase (ACC1 S1132A ) was overexpressed with the aim to increase the delivery of malonyl-CoA. Furthermore, the genes encoding for the pyruvate decarboxylase, aldehyde dehydrogenase and acetyl-CoA synthase, respectively, were overexpressed to enhance conversion of pyruvate into cytosolic acetyl-CoA, and the main gene responsible for the production of the by-product D-arabitol was deleted. Three different screening conditions were used to classify the performance of the different strains: 24-deep-well plates batch cultures, small-scale cultures in falcon tubes using FeedBeads® (i.e., slow release of glycerol over time), and mini bioreactor batch cultures. The best two strains from the FeedBeads® screening, PpHP8 and PpHP18, were tested in bioreactor fed-batch cultures using a pre-fixed exponentially increasing feeding rate. The strain PpHP18 produced up to 37.05 g L−1 of 3-HP at 0.712 g L−1 h−1 with a final product yield on glycerol of 0.194 Cmol−1 in fed-batch cultures. Remarkably, PpHP18 did not rank among the 2-top producer strains in small scale batch cultivations in deep-well plates and mini bioreactors, highlighting the importance of multiplexed screening conditions for adequate assessment of metabolic engineering strategies. These results represent a 50% increase in the product yield and final concentration, as well as over 30% increase in volumetric productivity compared to the previously obtained metrics for P. pastoris. Overall, the combination of glycerol as carbon source and a metabolically engineered P. pastoris strain resulted in the highest 3-HP concentration and productivity reported so far in yeast.

Deep Learning-Assisted Investigation of Electric Field-Dipole Effects on Catalytic Ammonia Synthesis.

External electric fields can modify binding energies of reactive surface species and enhance catalytic performance of heterogeneously catalyzed reactions. In this work, we used density functional theory (DFT) calculations-assisted and accelerated by a deep learning algorithm-to investigate the extent to which ruthenium-catalyzed ammonia synthesis would benefit from application of such external electric fields. This strategy allows us to determine which electronic properties control a molecule's degree of interaction with external electric fields. Our results show that (1) field-dependent adsorption/reaction energies are closely correlated to the dipole moments of intermediates over the surface, (2) a positive field promotes ammonia synthesis by lowering the overall energetics and decreasing the activation barriers of the potential rate-limiting steps (e.g., NH2 hydrogenation) over Ru, (3) a positive field (>0.6 V/Å) favors the reaction mechanism by avoiding kinetically unfavorable N≡N bond dissociation over Ru(1013), and (4) local adsorption environments (i.e., dipole moments of the intermediates in the gas phase, surface defects, and surface coverage of intermediates) influence the resulting surface adsorbates' dipole moments and further modify field-dependent reaction energetics. The deep learning algorithm developed here accelerates field-dependent energy predictions with acceptable accuracies by five orders of magnitudes compared to DFT alone and has the capacity of transferability, which can predict field-dependent energetics of other catalytic surfaces with high-quality performance using little training data.

Metabolic engineering of Clostridium ljungdahlii for the production of hexanol and butanol from CO2 and H2

BackgroundThe replacement of fossil fuels and petrochemicals with sustainable alternatives is necessary to mitigate the effects of climate change and also to counteract diminishing fossil resources. Acetogenic microorganisms such as Clostridium spp. are promising sources of fuels and basic chemical precursors because they efficiently utilize CO and CO2 as carbon source. However the conversion into high titers of butanol and hexanol is challenging.ResultsUsing a metabolic engineering approach we transferred a 17.9-kb gene cluster via conjugation, containing 13 genes from C. kluyveri and C. acetobutylicum for butanol and hexanol biosynthesis, into C. ljungdahlii. Plasmid-based expression resulted in 1075 mg L−1 butanol and 133 mg L−1 hexanol from fructose in complex medium, and 174 mg L−1 butanol and 15 mg L−1 hexanol from gaseous substrate (20% CO2 and 80% H2) in minimal medium. Product formation was increased by the genomic integration of the heterologous gene cluster. We confirmed the expression of all 13 enzymes by targeted proteomics and identified potential rate-limiting steps. Then, we removed the first-round selection marker using CRISPR/Cas9 and integrated an additional 7.8 kb gene cluster comprising 6 genes from C. carboxidivorans. This led to a significant increase in the hexanol titer (251 mg L−1) at the expense of butanol (158 mg L−1), when grown on CO2 and H2 in serum bottles. Fermentation of this strain at 2-L scale produced 109 mg L−1 butanol and 393 mg L−1 hexanol.ConclusionsWe thus confirmed the function of the butanol/hexanol biosynthesis genes and achieved hexanol biosynthesis in the syngas-fermenting species C. ljungdahlii for the first time, reaching the levels produced naturally by C. carboxidivorans. The genomic integration strain produced hexanol without selection and is therefore suitable for continuous fermentation processes.

Theoretical investigation of electrochemical reduction mechanism of CO2 on the Cu(1 1 1), Sn@Cu(1 1 1) and Sn(2 1 1) surfaces

The electrochemical reduction of CO2 is one of the important technologies for the effective utilization of CO2, and it is also a key step to produce value-added products. In the present study, density functional theory (DFT) is applied to study the reduction mechanism of CO2 to formic acid (HCOOH) and methanol (CH3OH) on the Cu(1 1 1), Sn@Cu(1 1 1) and Sn(2 1 1) catalysts. The adsorption structures of all possible intermediates are determined. It is found that for the HCOOH pathway, the rate-limiting step form the reduction of CO2 to form HCOO on the Cu(1 1 1) and Sn@Cu(1 1 1) surface is transformed to the reduction of HCOO forming HCOOH on the Sn(2 1 1) surface. The activity of three catalyst models towards HCOOH production follows: Sn@Cu(1 1 1) > Sn(2 1 1) > Cu(1 1 1). For the CH3OH pathway, the potential rate-limiting step on the Cu(1 1 1), Sn@Cu(1 1 1) and Sn(2 1 1) surfaces is the formation of COOH via CO2. The activity of three catalyst models towards CH3OH production follows: Sn@Cu(1 1 1) > Cu(1 1 1) > Sn(2 1 1). The results demonstrate that the doping of Sn onto Cu surface may be a potential strategy for the development of efficient CO2 electrocatalyst.

Green and eco-friendly montmorillonite clay for the removal of Cr(III) metal ion from aqueous environment

Pollutants found in the wastewater streams of industrial processes such as heavy metals are a significant concern worldwide. Clays are low-cost sorbents that have attracted extensive research interest due to their potential applications in removing heavy metals from wastewater. In this work, montmorillonite clay was collected from the Bengurir region; Morocco was used as an eco-friendly adsorbent for the removal of chromium (III) at various operating conditions. The used adsorbent was characterized using X-ray diffraction, X-ray fluorescence spectrometry, Fourier Transform Infrared Spectroscopy, and scanning electron microscope coupled with X-ray energy dispersion spectrometer analysis. Effects of adsorbent dose, initial concentration, and pH were investigated to optimize the adsorption process. The controlling mechanism and the potential rate-limiting steps were analyzed using Lagergren’s pseudo-first-order and pseudo-second-order models, but the experimental data were better fitted to the pseudo-second-order equation. The equilibrium isotherm data were analyzed by applying Langmuir and Freundlich’s isotherm models, and Langmuir showed good fits with the experimental data. A maximum Cr(III) adsorption capacity of 7.5 mg g−1 was found in the studied conditions.

Phenol adsorption mechanism on the zinc oxide surface: Experimental, cluster DFT calculations, and molecular dynamics simulations

Pollution by phenols is considered a major environmental issue. Therefore, their removal from wastewaters is of great practical significance and is directly related to human health. Herein, a porous zinc oxide (ZnO) was synthesized by a simple precipitation method and used as an adsorbent for the removal of phenol at different operating conditions. Besides experimental investigations, a comprehensive understanding of the mechanism of phenol adsorption on ZnO surface at the molecular level was achieved by using cluster Density Functional Theory (DFT) and molecular dynamics (MD) simulations. The prepared adsorbent was characterized using a variety of techniques, including X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), as well as the Brunauer Emmett Teller (BET) method. The effects of different variables, such as pH, pHpzc (pH at the point of zero charge), initial concentration, and temperature, were investigated to optimize and understand the adsorption process. Possible controlling mechanism and the potential rate-limiting steps were analyzed using Lagergren's pseudo-first-order and pseudo-second-order models, and the data were found to follow the pseudo-second-order equation. The maximum removal of the phenol molecule was observed at pH = 4 with adsorption capacities of 3.61 and 4.51 mg/g at 30° and 50°, respectively. Furthermore, Cluster DFT calculations and molecular dynamics simulations were used to get deeper mechanistic insights on the adsorption behavior of the phenol molecule onto zinc oxide. DFT results confirmed the predominance of chemisorption while those from MD simulations indicated an increased interaction of ZnO with phenol in the presence of solvent due to water-bridged H-bonds.

Insights into methyl orange adsorption behavior on a cadmium zeolitic-imidazolate framework Cd-ZIF-8: A joint experimental and theoretical study

Dyeing wastewater from textile industries is often contaminated with chemical pollutants, which causes severe water pollution problems that are challenging to treat. Recently, Zeolitic imidazolate frameworks (ZIFs) have attracted extensive research interest due to their potential applications in the removal of pollutants from wastewater. Herein, a Cd- zeolitic-imidazolate framework (Cd-ZIF-8) was synthesized and used as an adsorbent for the removal of methyl orange (MO) at different operating conditions. The prepared adsorbent was characterized using a variety of techniques, including X-ray diffraction (XRD), the Brunauer Emmett Teller (BET) method, FT-IR and scanning electron microscope (SEM). Its stability after adsorption was confirmed by XRD and pH tests. The effects of different variables, such as adsorbent dose, initial concentration, pH and temperature, were investigated to optimize the adsorption process. Adsorption parameters were determined by applying Langmuir and Freundlich models, and good fitting was observed for the Langmuir isotherm. Maximum MO adsorption capacities on Cd-ZIF-8 were 93.24 and 145.41 mg g−1 at 30° and 50°, respectively. Possible controlling mechanism and the potential rate-limiting steps were analyzed using Lagergren's pseudo-first-order and pseudo-second-order models, and the data were found to follow the pseudo-second-order equation. The prepared material was found to have good stability after the adsorption with no release of Cd ions over a wide range of pH. For a further explanation of the adsorption mechanism, interactions of MO molecules with the Cd and organic linkers of ZIF-8 were investigated using Density Functional Theory (DFT) cluster calculations.

Metabolic engineering of a fast-growing cyanobacterium Synechococcus elongatus PCC 11801 for photoautotrophic production of succinic acid

BackgroundCyanobacteria, a group of photosynthetic prokaryotes, are being increasingly explored for direct conversion of carbon dioxide to useful chemicals. However, efforts to engineer these photoautotrophs have resulted in low product titers. This may be ascribed to the bottlenecks in metabolic pathways, which need to be identified for rational engineering. We engineered the recently reported, fast-growing and robust cyanobacterium, Synechococcus elongatus PCC 11801 to produce succinate, an important platform chemical. Previously, engineering of the model cyanobacterium S. elongatus PCC 7942 has resulted in succinate titer of 0.43 g l−1 in 8 days.ResultsBuilding on the previous report, expression of α-ketoglutarate decarboxylase, succinate semialdehyde dehydrogenase and phosphoenolpyruvate carboxylase yielded a succinate titer of 0.6 g l−1 in 5 days suggesting that PCC 11801 is better suited as host for production. Profiling of the engineered strains for 57 intermediate metabolites, a number of enzymes and qualitative analysis of key transcripts revealed potential flux control points. Based on this, we evaluated the effects of overexpression of sedoheptulose-1,7-bisphosphatase, citrate synthase and succinate transporters and knockout of succinate dehydrogenase and glycogen synthase A. The final construct with seven genes overexpressed and two genes knocked out resulted in photoautotrophic production of 0.93 g l−1 succinate in 5 days.ConclusionWhile the fast-growing strain PCC 11801 yielded a much higher titer than the model strain, the efficient photoautotrophy of this novel isolate needs to be harnessed further for the production of desired chemicals. Engineered strains of S. elongatus PCC 11801 showed dramatic alterations in the levels of several metabolites suggesting far reaching effects of pathway engineering. Attempts to overexpress enzymes deemed to be flux controlling led to the emergence of other potential rate-limiting steps. Thus, this process of debottlenecking of the pathway needs to be repeated several times to obtain a significantly superior succinate titer.

Removal of phenol from aqueous solution by adsorption onto hematite (α-Fe2O3): Mechanism exploration from both experimental and theoretical studies

Iron oxides in general and especially hematite, α-Fe2O3 have been proved promising materials for efficient removal of various organic pollutants. Herein, we report a successful preparation of hematite (α-Fe2O3) by a facile precipitation method and its potential application in the removal of phenol from wastewater. The prepared material was subjected to extensive characterization using a variety of techniques such as scanning electron microscope coupled with energy-dispersive X-ray spectroscopy (SEM/EDX), X-ray diffraction (XRD), and the Brunauer Emmett Teller (BET) method. The operating conditions were optimized to improve the adsorption process efficiently. The adsorption analysis showed an adsorption capacity of 16.17 mg g−1 towards phenol at 30 °C. The reaction kinetics and potential rate-limiting steps were studied by Lagergren's pseudo-first-order and pseudo-second-order models, and it was found that the pseudo-second-order accurately described the adsorption kinetics. Freundlich and Langmuir adsorption isotherms models were applied, and the quality of the fittings clearly shows that the Langmuir model well describes the phenol adsorption on the hematite. The interaction mechanism between phenol and α-Fe2O3(0 0 1) surface was further addressed by Density Functional Theory (DFT) calculations and molecular dynamics (MD) simulations. Experimental and theoretical results indicate that there is strong evidence for the decisive effect of π–π interactions and H-bonds on the adsorption capacity.

Mechanism of simultaneous removal of aluminum and fluoride from aqueous solution by La/Mg/Si-activated carbon

La/Mg/Si-activated carbon derived from palm shell has been a suitable material for removal of aluminum and fluoride from aqueous solution. In the study, the mechanism of simultaneous removal of aluminum and fluoride by La/Mg/Si-activated carbon (La/Mg/Si-AC) was investigated to understand its high efficiency. It was found that the removal of aluminum and fluoride by La/Mg/Si-AC was favored at lower pH compared to the point of zero charge of La/Mg/Si-AC and high temperature. Adsorption capacity of Al(OH)4- was about 10 times higher than that of F- due to the strong binding affinity of Al(OH)4- on protonated surface and competition between F- and OH- toward charged adsorption site. Kinetics results showed that the aluminum and fluoride adsorption were explained using the pseudo-second-order kinetic model and intra-particle diffusion model. Adsorption process of Al(OH)4- and F- was driven by the potential rate-limiting step involved in mass transport process occurred on the boundary diffusion layer of porous adsorbent surface. Electrostatic interaction between protonated surface of La/Mg/Si-AC and negatively charged ions (i.e., Al(OH)4- and F-) as well as ion-exchange between hydroxide and ionic metal species were important mechanisms in the process of aluminum and fluoride adsorption. Driving forces for adsorption of individual Al(OH)4- and F- were not entirely different. Identifying the dominant mechanism will be helpful in understanding the adsorption process and developing new adsorbent.

Role of conserved arginine in the heme distal site of HutZ from Vibrio cholerae in the heme degradation reaction

HutZ from Vibrio cholerae is a dimeric enzyme that catalyzes degradation of heme. The highly conserved Arg92 residue in the HutZ family is proposed to interact with an iron-bound water molecule in the distal heme pocket. To clarify the specific role of Arg92 in the heme degradation reaction, the residue was substituted with alanine, leucine, histidine or lysine to modulate electrostatic interactions with iron-bound ligand. All four Arg92 mutants reacted with hydrogen peroxide to form verdoheme, a prominent intermediate in the heme degradation process. However, when ascorbic acid was used as an electron source, iron was not released even at pH 6.0 despite a decrease in the Soret band, indicating that non-enzymatic heme degradation occurred. Comparison of the rates of heme reduction, ligand binding and verdoheme formation suggested that proton transfer to the reduced oxyferrous heme, a potential rate-limiting step of heme degradation in HutZ, is hampered by mutation. In our previous study, we found that the increase in the distance between heme and Trp109 from 16 to 18 Å upon lowering the pH from 8.0 to 6.0 leads to activation of ascorbic acid-assisted heme degradation by HutZ. The distance in Arg92 mutants was >19 Å at pH 6.0, suggesting that subunit-subunit interactions at this pH are not suitable for heme degradation, similar to Asp132 and His63 mutants. These results suggest that interactions of Arg92 with heme-bound ligand induce alterations in the distance between subunits, which plays a key role in controlling the heme degradation activity of HutZ.

Comparative transcriptome and metabolome analysis suggests bottlenecks that limit seed and oil yields in transgenic Camelina sativa expressing diacylglycerol acyltransferase 1 and glycerol-3-phosphate dehydrogenase

BackgroundCamelina sativa has attracted much interest as alternative renewable resources for biodiesel, other oil-based industrial products and a source for edible oils. Its unique oil attributes attract research to engineering new varieties of improved oil quantity and quality. The overexpression of enzymes catalyzing the synthesis of the glycerol backbone and the sequential conjugation of fatty acids into this backbone is a promising approach for increasing the levels of triacylglycerol (TAG). In a previous study, we co-expressed the diacylglycerol acyltransferase (DGAT1) and glycerol-3-phosphate dehydrogenase (GPD1), involved in TAG metabolism, in Camelina seeds. Transgenic plants exhibited a higher-percentage seed oil content, a greater seed mass, and overall improved seed and oil yields relative to wild-type plants. To further increase seed oil content in Camelina, we utilized metabolite profiling, in conjunction with transcriptome profiling during seed development to examine potential rate-limiting step(s) in the production of building blocks for TAG biosynthesis.ResultsTranscriptomic analysis revealed approximately 2518 and 3136 transcripts differentially regulated at significant levels in DGAT1 and GPD1 transgenics, respectively. These transcripts were found to be involved in various functional categories, including alternative metabolic routes in fatty acid synthesis, TAG assembly, and TAG degradation. We quantified the relative contents of over 240 metabolites. Our results indicate major metabolic switches in transgenic seeds associated with significant changes in the levels of glycerolipids, amino acids, sugars, and organic acids, especially the TCA cycle and glycolysis intermediates.ConclusionsFrom the transcriptomic and metabolomic analysis of DGAT1, GPD1 and DGAT1 + GPD1 expressing lines of C. sativa, we conclude that TAG production is limited by (1) utilization of fixed carbon from the source tissues supported by the increase in glycolysis pathway metabolites and decreased transcripts levels of transcription factors controlling fatty acids synthesis; (2) TAG accumulation is limited by the activity of lipases/hydrolases that hydrolyze TAG pool supported by the increase in free fatty acids and monoacylglycerols. This comparative transcriptomics and metabolomics approach is useful in understanding the regulation of TAG biosynthesis, identifying bottlenecks, and the corresponding genes controlling these pathways identified as limitations, for generating Camelina varieties with improved seed and oil yields.

Enhanced butanol fermentation using metabolically engineered Clostridium acetobutylicum with ex situ recovery of butanol

In this study, metabolic target reactions for strain engineering were searched via intracellular coenzyme A (CoA) metabolite analysis. The metabolic reactions catalyzed by thiolase (AtoB) and aldehyde-alcohol dehydrogenase (AdhE1) were considered potential rate-limiting steps. In addition, CoA transferase (CtfAB) was highlighted as being important for the assimilation of organic acids, in order to achieve high butanol production. Based on this quantitative analysis, the BEKW_E1AB-atoB strain was constructed by overexpressing the thl (atoB), adhE1, and ctfAB genes in Clostridium acetobutylicum strain BEKW, which has the phosphotransacetylase (pta) and butyrate kinase (buk) genes knocked out. After 100h of continuous fermentation coupled with adsorptive ex situ butanol recovery, the concentrations found after considering desorption, yield, and productivity for the BEKW_E1AB-atoB strain were 55.7g/L, 0.38g/g, and 2.64g/L/h, respectively. The level of butanol production achieved (2.64g/L/h) represents the highest reported value obtained after adsorptive, long-term fermentation.

Hydrogeochemistry and coal-associated bacterial populations from a methanogenic coal bed

Biogenic coalbed methane (CBM), a microbially-generated source of natural gas trapped within coal beds, is an important energy resource in many countries. Specific bacterial populations and enzymes involved in coal degradation, the potential rate-limiting step of CBM formation, are relatively unknown. The U.S. Geological Survey (USGS) has established a field site, (Birney test site), in an undeveloped area of the Powder River Basin (PRB), with four wells completed in the Flowers-Goodale coal bed, one in the overlying sandstone formation, and four in overlying and underlying coal beds (Knoblach, Nance, and Terret). The nine wells were positioned to characterize the hydraulic conductivity of the Flowers-Goodale coal bed and were selectively cored to investigate the hydrogeochemistry and microbiology associated with CBM production at the Birney test site. Aquifer-test results indicated the Flowers-Goodale coal bed, in a zone from about 112 to 120m below land surface at the test site, had very low hydraulic conductivity (0.005m/d) compared to other PRB coal beds examined. Consistent with microbial methanogenesis, groundwater in the coal bed and overlying sandstone contain dissolved methane (46mg/L average) with low δ13C values (−67‰ average), high alkalinity values (22meq/kg average), relatively positive δ13C-DIC values (4‰ average), and no detectable higher chain hydrocarbons, NO3−, or SO42−. Bioassay methane production was greatest at the upper interface of the Flowers-Goodale coal bed near the overlying sandstone. Pyrotag analysis identified Aeribacillus as a dominant in situ bacterial community member in the coal near the sandstone and statistical analysis indicated Actinobacteria predominated coal core samples compared to claystone or sandstone cores. These bacteria, which previously have been correlated with hydrocarbon-containing environments such as oil reservoirs, have demonstrated the ability to produce biosurfactants to break down hydrocarbons. Identifying microorganisms involved in coal degradation and the hydrogeochemical conditions that promote their activity is crucial to understanding and improving in situ CBM production.