Galerina Marginata Research Articles

A Novel Unspecific Peroxygenase from Galatian marginata for Biocatalytic Oxyfunctionalization Reactions

Unspecific peroxygenases (UPOs, EC 1.11.2.1) are promising oxyfunctionalization catalysts because of their unique stereoselectivity. However, so far only a few UPOs have been reported. In this study, gene mining was used to identify a gene from Galerina marginata that coded for a novel UPO (GmaUPO). GmaUPO was expressed in Pichia pastoris X-33 by scale-up fermentation (the UPO activity of the culture supernatant was 118 U/L). GmaUPO exhibited a molecular weight of 40 kDa and exhibited highest activity at 35°C and pH 9, respectively. Furthermore, GmaUPO was demonstrated to catalyze the epoxidation, sulfoxidation, and hydroxylation of common substrates, particularly fatty acids such as tridecanoic acid. The molecular basis for GmaUPO regioselectivity for fatty acid hydroxylation was explored by molecular modelling. The regioselectivity was mostly governed by the architecture of the enzyme's active site.

Whole Genome Sequence of an Edible Mushroom Stropharia rugosoannulata (Daqiugaigu).

Stropharia rugosoannulata, also known as Daqiugaigu in China, is a well-known edible mushroom that has been widely cultivated in China in recent years. Many studies have focused on its nutrients, bioactive compounds, and lignin degradation capacity, although there are few molecular and genetic breeding studies due to the lack of genomic information. Here, we present the 47.9 Mb genome sequence of an S. rugosoannulata monokaryotic strain (A15), which has 20 contigs and an N50 of 3.64 Mb, which was obtained by a combination of Illumina and Nanopore sequencing platforms. Further analysis predicted 12,752 protein-coding genes, including 486 CAZyme-encoding genes. Phylogenetic analysis revealed a close evolutionary relationship between S. rugosoannulata and Hypholoma sublateritium, Psilocybe cyanescens, and Galerina marginata based on single-copy orthologous genes. Proteomic analysis revealed different protein expression profiles between the cap and the stipe of the S. rugosoannulata fruiting body. The proteins of the stipe associated with carbon metabolism, energy production, and stress-response-related biological processes had higher abundance, whereas proteins involved in fatty acid synthesis and mRNA splicing showed higher expression in the cap than in the stipe. The genome of S. rugosoannulata will provide valuable genetic resources not only for comparative genomic analyses and evolutionary studies among Basidiomycetes but also for alleviating the bottlenecks that restrict the molecular breeding of this edible mushroom.

Molecular cloning and the expression pattern of AePOPB involved in the α-amanitin biosynthesis in Amanita exitialis fruiting bodies

Amanita exitialis Zhu L. Yang & T. H. Li is the species responsible for the largest number of mushroom-associated human poisonings and fatalities in South China due to its lethal cyclic peptide toxins. Prolyl oligopeptidase B (POPB) is considered a key enzyme in the production of the highly toxic cyclic peptide α-amanitin. However, the POPB gene of A. exitialis has not been studied. In the present study we cloned and sequenced the full-length A. exitialis POPB (AePOPB) gene. The aim was to verify the gene structure and functions of AePOPB. The full-length sequence of AePOPB is 3144 bp, including 18 exons encoding 730 aa, and the advanced structure is very similar to that of the previously reported POPB in Galerina marginata (GmPOPB). The amino acid sequence of AePOPB is highly homologous with those from other amanitin-producing lethal mushrooms, implying that AePOPB may have a similar role in the biosynthesis of cyclic peptide toxins. Expression levels of AePOPB were detectable in all parts and developmental stages of the fruiting bodies, and AePOPB was expressed more strongly at early development stages (early and late elongation stages). At early and late elongation stages, the expression peaks occurred in the stipe, whereas at early and late mature stages, the expression peaks occurred in the pileus. The expression patterns of AePOPB in different stages and different parts of the fruiting bodies were highly consistent with those of Aeα-AMA, which is required for α-amanitin accumulation. These results indicate that AePOPB should be involved in the α-amanitin biosynthesis in A. exitialis.

Kinetic Landscape of a Peptide Bond-Forming Prolyl Oligopeptidase.

Prolyl oligopeptidase B from Galerina marginata (GmPOPB) has recently been discovered as a peptidase capable of breaking and forming peptide bonds to yield a cyclic peptide. Despite the relevance of prolyl oligopeptidases in human biology and disease, a kinetic analysis pinpointing rate-limiting steps for a member of this enzyme family is not available. Macrocyclase enzymes are currently exploited to produce cyclic peptides with potential therapeutic applications. Cyclic peptides are promising druglike molecules because of their stability and conformational rigidity. Here we describe an in-depth kinetic characterization of a prolyl oligopeptidase acting as a macrocyclase enzyme. By combining steady-state and pre-steady-state kinetics, we propose a kinetic sequence in which a step after macrocyclization limits steady-state turnover. Additionally, product release is ordered, where the cyclic peptide departs first followed by the peptide tail. Dissociation of the peptide tail is slow and significantly contributes to the turnover rate. Furthermore, trapping of the enzyme by the peptide tail becomes significant beyond initial rate conditions. The presence of a burst of product formation and a large viscosity effect further support the rate-limiting nature of a physical step occurring after macrocyclization. This is the first detailed description of the kinetic sequence of a macrocyclase enzyme from this class. GmPOPB is among the fastest macrocyclases described to date, and this work is a necessary step toward designing broad-specificity efficient macrocyclases.

Seven New Recorded Species in Five Genera of the Strophariaceae in Korea

Most known species in the Strophariaceae are decomposers and grow on various kind of organic matter. Approximately 18 genera and 1,316 species in the Strophariaceae have been reported worldwide. Through an ongoing survey of indigenous fungi in Korea, 29 specimens belonging to the Strophariaceae were collected from 2012 to 2016. These specimens were identified based on morphological characteristics and molecular analysis of internal transcribed spacer sequences. Fifteen taxa were confirmed, with eight species matching those previously recorded. Seven species in five genera were shown to be new records in Korea: Galerina marginata, Gymnopilus crociphyllus, Gymnopilus picreus, Hebeloma birrus, Hebeloma cavipes, Pholiota multicingulata, and Psilocybe thaizapoteca. In this study, we provide detailed morphological descriptions of these species and investigate their evolutionary relationships by constructing phylogenetic trees.

Production of (15)N-labeled α-amanitin in Galerina marginata.

α-Amanitin is the major causal constituent of deadly Amanita mushrooms that account for the majority of fatal mushroom poisonings worldwide. It is also an important biochemical tool for the study of its target, RNA polymerase II. The commercial supply of this bicyclic peptide comes from Amanita phalloides, the death cap mushroom, which is collected from the wild. Isotopically labeled amanitin could be useful for clinical and forensic applications, but α-amanitin has not been chemically synthesized and A. phalloides cannot be cultured on artificial medium. Using Galerina marginata, an unrelated saprotrophic mushroom that grows and produces α-amanitin in culture, we describe a method for producing (15)N-labeled α-amanitin using growth media containing (15)N as sole nitrogen source. A key to success was preparing (15)N-enriched yeast extract via a novel method designated "glass bead-assisted maturation." In the presence of the labeled yeast extract and (15)N-NH4Cl, α-amanitin was produced with >97% isotope enrichment. The labeled product was confirmed by HPLC, high-resolution mass spectrometry, and NMR.

Peptide Macrocyclization Catalyzed by a Prolyl Oligopeptidase Involved in α-Amanitin Biosynthesis

Amatoxins are ribosomally encoded and posttranslationally modified peptides that account for the majority of fatal mushroom poisonings of humans. Arepresentative amatoxin is the bicyclic octapeptideα-amanitin, formed via head-to-tail macrocyclization, which is ribosomally biosynthesized as a 35-amino acid propeptide in Amanita bisporigera and in the distantly related mushroom Galerina marginata. Although members of the prolyl oligopeptidase (POP) family of serine proteases have been proposed to play a role in α-amanitin posttranslational processing, the exact mechanistic details are not known. Here, we show that a specific POP (GmPOPB) is required for toxin maturation in G.marginata. Recombinant GmPOPB catalyzed two nonprocessive reactions: hydrolysis at an internal Pro to release the C-terminal 25-mer from the 35-mer propeptide and transpeptidation at the second Pro to produce the cyclic octamer. On the other hand, we show thatGmPOPA, the putative housekeeping POP of G.marginata, behaves like a conventional POP.

Silibinin for cyclopeptide mushroom poisonings

Much has been published in emergency medicine and clinical toxicology literature regarding mushroom poisoning management. However, few articles in pharmacy publications have addressed the management of such emergencies. Interestingly, a recently published comprehensive practical review of 37 antidotes, along with pharmacists' role in their storage, selection, and use, excluded mushroom poisoning antidotes.1 We wish to familiarize health-system pharmacists with silibinin, an investigational antidote for poisoning with cyclopeptide-containing mushrooms (CCMs). CCMs are the cause of most mushroom-related deaths worldwide.2 Ingestion of cyclopeptides—including amatoxins, phallotoxins, and virotoxins— can result in potentially fatal hepatotoxicity. CCMs are commonly called “destroying angel,” “angel of death,” and “death caps.” Examples of CCMs include Amanita phalloides, Amanita virosa, Galerina autumnalis, Galerina marginata, and Lepiota josserandi. Toxicity of cyclopeptides may result from a-amanitin, a bicyclic octapeptide, which inhibits RNA polymerase II and, therefore, DNA transcription. Organs and tissues primarily affected are those dependent on high rates of protein synthesis, notably the gastrointestinal tract, liver, and kidneys. The estimated human lethal dose of a-amatoxin is 0.1 mg/kg. Ingestion of one A. phalloides mushroom containing 5–8 mg of amatoxin is potentially fatal.

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations. GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of ˜10 pure enzymes requires less than 100 μg of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com). Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid. All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (β-glucosidase), EX3 (endo-β1,4-xylanase, GH10), and BX (β-xylosidase).

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of ˜10 pure enzymes requires less than 100 μg of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (β-glucosidase), EX3 (endo-β1,4-xylanase, GH10), and BX (β-xylosidase).

Amatoxins in wood-rotting Galerina marginata

Amatoxins, bicyclic octapeptide derivatives responsible for severe hepatic failure, are present in several Basidiomycota species belonging to four genera, i.e. Amanita, Conocybe, Galerina and Lepiota. DNA studies for G. autumnalis, G. marginata, G. oregonensis, G. unicolor and G. venenata (section Naucoriopsis) determined that these species are the same, supporting the concept of Galerina marginata complex. These mostly lignicolous species are designated as white-rot fungi having a broad host range and capable of degrading both hardwoods and softwoods. Twenty-seven G. marginata basidiomes taken from different sites and hosts (three sets) as well as 17 A. phalloides specimens (three sets) were collected in French locations. The 44 basidiomes were examined for amatoxins and phallotoxins using high-performance liquid chromatography. Toxinological data for the wood-rotting G. marginata and the ectomycorrhizal A. phalloides species were compared and statistically analyzed. The acidic and neutral phallotoxins were not detected in any G. marginata specimen, whereas the acidic (β-Ama) and neutral (α-Ama and γ-Ama) amanitins were found in all basidiomes from either Angiosperms or Gymnosperms hosts. The G. marginata amatoxin content varied from 78.17 to 243.61 μg.mg−1 of fresh weight and was elevated significantly in one set out of three. The amanitin amounts from certain Galerina specimens were higher than those from some A. phalloides basidiomes. Relationship between the amanitin distribution and the chemical composition of substrate was underlined and statistically validated for the white-rot G. marginata. Changes in nutritional components from decayed host due to enzymatic systems and genetic factors as well as environmental conditions seem to play a determinant role in the amanitin profile. Variability noticed in the amanitin distribution for the white-rot G. marginata basidiomes was not observed for the ectomycorrhizal A. phalloides specimens.

Amatoxins in Wood-Rotting Galerina marginata

Amatoxins in Wood-Rotting Galerina marginata

DNA studies in the Galerina marginata complex

The distinctiveness of the European Galerina marginata and the American G. autumnalis is tested with analyses of DNA sequences from the internal transcribed spacer (ITS-2) of the nuclear ribosomal repeat and RFLP analysis of the entire ITS region. European and North American material from six taxa described in Galerina subgenus Naucoriopsis are included along with out-group material of six taxa representing two other subgenera of Galerina. The results do not indicate any genetic difference between species by Smith & Singer (1964) referred to the two ‘stirpes’ Marginata and Autumnalis of Naucoriopsis, i.e. G. autumnalis, G. marginata, G. oregonensis, G. pseudomycenopsis, G. unicolor, and G. venenata and all except G. pseudomycenopsis are considered later synonyms of G. marginata. Galerina badipes (syn. G. cedretorum var. bispora of ‘stirps’ Cedretorum is well supported as a distinct species. Subgenus Naucoriopsis appears as a distinct infrageneric unit well separated from the infrageneric units represented by the out-group species.

Simultaneous assay for amatoxins and phallotoxins in Amanita phalloides Fr. by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method is described that allows the simultaneous determination of up to eight amatoxins and phallotoxins. The method identifies both neutral toxins (α- and γ-amanitin, phalloidin, phallisin and phalloin) and acidic toxins (β-amanitin, phallacidin, and phallisacin). Toxins were separated, identified and determined by gradient elution with 0.02 M aqueous ammonium acetate-acetonitrile and simultaneous monitoring of the absorbances at 214 and 295 nm. The assay was successfully applied to the analysis of the toxins in a crude extract of Amanita phalloides. The limit of detection for each toxin was 10 ng/ml of extraction medium. The assay was further validated by analysing the toxin content in Galerina marginata, a species containing only amatoxins. This relatively simple method should be suitable for the detection of amatoxins and phallotoxins in almost any species of mushrooms.