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Nature Communications Jan 2018Plastics, including poly(ethylene terephthalate) (PET), possess many desirable characteristics and thus are widely used in daily life. However, non-biodegradability,...
Plastics, including poly(ethylene terephthalate) (PET), possess many desirable characteristics and thus are widely used in daily life. However, non-biodegradability, once thought to be an advantage offered by plastics, is causing major environmental problem. Recently, a PET-degrading bacterium, Ideonella sakaiensis, was identified and suggested for possible use in degradation and/or recycling of PET. However, the molecular mechanism of PET degradation is not known. Here we report the crystal structure of I. sakaiensis PETase (IsPETase) at 1.5 Å resolution. IsPETase has a Ser-His-Asp catalytic triad at its active site and contains an optimal substrate binding site to accommodate four monohydroxyethyl terephthalate (MHET) moieties of PET. Based on structural and site-directed mutagenesis experiments, the detailed process of PET degradation into MHET, terephthalic acid, and ethylene glycol is suggested. Moreover, other PETase candidates potentially having high PET-degrading activities are suggested based on phylogenetic tree analysis of 69 PETase-like proteins.
Topics: Amino Acid Sequence; Bacterial Proteins; Biodegradation, Environmental; Burkholderiales; Catalytic Domain; Cloning, Molecular; Crystallography, X-Ray; Environmental Pollutants; Escherichia coli; Ethylene Glycol; Gene Expression; Hydrolases; Kinetics; Molecular Docking Simulation; Phthalic Acids; Polyethylene Terephthalates; Protein Binding; Protein Interaction Domains and Motifs; Protein Structure, Secondary; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity; Thermodynamics
PubMed: 29374183
DOI: 10.1038/s41467-018-02881-1 -
Cryobiology Dec 2015We describe here a new cryobiological and neurobiological technique, aldehyde-stabilized cryopreservation (ASC), which demonstrates the relevance and utility of advanced...
We describe here a new cryobiological and neurobiological technique, aldehyde-stabilized cryopreservation (ASC), which demonstrates the relevance and utility of advanced cryopreservation science for the neurobiological research community. ASC is a new brain-banking technique designed to facilitate neuroanatomic research such as connectomics research, and has the unique ability to combine stable long term ice-free sample storage with excellent anatomical resolution. To demonstrate the feasibility of ASC, we perfuse-fixed rabbit and pig brains with a glutaraldehyde-based fixative, then slowly perfused increasing concentrations of ethylene glycol over several hours in a manner similar to techniques used for whole organ cryopreservation. Once 65% w/v ethylene glycol was reached, we vitrified brains at -135 °C for indefinite long-term storage. Vitrified brains were rewarmed and the cryoprotectant removed either by perfusion or gradual diffusion from brain slices. We evaluated ASC-processed brains by electron microscopy of multiple regions across the whole brain and by Focused Ion Beam Milling and Scanning Electron Microscopy (FIB-SEM) imaging of selected brain volumes. Preservation was uniformly excellent: processes were easily traceable and synapses were crisp in both species. Aldehyde-stabilized cryopreservation has many advantages over other brain-banking techniques: chemicals are delivered via perfusion, which enables easy scaling to brains of any size; vitrification ensures that the ultrastructure of the brain will not degrade even over very long storage times; and the cryoprotectant can be removed, yielding a perfusable aldehyde-preserved brain which is suitable for a wide variety of brain assays.
Topics: Animals; Brain; Cryopreservation; Cryoprotective Agents; Ethylene Glycol; Glutaral; Rabbits; Swine; Tissue Banks; Vitrification
PubMed: 26408851
DOI: 10.1016/j.cryobiol.2015.09.003 -
Nature Communications Apr 2019The extreme durability of polyethylene terephthalate (PET) debris has rendered it a long-term environmental burden. At the same time, current recycling efforts still...
The extreme durability of polyethylene terephthalate (PET) debris has rendered it a long-term environmental burden. At the same time, current recycling efforts still lack sustainability. Two recently discovered bacterial enzymes that specifically degrade PET represent a promising solution. First, Ideonella sakaiensis PETase, a structurally well-characterized consensus α/β-hydrolase fold enzyme, converts PET to mono-(2-hydroxyethyl) terephthalate (MHET). MHETase, the second key enzyme, hydrolyzes MHET to the PET educts terephthalate and ethylene glycol. Here, we report the crystal structures of active ligand-free MHETase and MHETase bound to a nonhydrolyzable MHET analog. MHETase, which is reminiscent of feruloyl esterases, possesses a classic α/β-hydrolase domain and a lid domain conferring substrate specificity. In the light of structure-based mapping of the active site, activity assays, mutagenesis studies and a first structure-guided alteration of substrate specificity towards bis-(2-hydroxyethyl) terephthalate (BHET) reported here, we anticipate MHETase to be a valuable resource to further advance enzymatic plastic degradation.
Topics: Biodegradation, Environmental; Burkholderiales; Catalytic Domain; Enzymes; Ethylene Glycol; Fluorometry; Hydrolases; Hydrolysis; Ligands; Mutagenesis; Mutagenesis, Site-Directed; Phthalic Acids; Phylogeny; Plastics; Polyethylene Terephthalates; Protein Domains; Protein Folding; Protein Structure, Secondary; Substrate Specificity
PubMed: 30979881
DOI: 10.1038/s41467-019-09326-3 -
Applied and Environmental Microbiology Aug 2021Poly(ethylene terephthalate) (PET) is a commonly used synthetic plastic; however, its nonbiodegradability results in a large amount of waste accumulation that has a...
Development of a Targeted Gene Disruption System in the Poly(Ethylene Terephthalate)-Degrading Bacterium Ideonella sakaiensis and Its Applications to PETase and MHETase Genes.
Poly(ethylene terephthalate) (PET) is a commonly used synthetic plastic; however, its nonbiodegradability results in a large amount of waste accumulation that has a negative impact on the environment. Recently, a PET-degrading bacterium, Ideonella sakaiensis 201-F6 strain, was isolated, and the enzymes involved in PET digestion, PET hydrolase (PETase), and mono(2-hydroxyethyl) terephthalic acid (MHET) hydrolase (MHETase) were identified. Despite the great potentials of in bioremediation and biorecycling, approaches to studying this bacterium remain limited. In this study, to enable the functional analysis of PETase and MHETase genes , we have developed a gene disruption system in . The pT18-based disruption vector harboring directly connected 5'- and 3'-flanking regions of the target gene for homologous recombination was introduced into cells via conjugation. First, we deleted the orotidine 5'-phosphate decarboxylase gene () from the genome of the wild-type strain, producing the Δ strain with 5-fluoroorotic acid (5-FOA) resistance. Next, using the Δ strain as a parent strain and as a counterselection marker, we disrupted the genes for PETase and MHETase. The growth of both Δ and Δ strains on terephthalic acid (TPA; one of the PET hydrolytic products) was comparable to that of the parent strain. However, these mutant strains dramatically decreased the growth level on PET to that on a no-carbon source. Moreover, the Δ strain completely abolished PET degradation capacity. These results demonstrate that PETase and MHETase are essential for metabolism of PET. The poly(ethylene terephthalate) (PET)-degrading bacterium Ideonella sakaiensis possesses two unique enzymes able to serve in PET hydrolysis. PET hydrolase (PETase) hydrolyzes PET into mono(2-hydroxyethyl) terephthalic acid (MHET), and MHET hydrolase (MHETase) hydrolyzes MHET into terephthalic acid (TPA) and ethylene glycol (EG). These enzymes have attracted global attention, as they have potential to be used for bioconversion of PET. Compared to many studies, including biochemical and crystal structure analyses, few studies have been reported. Here, we developed a targeted gene disruption system in , which was then applied for constructing Δ and Δ strains. Growth of these disruptants revealed that PETase is the sole enzyme responsible for PET degradation in , while PETase and MHETase play essential roles in its PET assimilation.
Topics: Bacterial Proteins; Burkholderiales; Ethylene Glycol; Genes, Bacterial; Hydrolases; Hydrolysis; Metabolic Engineering; Phthalic Acids; Polyethylene Terephthalates; Recycling
PubMed: 34260304
DOI: 10.1128/AEM.00020-21 -
Critical Care (London, England) Feb 2023Ethylene glycol (EG) is metabolized into glycolate and oxalate and may cause metabolic acidemia, neurotoxicity, acute kidney injury (AKI), and death. Historically,...
Ethylene glycol (EG) is metabolized into glycolate and oxalate and may cause metabolic acidemia, neurotoxicity, acute kidney injury (AKI), and death. Historically, treatment of EG toxicity included supportive care, correction of acid-base disturbances and antidotes (ethanol or fomepizole), and extracorporeal treatments (ECTRs), such as hemodialysis. With the wider availability of fomepizole, the indications for ECTRs in EG poisoning are debated. We conducted systematic reviews of the literature following published EXTRIP methods to determine the utility of ECTRs in the management of EG toxicity. The quality of the evidence and the strength of recommendations, either strong ("we recommend") or weak/conditional ("we suggest"), were graded according to the GRADE approach. A total of 226 articles met inclusion criteria. EG was assessed as dialyzable by intermittent hemodialysis (level of evidence = B) as was glycolate (Level of evidence = C). Clinical data were available for analysis on 446 patients, in whom overall mortality was 18.7%. In the subgroup of patients with a glycolate concentration ≤ 12 mmol/L (or anion gap ≤ 28 mmol/L), mortality was 3.6%; in this subgroup, outcomes in patients receiving ECTR were not better than in those who did not receive ECTR. The EXTRIP workgroup made the following recommendations for the use of ECTR in addition to supportive care over supportive care alone in the management of EG poisoning (very low quality of evidence for all recommendations): i) Suggest ECTR if fomepizole is used and EG concentration > 50 mmol/L OR osmol gap > 50; or ii) Recommend ECTR if ethanol is used and EG concentration > 50 mmol/L OR osmol gap > 50; or iii) Recommend ECTR if glycolate concentration is > 12 mmol/L or anion gap > 27 mmol/L; or iv) Suggest ECTR if glycolate concentration 8-12 mmol/L or anion gap 23-27 mmol/L; or v) Recommend ECTR if there are severe clinical features (coma, seizures, or AKI). In most settings, the workgroup recommends using intermittent hemodialysis over other ECTRs. If intermittent hemodialysis is not available, CKRT is recommended over other types of ECTR. Cessation of ECTR is recommended once the anion gap is < 18 mmol/L or suggested if EG concentration is < 4 mmol/L. The dosage of antidotes (fomepizole or ethanol) needs to be adjusted during ECTR.
Topics: Humans; Antidotes; Fomepizole; Ethanol; Renal Dialysis; Glycolates; Ethylene Glycol; Poisoning
PubMed: 36765419
DOI: 10.1186/s13054-022-04227-2 -
Biotechnology Advances Dec 2023Microbial fermentation processes are expected to play an important role in reducing dependence on fossil-based raw materials for the production of everyday chemicals. In... (Review)
Review
Microbial fermentation processes are expected to play an important role in reducing dependence on fossil-based raw materials for the production of everyday chemicals. In order to meet the growing demand for biotechnological products in the future, alternative carbon sources that do not compete with human nutrition must be exploited. The chemical conversion of the industrially emitted greenhouse gas CO into microbially utilizable platform chemicals such as methanol represents a sustainable strategy for the utilization of an abundant carbon source and has attracted enormous scientific interest in recent years. A relatively new approach is the microbial synthesis of products from the C-compound ethylene glycol, which can also be synthesized from CO and non-edible biomass and, in addition, can be recovered from plastic waste. Here we summarize the main chemical routes for the synthesis of methanol and ethylene glycol from sustainable resources and give an overview of recent metabolic engineering work for establishing natural and synthetic microbial assimilation pathways. The different metabolic routes for C and C alcohol-dependent bioconversions were compared in terms of their theoretical maximum yields and their oxygen requirements for a wide range of value-added products. Assessment of the process engineering challenges for methanol and ethylene glycol-based fermentations underscores the theoretical advantages of new synthetic metabolic routes and advocates greater consideration of ethylene glycol, a C substrate that has received comparatively little attention to date.
Topics: Humans; Methanol; Carbon Dioxide; Ethylene Glycol; Biotechnology; Carbon; Metabolic Engineering
PubMed: 37918546
DOI: 10.1016/j.biotechadv.2023.108276 -
Journal of Medical Toxicology :... Jan 2022Fomepizole is an anti-metabolite therapy that is used to diminish the toxicity from methanol or ethylene glycol. Although its elimination kinetics have been well... (Clinical Trial)
Clinical Trial
INTRODUCTION
Fomepizole is an anti-metabolite therapy that is used to diminish the toxicity from methanol or ethylene glycol. Although its elimination kinetics have been well described in healthy human subjects, the elimination in poisoned patients have only been described in a few isolated cases. This study was designed to relate the elimination of fomepizole in a series of poisoned patients to that in healthy humans.
METHODS
Plasma samples from 26 patients in the clinical trials of the use of fomepizole for methanol and ethylene glycol poisoning were analyzed for fomepizole concentrations. The elimination of fomepizole was assessed after individual doses, both during and without intermittent hemodialysis.
RESULTS
In methanol- and ethylene glycol-poisoned patients, fomepizole had a volume of distribution of 0.66-0.68 L/kg. After repeated doses of fomepizole, the minimum trough concentration averaged 86-109 µmol/L, which is 10 times higher than the minimum therapeutic concentration. In healthy human subjects, fomepizole elimination follows Michaelis-Menten kinetics and has been calculated as zero-order elimination rates. Zero-order elimination rates averaged 13 and 17 μmol/L/h in methanol and ethylene glycol patients, respectively, compared to 6-19 μmol/L/h in healthy subjects. Elimination during intermittent hemodialysis followed first-order kinetics, with a half-life of 3 h.
CONCLUSIONS
Plasma concentrations during the repeated dosing confirmed that the recommended dosing schedule, with and without intermittent hemodialysis, maintained therapeutic concentrations throughout the treatments. Fomepizole elimination in poisoned patients at therapeutic plasma concentrations appears be similar to that reported previously in healthy human subjects.
Topics: Antidotes; Ethylene Glycol; Fomepizole; Humans; Methanol; Poisons; Pyrazoles
PubMed: 34697779
DOI: 10.1007/s13181-021-00862-3 -
Molecular Biology of the Cell Aug 2021With No Lysine (K) WNK kinases regulate electro-neutral cotransporters that are controlled by osmotic stress and chloride. We showed previously that autophosphorylation...
With No Lysine (K) WNK kinases regulate electro-neutral cotransporters that are controlled by osmotic stress and chloride. We showed previously that autophosphorylation of WNK1 is inhibited by chloride, raising the possibility that WNKs are activated by osmotic stress. Here we demonstrate that unphosphorylated WNK isoforms 3 and 1 autophosphorylate in response to osmotic pressure in vitro, applied with the crowding agent polyethylene glycol (PEG)400 or osmolyte ethylene glycol (EG), and that this activation is opposed by chloride. Small angle x-ray scattering of WNK3 in the presence and absence of PEG400, static light scattering in EG, and crystallography of WNK1 were used to understand the mechanism. Osmosensing in WNK3 and WNK1 appears to occur through a conformational equilibrium between an inactive, unphosphorylated, chloride-binding dimer and an autophosphorylation-competent monomer. An improved structure of the inactive kinase domain of WNK1, and a comparison with the structure of a monophosphorylated form of WNK1, suggests that large cavities, greater hydration, and specific bound water may participate in the osmosensing mechanism. Our prior work showed that osmolytes have effects on the structure of phosphorylated WNK1, suggestive of multiple stages of osmotic regulation in WNKs.
Topics: Autoradiography; Chromatography, Gel; Ethylene Glycol; Osmotic Pressure; Phosphorylation; Polyethylene Glycols; Protein Conformation; Protein Kinases; Protein Multimerization; Scattering, Small Angle; WNK Lysine-Deficient Protein Kinase 1; Water; X-Ray Diffraction
PubMed: 33689398
DOI: 10.1091/mbc.E20-01-0089 -
Applied Microbiology and Biotechnology Mar 2019Glycolic acid (GA) and ethylene glycol (EG) are versatile two-carbon organic chemicals used in multiple daily applications. GA and EG are currently produced by chemical... (Review)
Review
Glycolic acid (GA) and ethylene glycol (EG) are versatile two-carbon organic chemicals used in multiple daily applications. GA and EG are currently produced by chemical synthesis, but their biotechnological production from renewable resources has received a substantial interest. Several different metabolic pathways by using genetically modified microorganisms, such as Escherichia coli, Corynebacterium glutamicum and yeast have been established for their production. As a result, the yield of GA and EG produced from sugars has been significantly improved. Here, we describe the recent advancement in metabolic engineering efforts focusing on metabolic pathways and engineering strategies used for GA and EG production.
Topics: Corynebacterium glutamicum; Escherichia coli; Ethylene Glycol; Glycolates; Industrial Microbiology; Metabolic Engineering; Metabolic Networks and Pathways; Microorganisms, Genetically-Modified; Saccharomyces cerevisiae; Xylose
PubMed: 30707252
DOI: 10.1007/s00253-019-09640-2 -
Microbial Cell Factories Apr 2020Biological routes for ethylene glycol production have been developed in recent years by constructing the synthesis pathways in different microorganisms. However, no...
BACKGROUND
Biological routes for ethylene glycol production have been developed in recent years by constructing the synthesis pathways in different microorganisms. However, no microorganisms have been reported yet to produce ethylene glycol naturally.
RESULTS
Xylonic acid utilizing microorganisms were screened from natural environments, and an Enterobacter cloacae strain was isolated. The major metabolites of this strain were ethylene glycol and glycolic acid. However, the metabolites were switched to 2,3-butanediol, acetoin or acetic acid when this strain was cultured with other carbon sources. The metabolic pathway of ethylene glycol synthesis from xylonic acid in this bacterium was identified. Xylonic acid was converted to 2-dehydro-3-deoxy-D-pentonate catalyzed by D-xylonic acid dehydratase. 2-Dehydro-3-deoxy-D-pentonate was converted to form pyruvate and glycolaldehyde, and this reaction was catalyzed by an aldolase. D-Xylonic acid dehydratase and 2-dehydro-3-deoxy-D-pentonate aldolase were encoded by yjhG and yjhH, respectively. The two genes are part of the same operon and are located adjacent on the chromosome. Besides yjhG and yjhH, this operon contains four other genes. However, individually inactivation of these four genes had no effect on either ethylene glycol or glycolic acid production; both formed from glycolaldehyde. YqhD exhibits ethylene glycol dehydrogenase activity in vitro. However, a low level of ethylene glycol was still synthesized by E. cloacae ΔyqhD. Fermentation parameters for ethylene glycol and glycolic acid production by the E. cloacae strain were optimized, and aerobic cultivation at neutral pH were found to be optimal. In fed batch culture, 34 g/L of ethylene glycol and 13 g/L of glycolic acid were produced in 46 h, with a total conversion ratio of 0.99 mol/mol xylonic acid.
CONCLUSIONS
A novel route of xylose biorefinery via xylonic acid as an intermediate has been established.
Topics: Enterobacter cloacae; Ethylene Glycol; Glycolates; Xylose
PubMed: 32293454
DOI: 10.1186/s12934-020-01347-8