Fungus Coprinus Cinereus Research Articles

Production of α-cuprenene in Xanthophyllomyces dendrorhous: a step closer to a potent terpene biofactory

BackgroundThe red yeast Xanthophyllomyces dendrorhous is a natural producer of the carotenoid astaxanthin. Because of its high flux, the native terpene pathway leading to the production of the tetraterpene is of particular interest as it can be redirected toward the production of other terpene compounds. The genetic tools for the transformation of the yeast with the concurrent knock-out of genes involved in the astaxanthin biosynthesis are made available and here we show that the production of the sesquiterpene α-cuprenene is possible in mutant strains of X. dendrorhous transformed with the Cop6 gene originating from the fungus Coprinus cinereus. For the evaluation of the production levels, we chose to express the same gene and analyze the accumulation of α-cuprenene in Escherichia coli and Saccharomyces cerevisiae, as well. Here we propose that X. dendrorhous is a candidate in the search for the potential platform organism for the production of terpenes.ResultsAll three X. dendrorhous mutants functionally express the Cop6 gene and accumulate α-cuprenene. The production of α-cuprenene in the red yeast reached 80 mg/L, which represents a far higher concentration compared to the levels obtained in the E. coli and S. cerevisiae mutants. At this expression levels the pool of terpene precursors has not become a limiting factor in the X. dendrorhous mutants since the expression of the Cop6 gene in the genomic rDNA of the yeast allows production of both α-cuprenene and astaxanthin without affecting the growth or the accumulation levels of both compounds.ConclusionsWe have shown that X. dendrorhous can produce α-cuprenene, and the results here presented, next to the capability of accumulating at least two more non-native sesquiterpenes, demonstrates the high potential of this yeast to become an interesting terpene-based drugs producer.

Antineoplastic agents 582. Part 1: Isolation and structure of a cyclobutane-type sesquiterpene cancer cell growth inhibitor from Coprinus cinereus (Coprinaceae)

Bioassay-guided (murine P388 lymphocytic leukemia and human cancer cell lines) separation of an ethyl acetate extract prepared from the inky cap fungus Coprinus cinereus led to the isolation of three new sesquiterpenes, 7,7a-diepicoprinastatin 1 (1), 14-hydroxy-5-desoxy-2S,3S,9R-illudosin (2), and 4,5-dehydro-5-deoxyarmillol (3), together with the known armillol (4). The structure and relative configuration of 1 was determined by single-crystal X-ray diffraction experiments. The structures of compounds 2, 3, and 4 were each deduced by a combination of HRMS and 1D and 2D NMR techniques. Cyclobutane 2 led to modest inhibition of the murine P388 leukemia cell line.

Amino acid pool composition of the basidiomyceteCoprinus cinereus

The leaf-litter fungus Coprinus cinereus maintains a pool of free amino acid in its mycelium. When the organism is grown under conditions of high nitrogen availability with 13.2 mmol.L-1 L-asparagine as the nitrogen source, the primary constituents of this pool are glutamine, alanine, and glutamic acid. Together these 3 amino acids comprise approximately 70% of the pool. Nitrogen deprivation reduces the size of the free amino acid pool by 75%, and neither a high concentration of ammonium nor a protein nitrogen source support a similar pool size as L-asparagine. Nitrogen deprivation also reduces the concentration of glutamine to the pool while increasing glutamate. Concomitant with this shift is a marked increase in mycelial ammonium.

균류 Coprinus cinereus에서 DNA 회복에 관여하는 RAD3 유사유전자의 분리와 특성

본 연구는 출아형 효모 Saccharomyces cerevisiae에서 자외선의 상해 시 이를 정상으로 회복시키는 절제회복(excision repair) 유전자로 알려진 RADS의 특성 규명을 위하여 균류 Coprinus cinereus에서 이와 유사한 유전자를 분리하였다. RADS 유사 유전자를 분리하기 위하여 균류 C. cinereus의 염색체 DNA를 전기영동하여 분리한 다음 효모 RADS DNA를 probe로 하여 이와 hybridization하였다. 이 결과 RADS유사 유전자는 3.4kb의 insert DNA를 갖고 있었다. 또한 Southern hybridization으로 이 유사 유전자는 fungus C. cinereus의 염색체에 존재함을 확인하였다. 분리한 RADS 유사 유전자의 전사체 크기는 2.8kb 였으며, 자외선의 상해시 전혀 자외선에 대한 유도성이 없음을 Northern hybridization으로 확인하였다. 또한 유사유전자 부분을 삭제하였을 때 이 부분이 없는 세포는 전혀 생존을 못하였다. 이 결과 분리한 RADS 유사유전자는 세포의 생존에 필수적인 유전자임을 알 수 있었다. The RAD3 gene of Saccharomyces cerevisiae is essential for the incision step of UV-induced excision repair. An yeast RAD3 gene has been previously isolated by functional complementation. In order to identify the RAD3 homologous gene from fungus Coprinus cinereus, we have constructed cosmid libraries from electrophoretically separated chromosomes of the C. cinereus. The 13 C. cinereus chromosomes were resolved by pulse-field gel electrophoresis, hybridized with S. cerevisiae RAD3 DNA, and then isolated RAD3 homologous DNA from C. cinereus chromosome. The RAD3 homolog DNA was contained in 3.2 kb DNA fragment. Here, we report the results of characterization of a fungus C. cinereus homolog to the yeast RAD3 gene. Southern blot analysis confirmed that the C. cinereus chromosome contains the RAD3 homolog gene and this gene exists as a single copy in C. cinereus genome. When total RNA isolated from the C. cinereus cells were hybridized with the 3.4 kb PvuII DNA fragment of the S. cerevisiae RAD3 gene, transcripts size of 2.8 kb were detected. In order to investigate whether the increase of the amount of transcripts by DNA damaging agent, transcript levels were examined after treating agents to the cells. The level of transcripts were not increased by untraviolet light (UV). This result indicated that the RAD3 homologous gene is not UV inducible gene. Gene deletion experiments indicate that the HRD3 gene is essential for viability of the cells and DNA repair function. These observations suggest an evolutionary conservation of other protein components with which HRD3 interacts in mediating its DNA repair and viability functions.

Gene design, expression, crystallization and preliminary diffraction analysis of two isolectins from the fungus Coprinus cinereus: a model for studying functional diversification of galectins in the same organism and their evolutionary pathways.

It is the aim of comparative structural biology to define the evolutionarily important traits of protein function and the points of diversification. Consequently, structural analysis, especially of distant members in a family which in this case are lectins involved in cell adhesion and growth regulation in animals (i.e. galectins), is required. For this purpose, recent work has been focused on the first galectins known from outside the animal kingdom. These are the two isolectins from the basidiomycete Coprinus cinereus (inky cap mushroom), termed Cgl-1 and Cgl-2. Additionally, the close similarity (83% deduced amino-acid identity) but the pronounced difference in the expression patterns of these two fungal lectins during fruiting-body formation affords a suitable object for study of the relation of structural difference to the observed functional disparity in the same organism. Both galectins were crystallized after recombinant production. Crystals belong to either the orthorhombic space group C222(1) (Cgl-1) or the monoclinic space group P2(1) (Cgl-2). The latter crystals diffracted to 1.6 A resolution using synchrotron radiation. To solve the phasing problem, a selenomethionine-containing variant of Cgl-1 was designed. Crystals isomorphous to those of the native counterpart were obtained. Their structural analysis will also be crucial to solving the structure of Cgl-2.

균류 Coprinus cinereus에서 DNA 회복에 관여하는 RAD4 유사유전자의 분리와 특성

The RAD4 gene of Saccharomyces cerevisiae is essential for the incision step of UV-induced excision repair. A yeast RAD4 gene has been previously isolated by functional complementation. In order to identify the RAD4 homologous gene from fungus Coprinus cinereus, we have constructed cosmid libraries from electrophoretically separated chromosomes of the C. cinereus. The 13 C. cinereus chromosomes were resolved by pulse-field gel electrophoresis, hybridized with S. cerevisiae RAD4 DNA, and then isolated homologous C. cinereus chromosome. The insert DNA of the RAD4 homolog was contained 3.2 kb. Here, we report the characterization of fungus C. cinereus homolog of yeast RAD4 gene. Southern blot analysis confirmed that C. cinereus contains the RAD4 homolog gene and this gene exists as a single copy in C. cinereus genome. When total RNA isolated from C. cinereus cells was hybridized with the 1.2 kb PvuII DNA fragment of the S. cerevisiae RAD4 gene, a 2.5 kb of transcript was detected. In order to investigation whether the increase of transcripts by DNA damaging agent, transcripts levels were examined after treating the cells. The level of transcript did not increase by untraviolet light (UV). This result indicated that the RAD4 homologous gene is not UV inducible gene. Gene deletion experiments indicate that the RAD4 homologous gene is essential for cell viability.

CDNAs encoding large venom proteins from the parasitoid wasp Pimpla hypochondriaca identified by random sequence analysis

Venom from the parasitoid wasp Pimpla hypochondriaca contains numerous proteins, has potent in vitro anti-haemocytic properties, and disrupts host encapsulation responses. By sequencing 500 cDNAs randomly isolated from a venom gland library, we have identified 60 clones that encode proteins containing potential secretory signal sequences. To identify cDNAs encoding particular venom proteins, N-terminal amino acid sequences were determined for large (>30 kDa) venom proteins that had been separated using a combination of gel filtration and SDS-PAGE. We describe five of these cDNAs, which encoded residues that matched with the N-terminal sequences of previously undescribed venom proteins. cDNAs vpr1 and vpr3 encoded related proteins of approximately 32 kDa that were found in widely different fractions of gel filtration-separated venom. Neither vpr1 nor vpr3 were closely related to any other protein in the GenBank database, suggesting that they are highly specialised venom components. vpr2 encoded a 57-kDa polypeptide that was similar to a Drosophila protein, of unknown function, which lacks a signal sequence. A fourth clone, tre1, encoded a 61-kDa protein with extensive sequence similarity to trehalases. The 76-kDa sequence encoded by lac1 contained three regions which were very similar to histidine-rich copper-binding motifs, and could be aligned with the laccase from the fungus Coprinus cinereus. This study represents a significant step towards a holistic view of the molecular composition of a parasitoid wasp venom.

Structure of the laccase from Coprinus cinereus at 1.68 A resolution: evidence for different 'type 2 Cu-depleted' isoforms.

Laccases (E.C. 1.10.3.2; benzenediol oxygen oxidoreductases) couple the four-electron reduction of dioxygen to water to four one-electron oxidations of a reducing substrate. The three-dimensional structure of the 'blue' multi-copper oxidase laccase from the fungus Coprinus cinereus at 1.68 A reveals the structural basis for isoforms of the type 2 Cu-depleted species.

The Control of Meiosis Progression in the Fungus Coprinus cinereus by Light/Dark Cycles

Meiosis progression in Coprinus cinereus is controlled by light/dark cycles. Light is essential to propel basidia into karyogamy and light intensity determines the timing of meiotic events. The higher the light intensities, the faster the fruiting bodies enter karyogamy. The critical period when light has this influence is between 16 and 6 h before karyogamy. The control is highly stage specific. A 3-h dark period is essential for a Java dikaryon and the Japanese AmutBmut homokaryon to enter meiotic metaphase; without it the fruit body is permanently arrested at diffused diplotene. This arrest is light intensity-dependent (>20 hlx) and temperature-dependent (e.g., 27°C). The placement of the dark period is very stage specific; it has no effect when placed before karyogamy stage. A dikaryon of London origin is light blind and able to complete meiosis under continuous high light regime. Fruiting bodies arrested under a continuous high light can be rescued by a 3-h dark treatment, but there is always an 8-h lag time to enter meiotic metaphase. It is possible that the dark effect signals cellular processes leading to division events. Cytological studies of arrested fruiting bodies showed that chromosomes are normal in meiotic prophase through pachytene and diplotene, but are unable to undergo chromosome condensation. Genetic crosses between a monokaryon of Java stock J6;5.4 and a monokaryon BL55 or H5 of London stock showed that light-blindness is dominant, and is controlled by a single Mendelian gene.

The signature of balancing selection: fungal mating compatibility gene evolution.

A key problem in evolutionary biology has been distinguishing the contributions of current and historical processes to the maintenance of genetic variation. Because alleles at self-recognition genes are under balancing selection, they exhibit extended residence times in populations and thus may provide unique insight into population demographic history. However, evidence for balancing selection and extended residence times has almost exclusively depended on identification of transspecific polymorphisms; polymorphisms retained in populations through speciation events. We present a broadly applicable approach for detecting balancing selection and apply it to the b1 mating type gene in the mushroom fungus Coprinus cinereus. The comparison of neutral molecular variation within and between allelic classes was used to directly estimate the strength of balancing selection. Different allelic classes are defined as encoding different mating compatibility types and are thus potentially subject to balancing selection. Variation within an allelic class, where all alleles have the same mating compatibility type, provided an internal standard of neutral evolution. Mating compatibility in this organism is determined by the complex A mating type locus, and b1 is one of several redundantly functioning genes. Consequently, we conducted numerical simulations of a model with two subloci and varying levels of recombination to show that balancing selection should operate at each sublocus. Empirical data show that strong balancing selection has indeed occurred at the b1 locus. The widespread geographic distribution of identical b1 alleles suggests that their association with differing A mating types is the result of recent recombination events.

The divergence-homogenization duality in the evolution of the b1 mating type gene of Coprinus cinereus.

The A mating type locus of the fungus Coprinus cinereus is a complex, multigenic locus which regulates compatibility and subsequent sexual development. Genes within the A locus such as the b1 gene studied here exhibit extreme sequence variation. In this work, we asked how b1 alleles have evolved high levels of variation and, at the same time, conserved function. We compared sequence variation in 17 alleles characterized as belonging to seven different compatibility classes. Comparison of sequence variation between representatives of these seven classes shows that different regions of the b1 gene have been subject to varying levels of substitution, recombination, and structural/functional constraints. The N-terminal region of the encoded protein, which has been previously demonstrated to govern self/nonself recognition, exhibited hypervariability with levels of amino acid identity as low as 41%. We used a novel analysis of neutral mutations accumulating in this gene to rule out the possibility that the N-terminal region is hypermutable. In contrast, the C-terminal region displayed heterogeneous levels of variation, with functional motifs being better conserved. In fact, there is a duality in the b1 gene between variability and conservation; recombination events have homogenized the C-terminal region, while recombination events are undetectable in the N-terminal region. The ability to regulate sexual development is maintained in all of the mating compatibility alleles studied, and these data suggest that some functional motifs may tolerate high levels of substitution.

Crystallization and preliminary X-ray analysis of the laccase from Coprinus cinereus.

The laccase from the fungus Coprinus cinereus has been prepared and crystallized in a form suitable for X-ray diffraction analysis. Small plate-like crystals of an enzymatically deglycosylated form of the enzyme have been grown by the hanging-drop method using polyethylene glycol as precipitant. These crystals diffract to at least 2.2 A. They belong to the space group P2(1)2(1)2(1) with cell dimensions a = 45.4, b = 85.7, c = 143.1 A with a single molecule of laccase in the asymmetric unit.

Characterization of DNA ligase from the fungus Coprinus cinereus.

DNA ligase was highly purified from the fungus Coprinus cinereus at the miotic recombination stage, pachytene. The pachytene DNA ligase showed three polypeptides with molecular masses of 88, 84 and 80 kDa, as estimated by the [32P]AMP-labeling assay. These three polypeptides were susceptible to reaction with an mAb against a 16-amino-acid sequence in human DNA ligase I, which is conserved in C-terminal regions of mammalian, vaccinia virus and yeast DNA ligases. Since rapidly purified preparations from fresh pachytene cells exhibited a single polypeptide of DNA ligase with a molecular mass of 88 kDa, the smaller polypeptides seemed to be limited-degradation products of the 88-kDa polypeptide during the isolation and purification procedures. K(m) values for ATP and (dT)20 hybridized with (dA)n were 1.5 microM and 90 nM, respectively. This enzyme was capable of joining (dT)20.(rA)n and (rA)12-18 (dT)n as well as (dT)20.(dA)n and able to ligate blunt-ended DNA in the presence of poly(ethylene glycol) 6000. DNA ligases were also partially purified from zygotene cells at the meiotic pairing stage and mitotic mycelium cells. In their molecular mass, immuno-reactivity, K(m) value and substrate specificity, they were indistinguishable from pachytene DNA ligase. These results suggest that the fungus C. cinereus at the pachytene stage contains DNA ligase with a molecular mass of 88 kDa as a main or a single species, which is quite similar to DNA ligases from the zygotene and mycelium cells in molecular and catalytic properties.

Mating type control of sexual development inCoprinus cinereus

The multiallelic mating type genes of the hymenomycete fungus Coprinus cinereus determine mating compatibility by regulating a developmental sequence that converts an asexual monokaryon into a fertile dikaryon. The genes map to two loci, A and B, and mating compatibility requires different alleles of genes at both loci. The A genes encode two classes of proteins with conserved but dissimilar homeodomain DNA binding motifs (HD1 and HD2), which identify their role in development as transcriptional regulators. Transformation studies with cloned genes suggest that a compatible mating is sensed by combinatorial interactions between an HD1 and HD2 protein and that the N-terminal regions of these proteins are implicated in the specificity of this interaction. The B genes of C. cinereus have been cloned but their function is, as yet, unknown. In another species, Schizophyllum commune, the B genes encode pheromones and pheromone receptors. Although a pheromone response pathway is not apparent in cell fusion in hymenomycetes, it now seems likely to be involved in maintenance of dikaryotic growth. Key words: Coprinus, hymenomycete, mating type, homeodomain proteins, pheromones and receptors, sexual development.

A fungal mating type protein that regulates sexual and asexual development contains a POU-related domain.

The A mating type factor of the fungus Coprinus cinereus regulates essential steps in sexual development. Here we describe features of one of the four specificity genes of the A42 factor. By transformation we show that the gene regulates not only sexual development but also asexual sporulation. DNA sequence analysis shows that the gene beta 1-1, encodes a protein with a DNA binding motif and is thus likely to be a transcription factor. The DNA binding domain is an unusual homeodomain with D replacing the normally invariant N in the recognition helix and apparent absence of helix II. The homeodomain is linked to a helical region related to the POUs domain, which is part of a bipartite DNA binding domain of certain animal transcription factors. Like POU factors, the beta 1-1 protein has regions rich in serine, threonine and proline which are possible transactivation domains. Putative dimerization domains and sites for post-translational modification are described.

The effect of junlon PW110 and tween 80 on the production of cellulolytic enzymes by Coprinus cinereus

The effect of Junlon PW110 (an acrylic acid polymer) and of Tween 80 (a nonionic surfactant) on the production of cellulolytic and xylanolytic enzymes by the basidiomycete fungus Coprinus cinereus was investigated. The inclusion of each material in culture media resulted in significant increases in the production of extracellular cellulases and a mixture of the two was even more effective. Junlon PW110 also stimulated growth of C. cinereus and the production of xylanase and β-xylosidase. Tween 80 had no effect on xylanase production although it did increase β-xylosidase production. Junlon PW110 and Tween 80 in combination increased the rate of production of cellulase by cultures as well as its final yield.

Correlation of endonuclease activity at meiotic prophase and cofactor-induced genetic recombination in the basidiomycete Coprinus cinereus

An endonuclease was purified from the basidiocarps of the fungus Coprinus cinereus during late premeiotic S-phase, karyogamy, and pachytene stages of meiosis. This endonuclease causes single-strand nicks on supercoiled PM2 DNA. Its activity is dependent on cofactors such as Mg2+ and Ca2+, and is enhanced by hexaminecobalt chloride and dimethylsulfoxide. Treatments with increasing cofactor concentration elicit increasing enzyme activity in vitro and increasing genetic recombination in vivo. The percent recombination that can be induced by cofactor treatment during late premeiotic S-phase, karyogamy and pachytene is correlated with the amount of endonuclease that can be extracted from the respective stages. These results suggest that Mg2+ and Ca2+ induce genetic recombination by increasing the endonuclease activity that creates nicked and gapped DNA substrates for ensuing recombination events. We believe that the meiotic endonuclease of Coprinus is involved in the early phase of genetic recombination. Cold temperature treatment is known to retard repair activity at pachytene and causes a threefold increase in recombination. Double treatments involving Mg2+ and Ca2+ at karyogamy followed by cold temperature at pachytene result in an additive (more than fourfold) increase in recombination frequency. The peak concentration of the nicking endonuclease is found at late premeiotic S and early karyogamy, whereas the peak activity of the repairing DNA polymerase b is found at late pachytene. The staging of these two enzymes is in good accord with the genetic data.Key words: Coprinus, endonuclease, meiosis, genetic recombination, Z-DNA.

Gene cloning and transformation in the basidiomycete fungus Coprinus cinereus: Isolation and expression of the isocitrate lyase gene (acu-7)

The cloned isocitrate lyase structural gene of Aspergillus nidulans (acuD) was shown to hybridize under reduced stringency conditions to unique sequences in genomic DNA digests of the basidiomycete fungus Coprinus cinereus. A gene library of C. cinereus was constructed in the lambda replacement vector L47 and screened for sequences hybridizing to the A. nidulans gene. A recombinant phage was isolated which contained the hybridizing sequence on a 5.6-kb BamHI fragment. This fragment was subcloned into pUC13 to give plasmid pHIONA1 and shown to contain a functional C. cinereus isocitrate lyase gene (acu-7) by transformation of an acu-7 mutant. Direct selection for Acu+ transformants was not possible because of the toxicity of the acetate selection medium. Acu+ transformants were obtained as cotransformants by transforming an acu-7 trp-1 double mutant, having mutations in both the isocitrate lyase and tryptophan synthetase structural genes, with a plasmid containing the trp-1 gene and either pHIONA1 or the original lambda clone. Up to 47.5% of the selected Trp+ transformants were cotransformed to Acu+. A physical analysis of 40 Acu+ transformants showed that the acu-7 gene had integrated at non-homologous and often multiple sites in the genome. Meiotic stability of the integrated gene was demonstrated by genetic crosses.

Inversion of 5S ribosomal RNA genes within the genus Coprinus.

In the basidiomycete fungus Coprinus cinereus, the 5S ribosomal RNA (rRNA) genes are found in the rDNA repeat which encodes the other three rRNAs (18S, 5.8S, and 26S), and all genes are transcribed in the same direction (Cassidy et al. 1984). To facilitate examination of the rRNA gene organization in additional species, we developed a method to permit determination of the direction of transcription of the rRNA genes using crude preparations of genomic DNA. We used this method to determine the organization of the rDNA in five other members of the order Agaricales (Flammulina velutipes, Agaricus bisporus, Coprinus micaceus, Coprinus atramentarius, and Coprinus comatus). In four, the organization is the same as it is in Coprinus cinereus. However, in Coprinus comatus, the 5S RNA gene is present in the rDNA repeat, but is transcribed in the opposite direction. This alteration in rDNA organization within the genus Coprinus indicates that inverted 5S genes can arise and become propagated through the tandem array of genes. The presence of orientation-dependent promoter elements within the spacer DNA (Elion and Warner 1984) would be expected to place constraints on such inversions.

Biochemical analysis of the role of cytoplasmic ribosomes of Coprinus cinereus in cycloheximide resistance.

The development of an optimized in vitro polyuridylic acid-dependent polyphenylalanine-synthesizing system using cell-free extracts of the basidiomycete fungus Coprinus cinereus is described. The in vitro assay has been used to show that cycloheximide-resistant strains CY8.2, CY9.23 and Sp98, all mutant at the cy-2 locus, have cytoplasmic ribosomes which are more resistant to the drug than the corresponding sensitive strains, CY8, CY9 and CY3. Cycloheximide concentrations and molar ratios of cycloheximide to ribosomes required for 50% inhibition in vitro under standard assay conditions are presented for these strains. The molar ratio required for 50% inhibition in vitro is dependent on the concentration of ribosomes in the assay.