Ascocoryne Sarcoides Research Articles

Characterisation of ascocorynin biosynthesis in the purple jellydisc fungus Ascocoryne sarcoides

BackgroundNon-ribosomal peptide synthetase-like (NRPS-like) enzymes are highly enriched in fungal genomes and can be discriminated into reducing and non-reducing enzymes. Non-reducing NRPS-like enzymes possess a C-terminal thioesterase domain that catalyses the condensation of two identical aromatic α-keto acids under the formation of enzyme-specific substrate-interconnecting core structures such as terphenylquinones, furanones, butyrolactones or dioxolanones. Ascocoryne sarcoides produces large quantities of ascocorynin, which structurally resembles a terphenylquinone produced from the condensation of p-hydroxyphenylpyruvate and phenylpyruvate. Since the parallel use of two different substrates by a non-reducing NRPS-like enzyme appeared as highly unusual, we investigated the biosynthesis of ascocorynin in A. sarcoides.ResultsHere, we searched the genome of A. sarcoides for genes coding for non-reducing NRPS-like enzymes. A single candidate gene was identified that was termed acyN. Heterologous gene expression confirmed that AcyN is involved in ascocorynin production but only produces the non-hydroxylated precursor polyporic acid. Although acyN is embedded in an ascocorynin biosynthesis gene cluster, a gene encoding a monooxygenase required for the hydroxylation of polyporic acid was not present. Expression analyses of all monooxygenase-encoding genes from A. sarcoides identified a single candidate that showed the same expression pattern as acyN. Accordingly, heterologous co-expression of acyN and the monooxygenase gene resulted in the production of ascocorynin. Structural modelling of the monooxygenase suggests that the hydrophobic substrate polyporic acid enters the monooxygenase from a membrane facing entry site and is converted into the more hydrophilic product ascocorynin, which prevents its re-entry for a second round of hydroxylation.ConclusionThis study characterises the first naturally occurring polyporic acid synthetase from an ascomycete. It confirms the high substrate and product specificity of this non-reducing NRPS-like enzyme and highlights the requirement of a monooxygenase to produce the terphenylquinone ascocorynin.

In Silico Analysis of Functionalized Hydrocarbon Production Using Ehrlich Pathway and Fatty Acid Derivatives in an Endophytic Fungus.

Functionalized hydrocarbons have various ecological and industrial uses, from signaling molecules and antifungal/antibacterial agents to fuels and specialty chemicals. The potential to produce functionalized hydrocarbons using the cellulolytic, endophytic fungus, Ascocoryne sarcoides, was quantified using genome-enabled, stoichiometric modeling. In silico analysis identified available routes to produce these hydrocarbons, including both anabolic- and catabolic-associated strategies, and determined correlations between the type and size of the hydrocarbons and culturing conditions. The analysis quantified the limits of the wild-type metabolic network to produce functionalized hydrocarbons from cellulose-based substrates and identified metabolic engineering targets, including cellobiose phosphorylase (CP) and cytosolic pyruvate dehydrogenase complex (PDHcyt). CP and PDHcyt activity increased the theoretical production limits under anoxic conditions where less energy was extracted from the substrate. The incorporation of both engineering targets resulted in near-complete conservation of substrate electrons in functionalized hydrocarbons. The in silico framework was integrated with in vitro fungal batch growth experiments to support O2 limitation and functionalized hydrocarbon production predictions. The metabolic reconstruction of this endophytic filamentous fungus describes pathways for both specific and general production strategies of 161 functionalized hydrocarbons applicable to many eukaryotic hosts.

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Fig. S1. Consensus phylogram (50 % majority rule) resulting from a Bayesian analysis of the ITS sequence alignment of sequences generated in this study and reference sequences from NCBI GenBank. Bayesian posterior probabilities (PP) > 0.85 are shown at the nodes and thickened lines represent nodes with PP = 1.00. The scale bar represents the expected changes per site. Species and culture collection numbers are indicated. The tree was rooted to Ascocoryne sarcoides (culture NRRL 50072). Fig. S2. Consensus phylogram (50 % majority rule) resulting from a Bayesian analysis of the LSU sequence alignment of sequences generated in this study and reference sequences from NCBI GenBank. Bayesian posterior probabilities (PP) > 0.85 are shown at the nodes and thickened lines represent nodes with PP = 1.00. The scale bar represents the expected changes per site. Species and culture collection numbers are indicated. The tree was rooted to Ascocoryne sarcoides (culture NRRL 50072). Fig. S3. Consensus phylogram (50 % majority rule) resulting from a Bayesian analysis of the act sequence alignment of sequences generated in this study and reference sequences from NCBI GenBank. Bayesian posterior probabilities (PP) > 0.85 are shown at the nodes and thickened lines represent nodes with PP = 1.00. The scale bar represents the expected changes per site. Species and culture collection numbers are indicated. The tree was rooted to Ascocoryne sarcoides (culture NRRL 50072). Fig. S4. Consensus phylogram (50 % majority rule) resulting from a Bayesian analysis of the rpb1 sequence alignment of sequences generated in this study and reference sequences from NCBI GenBank. Bayesian posterior probabilities (PP) > 0.85 are shown at the nodes and thickened lines represent nodes with PP = 1.00. The scale bar represents the expected changes per site. Species and culture collection numbers are indicated. The tree was rooted to Ascocoryne sarcoides (culture NRRL 50072). Fig. S5. Consensus phylogram (50 % majority rule) resulting from a Bayesian analysis of the rpb2 sequence alignment of sequences generated in this study and reference sequences from NCBI GenBank. Bayesian posterior probabilities (PP) > 0.85 are shown at the nodes and thickened lines represent nodes with PP = 1.00. The scale bar represents the expected changes per site. Species and culture collection numbers are indicated. The tree was rooted to Ascocoryne sarcoides (culture NRRL 50072). Fig. S6. Consensus phylogram (50 % majority rule) resulting from a Bayesian analysis of the tef1 sequence alignment of sequences generated in this study and reference sequences from NCBI GenBank. Bayesian posterior probabilities (PP) > 0.85 are shown at the nodes and thickened lines represent nodes with PP = 1.00. The scale bar represents the expected changes per site. Species and culture collection numbers are indicated. The tree was rooted to Ascocoryne sarcoides (culture NRRL 50072). Fig. S7. Consensus phylogram (50 % majority rule) resulting from a Bayesian analysis of the full set 6-gene (ITS, LSU, act, tef1, rpb1, rpb2) sequence alignment. Bayesian posterior probabilities (PP) > 0.85 are shown at the nodes and thickened lines represent nodes with PP = 1.00. The scale bar represents the expected changes per site. Species and culture collection numbers are indicated. The tree was rooted to Ascocoryne sarcoides (culture NRRL 50072).Fig. S8. Consensus phylogram (50 % majority rule) resulting from a Bayesian analysis of the full set 4-gene (ITS, LSU, act, tef1) sequence alignment. Bayesian posterior probabilities (PP) > 0.85 are shown at the nodes and thickened lines represent nodes with PP = 1.00. The scale bar represents the expected changes per site. Species and culture collection numbers are indicated. The tree was rooted to Ascocoryne sarcoides (culture NRRL 50072).Fig. S9. Consensus phylogram (50 % majority rule) resulting from a Bayesian analysis of the full set 2-gene (rpb1, rpb2) sequence alignment. Bayesian posterior probabilities (PP) > 0.85 are shown at the nodes and thickened lines represent nodes with PP = 1.00. The scale bar represents the expected changes per site. Species and culture collection numbers are indicated. The tree was rooted to Ascocoryne sarcoides (culture NRRL 50072).

Endophytic Fungi: Prospects in Biofuel Production

Endophytic fungi are now being recognized as a yet unexplored source of volatile low molecular mass hydrocarbons and lipids for the production of next generation biofuels. These next generation biofuels are to be produced by more efficient and lesser number of biosynthetic steps directly from the microorganisms responsible and would have the preferred adaptability for immediate use in the current internal combustion engines without bringing about a radical change in automobile design. Research groups working across the world have reported isolation of endophytes having optimum lipid profiles for use as precursors for biofuel generation. Fungal endophytes have also been found to produce volatile organic compounds having similarity with conventional diesel fuel. The recent publication of the genome sequence of one such biofuel producing endophytic fungi Ascocoryne sarcoides will increase the understanding of the biosynthetic machinery by which endophytes are able achieve the complex feat of generating volatile hydrocarbons. Research on this important area of alternative bio-energy for ensuring India’s energy security in the near future has slowly started gaining momentum. The present article therefore aims to review the current status of research in the Indian perspective, in order to encourage and generate interest among research groups across India and the world for initiating and undertaking more vigorous and focused research activity on endophytic fungi from the Indian sub-continent having the potential to make fuel-related hydrocarbons.

Evaluation of Cellulose as a Substrate for Hydrocarbon Fuel Production by <i>Ascocoryne sarcoides</i> (NRRL 50072)

The fungal endophyte, Ascocoryne sarcoides, produced aviation, gasoline and diesel-relevant hydrocarbons when grown on multiple substrates including cellulose as the sole carbon source. Substrate, growth stage, culturing pH, temperature and medium composition were statistically significant factors for the type and quantity of hydrocarbons produced. Gasoline range (C_5-C_(12)), aviation range (C_8-C_(16)) and diesel range (C_9-C_(36)) organics were detected in all cultured media. Numerous non-oxygenated hydrocarbons were produced such as isopentane, 3,3-dimethyl hexane and d-limonene during exponential growth phase. Growth on cellulose at 23˚C and pH 5.8 produced the highest overall yield of fuel range organics (105 mg * g•biomass^(-1)). A change in metabolism was seen in late stationary phase from catabolism of cellulose to potential oxidation of hydrocarbons resulting in the production of more oxygenated compounds with longer carbon chain length and fewer fuel-related compounds. The results outline rational strategies for controlling the composition of the fuel-like compounds by changing culturing parameters.

우면산 일대의 균류상

Many fungi were collected at Mt. Umyeon from July, 2012 to Oct. 2012. They were identified. As the resulting, They were compposed of 3 divisions, 2 subdivisions, 5 classes, 3 subclasses, 17 orders, 5 families, 85 genera and 151 species. Among them dominant genus was Amanita. Anthrocobia, Ascocoryne, Erionema are newly genera to Korea. Following species are unrecorded in Korea : Calvatia giganeta, Amanita eliae, A. magniverrucata, A. sinensis, Inonotus tomentosus, Tylopilus rigens, Russula medullata, Anthrocobia maurilabra, Ascocoryne sarcoides, Erionema aureum and Fuligo candida. They were desinged Korean common names by authors.

Resolution of volatile fuel compound profiles from Ascocoryne sarcoides: a comparison by proton transfer reaction-mass spectrometry and solid phase microextraction gas chromatography-mass spectrometry

Volatile hydrocarbon production by Ascocoryne sacroides was studied over its growth cycle. Gas-phase compounds were measured continuously with a proton transfer reaction-mass spectrometry (PTR-MS) and at distinct time points with gas chromatography-mass spectrometry (GC-MS) using head space solid phase microextraction (SPME). The PTR-MS ion signal permitted temporal resolution of the volatile production while the SPME results revealed distinct compound identities. The quantitative PTR-MS results showed the volatile production was dominated by ethanol and acetaldehyde, while the concentration of the remainder of volatiles consistently reached 2,000 ppbv. The measurement of alcohols from the fungal culture by the two techniques correlated well. Notable compounds of fuel interest included nonanal, 1-octen-3-ol, 1-butanol, 3-methyl- and benzaldehyde. Abiotic comparison of the two techniques demonstrated SPME fiber bias toward higher molecular weight compounds, making quantitative efforts with SPME impractical. Together, PTR-MS and SPME GC-MS were shown as valuable tools for characterizing volatile fuel compound production from microbiological sources.

Genomic Analysis of the Hydrocarbon-Producing, Cellulolytic, Endophytic Fungus Ascocoryne sarcoides

The microbial conversion of solid cellulosic biomass to liquid biofuels may provide a renewable energy source for transportation fuels. Endophytes represent a promising group of organisms, as they are a mostly untapped reservoir of metabolic diversity. They are often able to degrade cellulose, and they can produce an extraordinary diversity of metabolites. The filamentous fungal endophyte Ascocoryne sarcoides was shown to produce potential-biofuel metabolites when grown on a cellulose-based medium; however, the genetic pathways needed for this production are unknown and the lack of genetic tools makes traditional reverse genetics difficult. We present the genomic characterization of A. sarcoides and use transcriptomic and metabolomic data to describe the genes involved in cellulose degradation and to provide hypotheses for the biofuel production pathways. In total, almost 80 biosynthetic clusters were identified, including several previously found only in plants. Additionally, many transcriptionally active regions outside of genes showed condition-specific expression, offering more evidence for the role of long non-coding RNA in gene regulation. This is one of the highest quality fungal genomes and, to our knowledge, the only thoroughly annotated and transcriptionally profiled fungal endophyte genome currently available. The analyses and datasets contribute to the study of cellulose degradation and biofuel production and provide the genomic foundation for the study of a model endophyte system.

Volatile organic compound production by organisms in the genus Ascocoryne and a re-evaluation of myco-diesel production by NRRL 50072

The Patagonian fungal endophyte NRRL 50072 is reported to produce a variety of medium-chain and highly branched volatile organic compounds (VOCs) that have been highlighted for their potential as fuel alternatives and are collectively termed myco-diesel. To assess the novelty of this observation, we determined the extent to which ten closely related Ascocoryne strains from commercial culture collections possess similar VOC production capability. DNA sequencing established a high genetic similarity between NRRL 50072 and each Ascocoryne isolate, consistent with its reassignment as Ascocoryne sarcoides. The Ascocoryne strains did not produce highly branched medium-chain-length alkanes, and efforts to reproduce the branched alkane production of NRRL 50072 were unsuccessful. However, we confirmed the production of 30 other products and expanded the list of VOCs for NRRL 50072 and members of the genus Ascocoryne. VOCs detected from the cultures consisted of short- and medium-chain alkenes, ketones, esters and alcohols and several sesquiterpenes. Ascocoryne strains NRRL 50072 and CBS 309.71 produced a more diverse range of volatiles than the other isolates tested. CBS 309.71 also showed enhanced production compared with other strains when grown on cellulose agar. Collectively, the members of the genus Ascocoryne demonstrated production of over 100 individual compounds, with a third of the short- and medium-chain compounds also produced when cultures were grown on a cellulose substrate. This comparative production analysis could facilitate future studies to identify and manipulate the biosynthetic machinery responsible for production of individual VOCs, including several that have a potential application as biofuels.

Endophyte Strain NRRL 50072 producing volatile organics is a species of Ascocoryne

A fungal endophyte (NRRL 50072) isolated from Eucryphia cordifolia from Patagonia that produces hydrocarbon derivatives is identified as an Ascocoryne sp. It was classified as Clonostachys rosea (Gliocaldium roseum) strain C-13 = NRRL 50072 primarily based upon its morphological characteristics. The fungus produced slimy clumped conidia on verticillated conidiophores and red- pigmented particles in culture. However, DNA sequence analysis (ITS rDNA) suggested a 99% identity to Ascocoryne sarcoides rather than to fungi assigned to the group of Gliocladium-like anamorphs. Comparative genetic, biological and morphological studies confirmed that the anamorphic stage of an authenticated field-collected culture of Ascocoryne sarcoides AV-70 shares many of the same morphological, and genetic features as NRRL 50072, but the latter is unable to produce synnematal masses unless it is grown on a proper substratum. These data suggest that NRRL 50072 is most closely related to the asexual stage of A. sarcoides, namely a Coryne sp. Serial transfer of the A. sarcoides AV-70 resulted in major cultural changes in the fungus especially in the production of aerial hyphae, pigment production and the number of synnemata being formed. This report appropriately sets the taxonomic framework for further molecular biological and biochemical work on NRRL 50072 and related fungi.

Relationship of root rot to black spruce windfall and mortality following strip clear-cutting

Root and butt rots are often implicated as causal factors influencing windfall and mortality of residual trees following partial cutting. Measurements of decay at stump level (i.e., the upward extension of root rot) were made on cross-sectional discs taken from windfallen and standing dead 100- to 130-year-old black spruce (Picea mariana (Mill.) BSP) near Nipigon in northwestern Ontario. Subsequently, causal fungi were identified using laboratory culturing procedures. The incidence and amount of decay in windfallen trees within leave strips following alternate strip clear-cutting was higher than the general stand levels but lower than that found in windfallen trees in uncut forest. The incidence and amount of decay was also higher in windfallen trees near the centres of the leave strips than in those near the edges and corners of these strips. These results indicate a strong association between root rot and windfall and suggest that for comparable windfirmness, trees near the edges of residual stands must have less decay than those in more sheltered locations. Decay levels tended to be lower on poorly drained sites than on well-drained sites. In uncut forest, and especially in the leave strips, more trees were uprooted than died standing or suffered stem breakage. The incidence and amount of decay tended to be lower in uprooted trees than in standing dead trees or those with stem breakage, although in uncut forest virtually all windfallen or standing dead trees had some degree of stump-level decay. Of the 21 wood-rotting Basidiomycetes isolated from windfallen and standing dead trees, Inonotus tomentosus (Fr.:Fr.) Teng was the most frequent, followed in order by Armillaria ostoyae (Romagn.) Herink, Coniophora puteana (Schum.:Fr.) Karst., and Scytinostroma galactinum (Fr.) Donk. The incidence of I. tomentosus, C. puteana, Xeromphalina campanella (Batsch.:Fr.) Kuhner & Maire, and Serpula himantioides (Fr.:Fr.) Karst., but not Armillaria ostoyae, Scytinostroma galactinum, and Sistotrema brinkmanii (Bres.) Erik., was greater in windfallen and standing dead trees from the leave strips than in the general stand populations. In the leave strips, I. tomentosus, Amylostereum chailletii (Pers.:Fr.) Boid., and Trichaptum abietinum (Dickson:Fr.) Ryv. tended to greater relative abundance in standing dead trees, while the relative abundance of C. puteana and Serpula himantioides was greater in trees with stem breakage. Armillaria ostoyae and Scytinostroma galactinum were as abundant in uprooted trees as in standing dead trees or those with stem breakage. Ascocoryne sarcoides (Jacq.:Fr.) G. & W., a staining fungus that may protect against decay fungi, was frequently isolated in this study.

Ascocorynin, a terphenylquinone from Ascocoryne Sarcoides

Ascocorynin, 2,5-dihydroxy-3-( p-hydroxyphenyl)-6-phenyl-1,4-benzoquinone, has been isolated from the fungus Ascocoryne sarcoides. The biogeneti

Ascocoryne species in living stems of Picea species A literature review

Ascocoryne sarcoides (Jacquin ex S.F. Gray) Groves and Wilson has been reported to be widely distributed and common in living stems of Picea spp. in Europe and Canada. A. cylichnium (L.-R. Tulasne) Korf has been reported relatively seldom from such stems in Europe and apparently not at all in Canada. However, the identifications can usually not be verified because descriptions of cultures are rarely given. A. cylichnium may be common in spruce stems in both regions. Ways of infection and distribution in the stem are mentioned, and data on enzyme production and effect on wood are given.

A bilayer plate technique to detect broad-spectrum antagonism in microorganisms and its application to wood-inhabiting fungi.

A simple method is described for measuring antagonistic activity in microorganisms based on the mycelial growth response of up to 10 isolates of the bioassay fungus in a single agar plate. The technique overcomes disadvantages of existing methods by providing quantitative estimates of interstrain variability in the bioassay fungus as well as the antagonist. Application of the method to a wide range of isolates of Ascocoryne sarcoides (Jacq. ex Gray) Groves and Wilson against three species of decay fungi has permitted the rapid detection of broad-spectrum strains of this fungus which would not have been practical by conventional bioassays.

Heart rot of black spruce in Ontario. II. The mycoflora in defective and normal wood of living trees

The fungi most frequently associated with heart rot in black spruce stems were the Basidiomycetes Fomes pini (Fr.) Karst., Scytinostroma galactina (Fr.) Donk, Polyporus tomentosus Fr., Peniophora septentrionalis Laurila, and Coniophora puteana (Schum. ex Fr.) Karst. Factors related to the occurrence of these and other Basidiomycetes are assessed. An Ascomycete, Ascocoryne sarcoides (Jacq. ex Gray) Groves and Wilson, was frequently isolated from both clear and defective wood in living black spruce (Picea mariana (Mill.) B.S.P.) stems older than 75 years of age. In laboratory tests this fungus invaded but did not appreciably alter black spruce heartwood, and provided some protection against subsequent decay by F. pini. Evidence is presented that the widespread occurrence of A. sarcoides in mature black spruce stems partly explains the relatively low incidence of defect in this species. The potential use of A. sarcoides as an agent for the biological control of decay in black spruce and other conifers is discussed.