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Frontiers in Genetics 2022Trehalose is commonly used as an impermeable cryoprotectant for cryopreservation of cells, but its cryoprotective mechanism has now not but been determined. This study...
Trehalose is commonly used as an impermeable cryoprotectant for cryopreservation of cells, but its cryoprotective mechanism has now not but been determined. This study investigated the cryopreservation impact of trehalose on buck semen cryopreservation and finished metabolic profiling of freeze-thawed media by way of the GC-MS-based metabolomics for the first time. Metabolic pattern recognition and metabolite identification by means of principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and metabolic pathway topology analysis revealed the results of trehalose on buck sperm metabolism at some point of cryopreservation. The results confirmed that trehalose drastically progressed sperm motility parameters and structural integrity after thawing. PCA and PLS-DA analysis discovered that the metabolic patterns of the freezing-thawing media of buck semen cryopreserved with trehalose (T group) or without trehalose (G group, Control) were certainly separated. Using screening conditions of VIP >1.5 and vaule <0.05, a total of 48 differential metabolites have been recognized, whithin l-isoleucine, L-leucine, L-threonine, and dihydroxyacetone were notably enriched in valine, leucine and isoleucine biosynthesis, glycerolipid metabolism, and aminoacyl-tRNA biosynthesis pathways. In brief, trehalose can efficiently improve membrane structural integrity and motion parameters in buck sperm after thawing, and it exerts a cryoprotective impact with the aid of changing sperm amino acid synthesis and the glycerol metabolism pathway.
PubMed: 35991557
DOI: 10.3389/fgene.2022.938622 -
The Journal of General and Applied... Jun 2023Corynebacterium glutamicum was metabolically engineered to produce phenylalanine, a valuable aromatic amino acid that can be used as a raw material in the food and...
Corynebacterium glutamicum was metabolically engineered to produce phenylalanine, a valuable aromatic amino acid that can be used as a raw material in the food and pharmaceutical industries. First, a starting phenylalanine-producer was constructed by overexpressing tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase and phenylalanine- and tyrosine-insensitive bifunctional enzyme chorismate mutase prephenate dehydratase from Escherichia coli, followed by the inactivation of enzymes responsible for the formation of dihydroxyacetone and the consumption of shikimate pathway-related compounds. Second, redirection of the carbon flow from tyrosine to phenylalanine was attempted by deleting of the tyrA gene encoding prephenate dehydrogenase, which catalyzes the committed step for tyrosine biosynthesis from prephenate. However, suppressor mutants were generated, and two mutants were isolated and examined for phenylalanine production and genome sequencing. The suppressor mutant harboring an amino acid exchange (L180R) on RNase J, which was experimentally proven to lead to a loss of function of the enzyme, showed significantly enhanced production of phenylalanine. Finally, modifications of phosphoenolpyruvate-pyruvate metabolism were investigated, revealing that the inactivation of either phosphoenolpyruvate carboxylase or pyruvate carboxylase, which are enzymes of the anaplerotic pathway, is an effective means for improving phenylalanine production. The resultant strain, harboring a phosphoenolpyruvate carboxylase deficiency, synthesized 50.7 mM phenylalanine from 444 mM glucose. These results not only provided new insights into the practical mutations in constructing a phenylalanine-producing C. glutamicum but also demonstrated the creation of a potential strain for the biosynthesis of phenylalanine-derived compounds represented by plant secondary metabolites.
Topics: Phenylalanine; Corynebacterium glutamicum; Metabolic Engineering; Tyrosine; Escherichia coli
PubMed: 35989300
DOI: 10.2323/jgam.2022.08.002 -
FEMS Yeast Research Aug 2022In response to osmotic dehydration cells sense, signal, alter gene expression, and metabolically counterbalance osmotic differences. The main compatible solute/osmolyte... (Review)
Review
In response to osmotic dehydration cells sense, signal, alter gene expression, and metabolically counterbalance osmotic differences. The main compatible solute/osmolyte that accumulates in yeast cells is glycerol, which is produced from the glycolytic intermediate dihydroxyacetone phosphate. This review covers recent advancements in understanding mechanisms involved in sensing, signaling, cell-cycle delays, transcriptional responses as well as post-translational modifications on key proteins in osmoregulation. The protein kinase Hog1 is a key-player in many of these events, however, there is also a growing body of evidence for important Hog1-independent mechanisms playing vital roles. Several missing links in our understanding of osmoregulation will be discussed and future avenues for research proposed. The review highlights that this rather simple experimental system-salt/sorbitol and yeast-has developed into an enormously potent model system unravelling important fundamental aspects in biology.
Topics: Glycerol; Osmoregulation; Osmotic Pressure; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Water-Electrolyte Balance
PubMed: 35927716
DOI: 10.1093/femsyr/foac035 -
PloS One 2022Variation in the antibacterial potency of manuka honey has been reported in several published studies. However, many of these studies examine only a few honey samples,...
Correlation of the antibacterial activity of commercial manuka and Leptospermum honeys from Australia and New Zealand with methylglyoxal content and other physicochemical characteristics.
Variation in the antibacterial potency of manuka honey has been reported in several published studies. However, many of these studies examine only a few honey samples, or test activity against only a few bacterial isolates. To address this deficit, a collection of 29 manuka/Leptospermum honeys was obtained, comprising commercial manuka honeys from Australia and New Zealand and several Western Australian Leptospermum honeys obtained directly from beekeepers. The antibacterial activity of honeys was quantified using several methods, including the broth microdilution method to determine minimum inhibitory concentrations (MICs) against four species of test bacteria, the phenol equivalence method, determination of antibacterial activity values from optical density, and time kill assays. Several physicochemical parameters or components were also quantified, including methylglyoxal (MGO), dihydroxyacetone (DHA), hydroxymethylfurfural (HMF) and total phenolics content as well as pH, colour and refractive index. Total antioxidant activity was also determined using the DPPH* (2,2-diphenyl-1-picrylhydrazyl) and FRAP (ferric reducing-antioxidant power) assays. Levels of MGO quantified in each honey were compared to the levels stated on the product labels, which revealed mostly minor differences. Antibacterial activity studies showed that MICs varied between different honey samples and between bacterial species. Correlation of the MGO content of honey with antibacterial activity showed differing relationships for each test organism, with Pseudomonas aeruginosa showing no relationship, Staphylococcus aureus showing a moderate relationship and both Enterococcus faecalis and Escherichia coli showing strong positive correlations. The association between MGO content and antibacterial activity was further investigated by adding known concentrations of MGO to a multifloral honey and quantifying activity, and by also conducting checkerboard assays. These investigations showed that interactions were largely additive in nature, and that synergistic interactions between MGO and the honey matrix did not occur.
Topics: Anti-Bacterial Agents; Australia; Escherichia coli; Honey; Leptospermum; Magnesium Oxide; New Zealand; Pyruvaldehyde
PubMed: 35901185
DOI: 10.1371/journal.pone.0272376 -
Bioresources and Bioprocessing Jul 2022Catalytic valorization of raw glycerol derived from biodiesel into high-value chemicals has attracted great attention. Here, we report chemoenzymatic cascade reactions...
Catalytic valorization of raw glycerol derived from biodiesel into high-value chemicals has attracted great attention. Here, we report chemoenzymatic cascade reactions that convert glycerol to lactic acid and glycolic acid. In the enzymatic step, a coenzyme recycling system was developed to convert glycerol into 1,3-dihydroxyacetone (DHA) with a yield of 92.3% in potassium phosphate buffer (300 mM, pH 7.1) containing 100 mM glycerol, 2 mM NAD, 242 U/mL glycerol dehydrogenase-GldA and NADH oxidase-SpNox at 30 °C. Subsequently, NaOH or NaClO catalyzes the formation of lactic acid and glycolic acid from DHA. The high yield of lactic acid (72.3%) and glycolic acid (78.2%) verify the benefit of the chemoenzymatic approaches.
PubMed: 38647569
DOI: 10.1186/s40643-022-00561-z -
Cellular and Molecular Life Sciences :... Jul 2022Transaminases play key roles in central metabolism, transferring the amino group from a donor substrate to an acceptor. These enzymes can often act, with low efficiency,...
Transaminases play key roles in central metabolism, transferring the amino group from a donor substrate to an acceptor. These enzymes can often act, with low efficiency, on compounds different from the preferred substrates. To understand what might have shaped the substrate specificity of this class of enzymes, we examined the reactivity of six human cytosolic transaminases towards amino acids whose main degradative pathways do not include any transamination. We also tested whether sugars and sugar phosphates could serve as alternative amino group acceptors for these cytosolic enzymes. Each of the six aminotransferases reacted appreciably with at least three of the alternative amino acid substrates in vitro, albeit at usually feeble rates. Reactions with L-Thr, L-Arg, L-Lys and L-Asn were consistently very slow-a bias explained in part by the structural differences between these amino acids and the preferred substrates of the transaminases. On the other hand, L-His and L-Trp reacted more efficiently, particularly with GTK (glutamine transaminase K; also known as KYAT1). This points towards a role of GTK in the salvage of L-Trp (in cooperation with ω-amidase and possibly with the cytosolic malate dehydrogenase, MDH1, which efficiently reduced the product of L-Trp transamination). Finally, the transaminases were extremely ineffective at utilizing sugars and sugar derivatives, with the exception of the glycolytic intermediate dihydroxyacetone phosphate, which was slowly but appreciably transaminated by some of the enzymes to yield serinol phosphate. Evidence for the formation of this compound in a human cell line was also obtained. We discuss the biological and evolutionary implications of our results.
Topics: Amino Acids; Cytosol; Humans; Kinetics; Substrate Specificity; Sugars; Transaminases
PubMed: 35834009
DOI: 10.1007/s00018-022-04439-3 -
Analytical Biochemistry Sep 2022For many years, Shiliu Buxue Syrup (SLBXS) has been used in the treatment of anemia in Xinjiang, China. However, the potential therapeutic mechanism of SLBXS in the...
For many years, Shiliu Buxue Syrup (SLBXS) has been used in the treatment of anemia in Xinjiang, China. However, the potential therapeutic mechanism of SLBXS in the treatment of anemia remains unclear. We qualitatively analyzed the ingredients of SLBXS and predicted the underlying mechanisms by network pharmacology. A mice model of anemia was established by subcutaneous injection of 1-Acetyl-2-phenylhydrazine (APH). Spleen metabolomics was performed to screen potential biomarkers and pathways related to anemia. Furthermore, core targets of crucial pathways were experimentally validated. Finally, molecular docking was used for predicting interactions between compositions and targets. Network pharmacology indicated that the 230 SLBXS ingredients may affect 141 target proteins to regulate the PI3K/AKT and HIF-1 signaling pathways. Metabolomics revealed that SLBXS could mediate 30 biomarkers, such as phosphoric acid, l-pyroglutamic acid, alpha-Tocopherol, 1-stearoyl-rac-glycerol, and dihydroxyacetone phosphate, to regulate drug metabolism-other enzymes, glutathione metabolism, glycolysis or gluconeogenesis, nicotinate and nicotinamide metabolism, nitrogen metabolism, and purine metabolism. Western blot indicated that SLBXS can regulate the protein expression levels of AKT1, Bcl2, Caspase3, HIF-1α, VEGF-A, and NOS2. The molecular docking revealed that most of the compositions had a good binding ability to the core targets. Based on these findings, we speculate that SLBXS treats anemia mainly by modulating the PI3K/AKT and HIF-1 pathways and glutathione and glycolytic metabolisms.
Topics: Anemia; Animals; Biomarkers; Drugs, Chinese Herbal; Glutathione; Metabolomics; Mice; Molecular Docking Simulation; Network Pharmacology; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt
PubMed: 35690102
DOI: 10.1016/j.ab.2022.114774 -
World Journal of Microbiology &... Jun 2022Gluconobacter oxydans is a well-known acetic acid bacterium that has long been applied in the biotechnological industry. Its extraordinary capacity to oxidize a variety... (Review)
Review
Gluconobacter oxydans is a well-known acetic acid bacterium that has long been applied in the biotechnological industry. Its extraordinary capacity to oxidize a variety of sugars, polyols, and alcohols into acids, aldehydes, and ketones is advantageous for the production of valuable compounds. Relevant G. oxydans industrial applications are in the manufacture of L-ascorbic acid (vitamin C), miglitol, gluconic acid and its derivatives, and dihydroxyacetone. Increasing efforts on improving these processes have been made in the last few years, especially by applying metabolic engineering. Thereby, a series of genes have been targeted to construct powerful recombinant strains to be used in optimized fermentation. Furthermore, low-cost feedstocks, mostly agro-industrial wastes or byproducts, have been investigated, to reduce processing costs and improve the sustainability of G. oxydans bioprocess. Nonetheless, further research is required mainly to make these raw materials feasible at the industrial scale. The current shortage of suitable genetic tools for metabolic engineering modifications in G. oxydans is another challenge to be overcome. This paper aims to give an overview of the most relevant industrial G. oxydans processes and the current strategies developed for their improvement.
Topics: Acetic Acid; Biotechnology; Fermentation; Gluconobacter oxydans; Metabolic Engineering
PubMed: 35688964
DOI: 10.1007/s11274-022-03310-8 -
Frontiers in Microbiology 2022Acetic acid bacteria (AAB) are a group of Gram-negative, strictly aerobic bacteria, including 19 reported genera until 2021, which are widely found on the surface of... (Review)
Review
Acetic acid bacteria (AAB) are a group of Gram-negative, strictly aerobic bacteria, including 19 reported genera until 2021, which are widely found on the surface of flowers and fruits, or in traditionally fermented products. Many AAB strains have the great abilities to incompletely oxidize a large variety of carbohydrates, alcohols and related compounds to the corresponding products mainly including acetic acid, gluconic acid, gulonic acid, galactonic acid, sorbose, dihydroxyacetone and miglitol the membrane-binding dehydrogenases, which is termed as AAB oxidative fermentation (AOF). Up to now, at least 86 AOF products have been reported in the literatures, but no any monograph or review of them has been published. In this review, at first, we briefly introduce the classification progress of AAB due to the rapid changes of AAB classification in recent years, then systematically describe the enzymes involved in AOF and classify the AOF products. Finally, we summarize the application of molecular biology technologies in AOF researches.
PubMed: 35685922
DOI: 10.3389/fmicb.2022.879246 -
Molecules (Basel, Switzerland) May 2022New carriers of phytosterols; acylglycerols containing natural myristic acid at -1 and -3 positions and stigmasterol residue linked to -2 position by carbonate and...
New carriers of phytosterols; acylglycerols containing natural myristic acid at -1 and -3 positions and stigmasterol residue linked to -2 position by carbonate and succinate linker have been designed and synthesized in three-step synthesis from dihydroxyacetone (DHA). The synthetic pathway involved Steglich esterification of DHA with myristic acid; reduction of carbonyl group of 1,3-dimyristoylpropanone and esterification of 1,3-dimyristoylglicerol with stigmasterol chloroformate or stigmasterol hemisuccinate. The structure of the obtained hybrids was established by the spectroscopic methods (NMR; IR; HRMS). Obtained hybrid molecules were used to form new liposomes in the mixture with model phospholipid and their effect on their physicochemical properties was determined, including the polarity, fluidity, and main phase transition of liposomes using differential scanning calorimetry and fluorimetric methods. The results confirm the significant effect of both stigmasterol-containing acylglycerols on the hydrophilic and hydrophobic region of liposome membranes. They significantly increase the order in the polar heads of the lipid bilayer and increase the rigidity in the hydrophobic region. Moreover, the presence of both acylglycerols in the membranes shifts the temperature of the main phase transition towards higher temperatures. Our results indicate stabilization of the bilayer over a wide temperature range (above and below the phase transition temperature), which in addition to the beneficial effects of phytosterols on human health makes them more attractive components of novel lipid nanocarriers compared to cholesterol.
Topics: Calorimetry, Differential Scanning; Glycerides; Humans; Lipid Bilayers; Liposomes; Myristic Acid; Phytosterols; Stigmasterol
PubMed: 35684341
DOI: 10.3390/molecules27113406