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The cell energy fraction that powered maintenance and expression of genes encoding pro-phage elements, pta- ack cluster, early sporulation, sugar ABC transporter periplasmic proteins, 6-phosphofructokinase, pyruvate kinase, and fructose-1,6-disphosphatase in acetogen Clostridium sp. MT871 was re-directed to power synthetic operon encoding isobutanol biosynthesis at the expense of these genes achieved via their elimination. Genome tailoring decreased cell duplication time by 7.0 ± 0.1 min ( p < 0.05) compared to the parental strain, with intact genome and cell duplication time of 68 ± 1 min ( p < 0.05). Clostridium sp. MT871 with tailored genome was UVC-mutated to withstand 6.1 % isobutanol in fermentation broth to prevent product inhibition in an engineered commercial biocatalyst producing 5 % (674.5 mM) isobutanol during two-step continuous fermentation of CO/H gas blend. Biocatalyst Clostridium sp. MT871RGIB6 was engineered to express six copies of synthetic operon comprising optimized synthetic format dehydrogenase, pyruvate formate lyase, acetolactate synthase, acetohydroxyacid reductoisomerase, 2,3-dihydroxy-isovalerate dehydratase, branched-chain alpha-ketoacid decarboxylase gene, aldehyde dehydrogenase, and alcohol dehydrogenase, regaining cell duplication time of 68 ± 1 min ( p < 0.05) for the parental strain. This is the first report on isobutanol production by an engineered acetogen biocatalyst suitable for commercial manufacturing of this chemical/fuel using continuous fermentation of CO/H blend thus contributing to the reversal of global warming. [ABSTRACT FROM AUTHOR]

A time- and cost-efficient two-step gene elimination procedure was used for acetogen Clostridium sp. MT1834 capable of fermenting CO/H blend to 245 mM acetate ( p < 0.005). The first step rendered the targeted gene replacement without affecting the total genome size. We replaced the acetate pta- ack cluster with synthetic bi-functional acetaldehyde-alcohol dehydrogenase ( al- adh). Replacement of pta- ack with al- adh rendered initiation of 243 mM ethanol accumulation at the expense of acetate production during CO/H blend continuous fermentation ( p < 0.005). At the second step, al- adh was eliminated to reduce the genome size. Resulting recombinants accumulated 25 mM mevalonate in fermentation broth ( p < 0.005). Cell duplication time for recombinants with reduced genome size decreased by 9.5 % compared to Clostridium sp. MT1834 strain under the same fermentation conditions suggesting better cell energy pool management in the absence of the ack- pta gene cluster in the engineered biocatalyst. If the first gene elimination step was used alone for spo0A gene replacement with two copies of synthetic formate dehydrogenase in recombinants with a shortened genome, mevalonate production was replaced with 76.5 mM formate production in a single step continuous CO/H blend fermentation ( p < 0.005) with cell duplication time almost nearing that of the wild strain. [ABSTRACT FROM AUTHOR]

Aims To engineer acetogen biocatalyst selectively overproducing ethanol from synthesis gas or CO2/ H2 as the only liquid carbonaceous product. Methods and Results Ethanol-resistant mutant originally capable of producing only acetate from CO2/ CO was engineered to eliminate acetate production and spore formation using our proprietary Cre- lox66/ lox71-system. Bi-functional aldehyde/alcohol dehydrogenase was inserted into the chromosome of the engineered mutant using Tn7-based approach. Recombinants with three or six copies of the inserted gene produced 525 mmol l−1 and 1018 mmol l−1 of ethanol, respectively, in five independent single-step fermentation runs 25 days each ( P < 0·005) in five independent repeats using syngas blend 60% CO and 40% H2. Ethanol production was 64% if only CO2 + H2 blend was used compared with syngas blend ( P < 0·005). Conclusions Elimination of genes unnecessary for syngas fermentation can boost artificial integrated pathway performance. Significance and Impact of the Study Cell energy released via elimination of phosphotransacetylase, acetate kinase and early-stage sporulation genes boosted ethanol production. Deletion of sporulation genes added theft-proof feature to the engineered biocatalyst. Production of ethanol from CO2/ H2 blend might be utilized as a tool to mitigate global warming proportional to CO2 fermentation scale. [ABSTRACT FROM AUTHOR]

An optimality principle is applied to determine kinetic constants for biochemical (enzymatic and second-order) reactions involved in a physiological system, a minimum protein consumption criterion being adopted. A direct optimization problem is to determine optimal zymogen concentrations. An 'inverse' optimization problem is also considered; this problem is to determine the kinetic constants for the biochemical reactions involved in a physiological system such that the optimal and observed zymogen and procofactor concentrations coincide. In solving an 'inverse' optimization problem we assume that the scheme of biochemical reactions and zymogen and procofactor concentrations are known. Good agreement is observed between the model results for the extrinsic blood coagulation pathway and relevant biochemical data. The proposed method is also applicable to determining kinetic constants for other physiological biochemical-reaction systems. [ABSTRACT FROM AUTHOR]

Focuses on the use of a high-voltage radio frequency modulated square pulses for the electrotransformation of Clostridium acetylbutylicum ATCC 824 cells in the United States. Determination of frequency at 100 kilohertz; Change of clostridial cells with pSOS84 plasmid DNA; Possibility of membrane structures to affect transformation yield.

In this work, we have examined the radiation-induced synthetic chemistry occurring in an astrochemically important C2H2–CO system at the molecular level using a matrix isolation approach. The 1:1 C2H2···CO intermolecular complex of linear structure was obtained in the solid low-temperature (5 K) noble gas matrices by deposition of the C2H2/CO/Ng (Ng = Ar, Kr, Xe) gaseous mixtures and characterized by Fourier-transform infrared spectroscopy. It was found that the X-ray radiolysis of the C2H2···CO complex resulted in formation of C3O (tricarbon monoxide), HCCCHO (propynal), c-H2C3O (cyclopropenone), H2CCCO (propadienone), and HC3O (oxoprorynyl radical). This means that the studied complex may be considered as the simplest building block (or minimal size of intermolecular reactor) for cold astrochemistry occurring in mixed interstellar ices. Remarkably, the discovered transformations of the complex actually represent synthetic routes leading to various C3 species, whereas the acetylenic C–H bond cleavage yielding ethynyl radical appears to be a minor process. Prolonged irradiation results in dehydrogenation, while the C3 skeleton is retained. The interpretation of the C2H2···CO radiolysis mechanism (possible reactions pathways) is provided based on the analysis of kinetic curves and matrix effect. Astrochemical implications of the results are discussed. [ABSTRACT FROM AUTHOR]

Investigations of the low-temperature radiation-induced transformations in the C2H2–H2O system are relevant to the chemistry occurring in interstellar and cometary ices. In this work, we applied a matrix isolation technique to study the radiation-driven evolution of this system at molecular level in order to get new mechanistic insight. The 1:1 C2H2⋅ ⋅ ⋅H2O complexes were prepared in various solid noble-gas matrices (Ar, Kr, Xe) and these icy matrices were subjected to X-ray irradiation at 5 K. Decomposition of initial complex and formation of products were monitored by Fourier-transform infrared (FTIR) spectroscopy. It was found that complexation with H2O resulted in significant enhancement of the radiolytic decay of C2H2 molecules and provided new channels for its radiation-induced transformations. Ketene (both isolated H2CCO and in the form of H2CCO–H2 pair), ketenyl radical (HCCO), carbon monoxide (CO), and methane (CH4) were observed as main products of the C2H2⋅ ⋅ ⋅H2O radiolysis. In addition, vinyl alcohol (CH2CHOH) was detected in an Ar matrix. The mechanistic interpretation (reaction routes leading to formation of these products) is discussed on the basis of consideration of kinetic dependences and matrix effects. Conversion of C2H2⋅ ⋅ ⋅H2O to CH4 is a prominent example of water-mediated cleavage of the C≡C triple bond, which may occur under prolonged irradiation in rigid media. Possible astrochemical implications of the obtained results are highlighted. [ABSTRACT FROM AUTHOR]

Veratrum poisonings are described in the toxicology literature as multiple Veratrum species grow in different parts of the Northern Hemisphere and are occasionally ingested by mistake. Veratrum toxicity is attributed to the steroidal alkaloids contained in all parts of the plant. In Russia, Veratrum poisonings are more common since there is an over-the-counter Veratrum lobelianum -based tincture, Veratrum Aqua (VA), which is topically used for the treatment of lice infestation. Despite its toxicity, VA is misused in traditional medicine as a remedy for alcohol use disorder. We describe four cases of VA poisoning that occurred in Moscow, Russia. Three main V. lobelianum alkaloids (jervine, protoveratrine A (proA) and protoveratrine B) were determined in patient plasma and urine samples using liquid chromatography–tandem mass spectrometry (LC–MS-MS). Here, we describe a novel validated LC–MS-MS method for jervine and proA quantification. A simple and rapid liquid–liquid extraction with methyl tert -butyl ether was utilized for analyte extraction. Chromatographic separation was achieved using a Poroshell 120 EC-C18 column, and the total run time was 14 min. The lower limit of quantification was 0.1 ng/mL for jervine and proA in both plasma and urine. Biological samples were obtained upon hospital admission and during treatment, thus enabling to get a better understanding of the alkaloid elimination profile. Upon admission, plasma concentrations of jervine (concentration range: 0.10–5.01 ng/mL) prevailed over proA (concentration range: 0–0.67 ng/mL). At this time, proA already reached maximum concentrations in urine (concentration range: 0.15–37.70 ng/mL). Maximum concentrations of jervine in urine were observed 24 h after admission (concentration range: 0.10–9.55 ng/mL). In all cases, plasma concentrations of Veratrum alkaloids correlated with condition severity. Since none of the patients confirmed VA intake, instrumental analysis was the basis for the definitive diagnosis of VA poisoning. [ABSTRACT FROM AUTHOR]

Aims: To engineer acetogen biocatalyst capable of fermenting synthesis gas blend to acetone as the only liquid carbonaceous product. Methods and Results: The metabolic engineering comprised inactivation of phosphotransacetylase via integration of a cassette comprising synthetic genes erm(B), thiolase and HMG-CoA synthase. Acetaldehyde dehydrogenase was inactivated via integration of a cassette consisting of synthetic genes cat, HMG-CoA lyase and acetoacetate decarboxylase. The engineered biocatalyst Clostridum sp. MAceT113 lost production of 253 mmol l−1 ethanol and 296 mmol l−1 acetate and started producing 1·8 mol l−1 acetone in single-stage continuous syngas fermentation. Conclusions: The acetone concentration in culture broth is economical for bulk manufacture because it is about twenty times of that achieved with known acetone-butanol-ethanol fermentation of sugars. Significance and Impact of the Study: The process shows the opportunity to produce acetone from synthesis gas at concentrations comparable with production of acetone from products of petroleum cracking. This is the first report on elimination of acetate and acetaldehyde production and directing carbon flux from Acetyl-CoA to acetone via a non-naturally occurring in acetogen acetone biosynthesis pathway identified in eukaryotic organisms. [ABSTRACT FROM AUTHOR]