C2 Carbon Research Articles

Draft Genome Sequence of Mycobacterium sp. Strain JC1 DSM 3803.

Mycobacterium sp. strain JC1 DSM 3803 is one of the few known bacteria predicted to possess the xylulose monophosphate (XuMP) pathway of C1 assimilation. The draft genome is 7,921,603 bp with a GC content of 66.88% and will allow more in-depth investigation of this bacterium's unique metabolism.

Production of the biocommodities butanol and acetone from methanol with fluorescent FAST-tagged proteins using metabolically engineered strains of Eubacterium limosum

BackgroundThe interest in using methanol as a substrate to cultivate acetogens increased in recent years since it can be sustainably produced from syngas and has the additional benefit of reducing greenhouse gas emissions. Eubacterium limosum is one of the few acetogens that can utilize methanol, is genetically accessible and, therefore, a promising candidate for the recombinant production of biocommodities from this C1 carbon source. Although several genetic tools are already available for certain acetogens including E. limosum, the use of brightly fluorescent reporter proteins is still limited.ResultsIn this study, we expanded the genetic toolbox of E. limosum by implementing the fluorescence-activating and absorption shifting tag (FAST) as a fluorescent reporter protein. Recombinant E. limosum strains that expressed the gene encoding FAST in an inducible and constitutive manner were constructed. Cultivation of these recombinant strains resulted in brightly fluorescent cells even under anaerobic conditions. Moreover, we produced the biocommodities butanol and acetone from methanol with recombinant E. limosum strains. Therefore, we used E.limosum cultures that produced FAST-tagged fusion proteins of the bifunctional acetaldehyde/alcohol dehydrogenase or the acetoacetate decarboxylase, respectively, and determined the fluorescence intensity and product concentrations during growth.ConclusionsThe addition of FAST as an oxygen-independent fluorescent reporter protein expands the genetic toolbox of E. limosum. Moreover, our results show that FAST-tagged fusion proteins can be constructed without negatively impacting the stability, functionality, and productivity of the resulting enzyme. Finally, butanol and acetone can be produced from methanol using recombinant E.limosum strains expressing genes encoding fluorescent FAST-tagged fusion proteins.

The detection, purity and structural properties of partially soluble mushroom and cereal β-D-glucans: A solid-state NMR study

β-D-glucans are proposed to have many health benefits. It is therefore important to have methods which can distinguish these from other carbohydrates present in natural products, as well as giving glucan content and structural information. Correlations between features in the CP/MAS spectra of β-D-glucans and enzyme assay determined β-D-glucan content were generally found to be poor. The β-D-glucan in dry and hydrated forms of the mushroom Ganoderma lucidum was investigated in detail by spectral peak fitting to the anomeric carbon C1 region in CP/MAS NMR spectra. Hydrated samples gave spectra with enhanced resolution and suggested that a clear distinction between β-D-glucans and other carbohydrates could be possible in the anomeric carbon C1 region. Chemical shift values for a range of carbohydrate polymers, which can be found alongside β-D-glucans, as well as the values for various linkages are given. Contamination by other carbohydrates and buffer salts is discussed.

Analysis of boswellic acids in dietary supplements containing Indian frankincense (Boswellia serrata) by Supercritical Fluid Chromatography

Boswellic acids, a class of triterpenes, are the bioactive constituents in Indian frankincense, an herbal drug with pronounced anti-inflammatory activity. In this study their separation and quantification in B. serrata extracts is reported for the first time by using Supercritical Fluid Chromatography. Under optimized conditions, i.e. a Viridis HSS C18 SB column and carbon dioxide, methanol, acetonitrile and ammonium hydroxide as mobile phase, six boswellic acids could be separated in under 6 min. The assay fulfilled all validation criteria with coefficients of determination higher than 0.999, a wide linear range (30–1000 μg/mL), recovery rates from 97.1–103.0 %, excellent precision, and detection limits typical for SFC with UV-detection (≤ 5.5 μg/mL). The method could easily be hyphenated to mass spectrometry, which was helpful to tentatively assign further compounds (mainly derivatives of tirucallic acid) and to increase the assay’s sensitivity. Its practical applicability was confirmed by analyzing several commercial products, which mainly contained β-boswellic acid as dominant triterpene, yet in extremely variable amounts ranging from 0.9 to 16.9 %.

Coumarin-Chalcone Hybrids as Inhibitors of MAO-B: Biological Activity and In Silico Studies.

Fourteen coumarin-derived compounds modified at the C3 carbon of coumarin with an α,β-unsaturated ketone were synthesized. These compounds may be designated as chalcocoumarins (3-cinnamoyl-2H-chromen-2-ones). Both chalcones and coumarins are recognized scaffolds in medicinal chemistry, showing diverse biological and pharmacological properties among which neuroprotective activities and multiple enzyme inhibition, including mitochondrial enzyme systems, stand out. The evaluation of monoamine oxidase B (MAO-B) inhibitors has aroused considerable interest as therapeutic agents for neurodegenerative diseases such as Parkinson’s. Of the fourteen chalcocumarins evaluated here against MAO-B, ChC4 showed the strongest activity in vitro, with IC50 = 0.76 ± 0.08 µM. Computational docking, molecular dynamics and MM/GBSA studies, confirm that ChC4 binds very stably to the active rMAO-B site, explaining the experimental inhibition data.

Synthesis and Theoretical Calculations of 2-(p-Tolyl)-2,3-Dihydro-1H-Perimidine using Density Functional Theory

In the present study, 2-(p-tolyl)-2,3-dihydro-1H-perimidine (TDHP) is synthesized from 1,8-naphthalenediamine and 4-methylbenzaldehyde by embedding a one-carbon unit between the nitrogen followed by ring closure using green chemistry approach. 1H NMR and 13C NMR spectral techniques were used to validate the structure of the TDHP. The synthesized perimidine TDHP is studied using density functional theory (DFT) to provide valuable insights into structural, chemical, and thermochemical study.The structural and chemical properties of TDHP were computed using the DFT method on the B3LYP/6-311G(d,p) basis package. Bond lengths were predicted from the optimised molecular structure, and the physical and chemical properties of the molecules were inferred as a consequence. The HOMO and LUMO are computed, and quantum chemical parameters are determined using electronic energies. The calculated HOMO-LUMO energy gap is 4.25 eV indicating charge transfer phenomenon within the molecule. The electron density and chemical behaviour of the TDHP was predicted using Mulliken atomic charges and the molecular electrostatic surface potential plot.Amongst all carbon atoms, the C8 carbon as more positive and C27 as more negative carbon atoms. The high global electrophilicity index suggests electrophilic character of the TDHP.The harmonic vibrational frequencies were used to measure total energy, total molar entropy, and molar heat capacity.

D-Glucose Isomerization with PAMAM Dendrimers as Environmentally Friendly Catalysts.

The isomerization of d-glucose to d-fructose plays a key role in the biochemical and chemical conversion of biomass, and it is therefore desirable to develop and improve catalysts for this reaction. In this study, the environmentally friendly polymer poly(amidoamine) (PAMAM) dendrimer's properties as catalysts for this isomerization are investigated. The experimental results showed that the PAMAM dendrimers, which have basic terminal groups, can effectively promote the d-glucose isomerization reaction. Under the optimized reaction conditions, d-fructose was generated with a 20% maximum yield and above 90% selectivity. 13C and 2H isotope experiments by NMR were carried out to explore the reaction mechanism. When the reaction was performed in D2O, the C1 signal of d-fructose changed to a triplet, which confirmed that the C1 carbon binds to a deuterium atom, i.e., isotopic exchange. It was also found that the deuterium atom at the C2 position of d-glucose-2-d1 cannot transfer to d-fructose. These data indicate that PAMAM dendrimers catalyze d-glucose isomerization through a mechanism, which includes deprotonation, formation of ene-diol intermediate, and proton exchange with the solvent.

Biodegradation of C20 carbon clusters from Diesel Fuel by Coriolopsis gallica: optimization, metabolic pathway, phytotoxicity.

The online version contains supplementary material available at 10.1007/s13205-021-02769-w.

Experimental Study on CH+ Formation from Doubly Charged Carbon and Molecular Hydrogen

Abstract We studied the reaction of doubly charged carbon C2+ (C iii) with molecular hydrogen, a possible source of the high, unexplained abundances of interstellar CH+. The experiment was carried out using the cryogenic linear 22-pole radio frequency ion trap. The measured reaction rate coefficient amounts to (1.5 ± 0.2) × 10−10 cm3 s−1, nearly independently of the covered temperature range from 15 to 300 K. In the product distribution study, the C+ ion was identified as the dominant product of the reaction. For the CH+ production, we determine an upper limit for the reaction rate coefficient at 2 × 10−12 cm3 s−1.

Is it possible to differentiate between 2-phenylaminodihydro-1,3-thiazine from 2-phenyliminotetrahydro-1,3-thiazine by spectral methods? New glance to the old problem

Several studies have reported the presence of amine and imine tautomeric forms for hydrogenated 1,3-thiazine derivatives. However, identification of their tautomeric forms by UV, FTIR and mass-spectral methods does not yield expected results. Here, we report the synthesis of 2-phenylaminodihydro-1,3-thiazine and 2-phenyliminotetrahydro-1,3-thiazine and the analysis of their UV, FTIR and NMR (1H and 13C) spectral data. An identical picture of UV spectra was recorded for both compounds. However, distinctive characteristics were found in the FTIR, 1H and 13C NMR spectra. The C=N band of amine form was observed in higher frequency region relative to imine form. The signal of C2 carbon of amine form in 13C NMR spectrum was occurred in more downfield (δ 165.3 ppm) relative to C2 signal of imine form (δ 152.1 ppm). In addition, the difference between C2 and C8 carbon signals of amine form was very high (Δδ = 30.6 ppm) relative to imine form (δ 5.4 ppm). The position of C2 and C8 signals and the difference between them in 13C NMR spectrum was found to be more promising in identification of tautomeric forms in case of hydrogenated 1,3-thiazine derivatives.

Electrodeposited Hybrid Biocathode-Based CO2 Reduction via Microbial Electro-Catalysis to Biofuels.

Microbial electrosynthesis is a new approach to converting C1 carbon (CO2) to more complex carbon-based products. In the present study, CO2, a potential greenhouse gas, was used as a sole carbon source and reduced to value-added chemicals (acetate, ethanol) with the help of bioelectrochemical reduction in microbial electrosynthesis systems (MES). The performance of MES was studied with varying electrode materials (carbon felt, stainless steel, and cobalt electrodeposited carbon felt). The MES performance was assessed in terms of acetic acid and ethanol production with the help of gas chromatography (GC). The electrochemical characterization of the system was analyzed with chronoamperometry and cyclic voltammetry. The study revealed that the MES operated with hybrid cobalt electrodeposited carbon felt electrode yielded the highest acetic acid (4.4 g/L) concentration followed by carbon felt/stainless steel (3.7 g/L), plain carbon felt (2.2 g/L), and stainless steel (1.87 g/L). The alcohol concentration was also observed to be highest for the hybrid electrode (carbon felt/stainless steel/cobalt oxide is 0.352 g/L) as compared to the bare electrodes (carbon felt is 0.22 g/L) tested, which was found to be in correspondence with the pH changes in the system. Electrochemical analysis revealed improved electrotrophy in the hybrid electrode, as confirmed by the increased redox current for the hybrid electrode as compared to plain electrodes. Cyclic voltammetry analysis also confirmed the role of the biocatalyst developed on the electrode in CO2 sequestration.

Reduction of Grazing Capacity in High-Elevation Rangelands After Black Locust Invasion in South Africa

The growing invasion of ecosystems by invasive alien plants (IAPs) has substantially affected biodiversity worldwide, compromising provision of ecosystem services. In this study, we present evidence of the impacts of an IAP, Robinia pseudoacacia L., on native plant diversity in montane rangelands of South Africa and its threats to grazing, an ecosystem service. We assessed stand characteristics, understory vegetation composition and rangeland condition similarities in invaded and uninvaded sites. We observed a shift in grass communities after invasion by R. pseudoacacia as invaded communities differed by 96% from uninvaded rangeland. Invaded habitat was dominated by nitrophilous, shade-tolerant alien ruderals that follow the primitive C3 carbon fixation pathway. Nitrogen fixation and light-demanding properties of R. pseudoacacia are likely to be the main factors driving these changes. As a result, range condition was significantly lower in invaded habitats with smaller and dense trees (180 ± 24.3) (mean ± standard error) when compared with adjacent uninvaded habitat (401 ± 24.3). These preliminary findings support an urgent need for sustainable control of R. pseudoacacia as an effective approach to stop further reduction in grazing capacity and losses in livestock production.

Two homologs of the Cat8 transcription factor are involved in the regulation of ethanol utilization in Komagataella phaffii

The transcription factors Cat8 and Sip4 were described in Saccharomyces cerevisiae and Kluyveromyces lactis to have very similar DNA binding domains and to be necessary for derepression of a variety of genes under non-fermentative growth conditions via binding to the carbon source responsive elements (CSREs). The methylotrophic yeast Komagataella phaffii (syn Pichia pastoris) has two transcription factors (TFs), which are putative homologs of Cat8 based on sequence similarity, termed Cat8-1 and Cat8-2. It is yet unclear in which cellular processes they are involved and if one of them is actually the homolog of Sip4. To study the roles of the Cat8 homologs in K. phaffii, overexpression or deletion strains were generated for the two TFs. The ability of these mutant strains to grow on different carbon sources was tested, and transcript levels of selected genes from the carbon metabolism were quantified. Our experiments showed that the TFs are required for the growth of K. phaffii on C2 carbon sources, but not on glucose, glycerol or methanol. K. phaffii deleted for Cat8-1 showed impaired growth on acetate, while both Cat8-1 and Cat8-2 are involved in the growth of K. phaffii on ethanol. Correspondingly, both TFs are participating in the activation of ADH2, ALD4 and ACS1, three genes encoding enzymes important for the assimilation of ethanol. Different from S. cerevisiae and K. lactis, Cat8-1 is not regulating the transcription of the putative Sip4-family member Cat8-2 in K. phaffii. Furthermore, Cat8-1 is necessary for the activation of genes from the glyoxylate cycle, whereas Cat8-2 is necessary for the activation of genes from the carnitine shuttle. Neither Cat8-1 nor Cat8-2 are required for the activation of gluconeogenesis genes. Finally, the CAT8-2 gene is repressed by the Mig1-2 transcription factor on glucose and autorepressed by the Cat8-2 protein on all tested carbon sources. Our study identified the involvement of K. phaffii Cat8-1 and Cat8-2 in C2-metabolism, and highlighted similarities and differences to their homologs in other yeast species.

Influence of fullerenes C60 and single-walled carbon nanotubes on the Carr – Helfrich effect in nematic liquid crystal

Influence of fullerenes C60 and single-walled carbon nanotubes (SWCNTs) with concentration of 0.5 wt.% on the Carr-Helfrich electrohydrodynamic instability in nematic liquid crystal 4-methoxybenzylidene-4′-butylaniline (MBBA) is investigated. It is shown that the inclusion of these dopants into liquid crystal leads to decreasing of the threshold voltage of Williams domains formation. Critical frequency of disappearance of the EHDI decreases for the MBBA + fullerenes colloid and it increases for the MBBA + SWCNTs colloid with respect to the pure LC. A decrease of time characteristics in the MBBA + fullerenes colloid and their increase in the MBBA + SWCNTs colloid compared with the pure MBBA are observed. The experimental results are explained on the base of the Carr-Helfrich theory for electrohydrodynamic instability in nematic liquid crystal.

NHC Organocatalysis in D2O for the Highly Diastereoselective Synthesis of Deuterated Spiropyran Analogues

AbstractMetal‐free N‐heterocyclic carbene (NHC) organocatalysis in D2O medium was elegantly utilised for the synthesis of highly diastereoselective deuterated spiropyran derivatives. Selective deuteration was exclusively achieved on the C5 carbon atom of the pyran ring. Quantum chemical calculations were performed to prove the preferential formation of a single diastereomer from the possible four diastereomers. The scope of NHC organocatalysis in D2O was also extended for the synthesis of deuterated spirooxindole derivative.

Metabolic engineering of Methylorubrum extorquens AM1 for poly (3-hydroxybutyrate-co-3-hydroxyvalerate) production using formate

Formate is a promising environmentally friendly and sustainable feedstock synthesized from syngas or carbon dioxide. Methylorubrum extorquens is a type II methylotroph that can use formate as a carbon source. It accumulates polyhydroxyalkanoates (PHAs) inside the cell, mainly producing poly-3-hydroxybutyrate (PHB), a degradable biopolymer. Owing to its high melting point and stiff nature, however, mechanical property improvement is warranted in the form of copolymerization. To produce the PHA copolymer, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the endogenous gene phaC was deleted and the pathway genes bktB, phaJ1, and phaC2, with broader substrate specificities, were heterologously expressed. To improve the incorporation of 3-hydroxyvalerate (3HV), the expression level of bktB was improved by untranslated region (UTR) engineering, and the endogenous gene phaA was deleted. The engineered M. extorquens produced PHBV with 8.9% 3HV using formate as the sole carbon source. In addition, when propionate and butyrate were supplemented, PHBVs with 3HV portions of up to 70.6% were produced. This study shows that a PHBV copolymer with a high proportion of 3HV can be synthesized using formate, a C1 carbon source, through metabolic engineering and supplementation with short-chain fatty acids.

The Entner-Doudoroff Pathway Is an Essential Metabolic Route for Methylotuvimicrobium buryatense 5GB1C.

Methylotuvimicrobium buryatense 5GB1C, a fast-growing gammaproteobacterial methanotroph, is equipped with two glycolytic pathways, the Entner-Doudoroff (ED) pathway and the Embden-Meyerhof-Parnas (EMP) pathway. Metabolic flux analysis and 13C-labeling experiments have shown the EMP pathway is the principal glycolytic route in M. buryatense 5GB1C, while the ED pathway appears to be metabolically and energetically insignificant. However, it has not been possible to obtain a null mutant in the edd-eda genes encoding the two unique enzymatic reactions in the ED pathway, suggesting the ED pathway may be essential for M. buryatense 5GB1C growth. In this study, the inducible P BAD promoter was used to manipulate gene expression of edd-eda, and in addition, the expression of these two genes was separated from that of a downstream gltA gene. The resulting strain shows arabinose-dependent growth that correlates with ED pathway activity, with normal growth achieved in the presence of ∼0.1 g/liter arabinose. Flux balance analysis shows that M. buryatense 5GB1C with a strong ED pathway has a reduced energy budget, thereby limiting the mutant growth at a high concentration of arabinose. Collectively, our study demonstrates that the ED pathway is essential for M. buryatense 5GB1C. However, no known mechanism can directly explain the essentiality of the ED pathway, and thus, it may have a yet unknown regulatory role required for sustaining a healthy and functional metabolism in this bacterium.IMPORTANCE The gammaproteobacterial methanotrophs possess a unique central metabolic architecture where methane and other reduced C1 carbon sources are assimilated through the ribulose monophosphate cycle. Although efforts have been made to better understand methanotrophic metabolism in these bacteria via experimental and computational approaches, many questions remain unanswered. One of these is the essentiality of the ED pathway for M. buryatense 5GB1C, as current results appear contradictory. By creating a construct with edd-eda and gltA genes controlled by P BAD and P J23101 , respectively, we demonstrated the essentiality of the ED pathway for this obligate methanotroph. It is also demonstrated that these genetic tools are applicable to M. buryatense 5GB1C and that expression of the target genes can be tightly controlled across an extensive range. Our study adds to the expanding knowledge of methanotrophic metabolism and practical approaches to genetic manipulation for obligate methanotrophs.

Spectral Probe for Electron Transfer and Addition Reactions of Azide Radicals with Substituted Quinoxalin-2-Ones in Aqueous Solutions.

The azide radical (N3●) is one of the most important one-electron oxidants used extensively in radiation chemistry studies involving molecules of biological significance. Generally, it was assumed that N3● reacts in aqueous solutions only by electron transfer. However, there were several reports indicating the possibility of N3● addition in aqueous solutions to organic compounds containing double bonds. The main purpose of this study was to find an experimental approach that allows a clear assignment of the nature of obtained products either to its one-electron oxidation or its addition products. Radiolysis of water provides a convenient source of one-electron oxidizing radicals characterized by a very broad range of reduction potentials. Two inorganic radicals (SO4●−, CO3●−) and Tl2+ ions with the reduction potentials higher, and one radical (SCN)2●− with the reduction potential slightly lower than the reduction potential of N3● were selected as dominant electron-acceptors. Transient absorption spectra formed in their reactions with a series of quinoxalin-2-one derivatives were confronted with absorption spectra formed from reactions of N3● with the same series of compounds. Cases, in which the absorption spectra formed in reactions involving N3● differ from the absorption spectra formed in the reactions involving other one-electron oxidants, strongly indicate that N3● is involved in the other reaction channel such as addition to double bonds. Moreover, it was shown that high-rate constants of reactions of N3● with quinoxalin-2-ones do not ultimately prove that they are electron transfer reactions. The optimized structures of the radical cations (7-R-3-MeQ)●+, radicals (7-R-3-MeQ)● and N3● adducts at the C2 carbon atom in pyrazine moiety and their absorption spectra are reasonably well reproduced by density functional theory quantum mechanics calculations employing the ωB97XD functional combined with the Dunning’s aug-cc-pVTZ correlation-consistent polarized basis sets augmented with diffuse functions.

Understanding the reaction mechanism of the regioselective piperidinolysis of aryl 1-(2,4-dinitronaphthyl) ethers in DMSO: Kinetic and DFT studies

Reactions of aryl 1-(2,4-dinitronaphthyl) ethers with piperidine in dimethyl sulfoxide at 25oC resulted in substitution of the aryloxy group at the ipso carbon atom. The reaction was measured spectrophotochemically and the kinetic studies suggested that the titled reaction is accurately third order. The mechanism is began by fast nucleophilic attack of piperidine on C1 to form zwitterion intermediate (I) followed by deprotonation of zwitterion intermediate (I) to the Meisenheimer ion (II) in a slow step, that is, SB catalysis. The regular variation of activation parameters suggested that the reaction proceeded through a common mechanism. The Hammett equation using reaction constant σo values and Brønsted coefficient value showed that the reaction is poorly dependent on aryloxy substituent and the reaction was significantly associative and Meisenheimer intermediate-like. The mechanism of piperidinolysis has been theoretically investigated using density functional theory method using B3LYP/6-311G(d,p) computational level. The combination between experimental and computational studies predicts what mechanism is followed either through uncatalyzed or catalyzed reaction pathways, that is, SB and SB-GA. The global parameters of the reactants, the proposed activated complexes, and the local Fukui function analysis explained that C1 carbon atom is the most electrophilic center of ether. Also, kinetics and theoretical calculation of activation energies indicated that the mechanism of the piperidinolysis passed through a two-step mechanism and the proton transfer process was the rate determining step.

Palladium-mediated C–O bond activation of benzopyrone in 4-oxo-4H-chromone-3-carbaldehyde-4(N)-substituted thiosemicarbazone: synthesis, structure, nucleic acid/albumin interaction, DNA cleavage, antioxidant and cytotoxic studies

Palladium ion-mediated C–O activation at the C2 carbon of the benzopyrone moiety of 3-formylchromone-4(N)-substituted thiosemicarbazone (HL1–4) has been observed in square-planar palladium(ii) complexes.