Similar Datasets
Project description:Differential expression analysis of overall transcriptome in response to varying carbon source
2017-10-01 | GSE95479 | GEO
Project description:Substrate-based regulation in gut fungi
| PRJNA377241 | ENA
Project description:While the Pycnoporus cinnabarinus laccase (PcL) is one of the most promising high-redox-potential enzymes for environmental biocatalysis, its practical use has to date remained limited due to the lack of directed evolution platforms with which to improve its features. Here, we describe the construction of a PcL fusion gene and the optimization of conditions to induce its functional expression in Saccharomyces cerevisiae, facilitating its directed evolution and semirational engineering. The native PcL signal peptide was replaced by the ?-factor preproleader, and this construct was subjected to six rounds of evolution coupled to a multiscreening assay based on the oxidation of natural and synthetic redox mediators at more neutral pHs. The laccase total activity was enhanced 8,000-fold: the evolved ?-factor preproleader improved secretion levels 40-fold, and several mutations in mature laccase provided a 13.7-fold increase in k(cat). While the pH activity profile was shifted to more neutral values, the thermostability and the broad substrate specificity of PcL were retained. Evolved variants were highly secreted by Aspergillus niger (?23 mg/liter), which addresses the potential use of this combined-expression system for protein engineering. The mapping of mutations onto the PcL crystal structure shed new light on the oxidation of phenolic and nonphenolic substrates. Furthermore, some mutations arising in the evolved preproleader highlighted its potential for heterologous expression of fungal laccases in yeast (S. cerevisiae).
| S-EPMC3294479 | biostudies-literature
Project description:An efficient transformation and expression system was developed for the industrially relevant basidiomycete Pycnoporus cinnabarinus. This was used to transform a laccase-deficient monokaryotic strain with the homologous lac1 laccase gene placed under the regulation of its own promoter or that of the SC3 hydrophobin gene or the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of Schizophyllum commune. SC3-driven expression resulted in a maximal laccase activity of 107 nkat ml(-1) in liquid shaken cultures. This value was about 1.4 and 1.6 times higher in the cases of the GPD and lac1 promoters, respectively. lac1-driven expression strongly increased when 25 g of ethanol liter(-1) was added to the medium. Accordingly, laccase activity increased to 1,223 nkat ml(-1). These findings agree with the fact that ethanol induces laccase gene expression in some fungi. Remarkably, lac1 mRNA accumulation and laccase activity also strongly increased in the presence of 25 g of ethanol liter(-1) when lac1 was expressed behind the SC3 or GPD promoter. In the latter case, a maximal laccase activity of 1,393 nkat ml(-1) (i.e., 360 mg liter(-1)) was obtained. Laccase production was further increased in transformants expressing lac1 behind its own promoter or that of GPD by growth in the presence of 40 g of ethanol liter(-1). In this case, maximal activities were 3,900 and 4,660 nkat ml(-1), respectively, corresponding to 1 and 1.2 g of laccase per liter and thus representing the highest laccase activities reported for recombinant fungal strains. These results suggest that P. cinnabarinus may be a host of choice for the production of other proteins as well.
| S-EPMC525127 | biostudies-literature
Project description:Industrial effluents of textile, paper, and leather industries contain various toxic dyes as one of the waste material. It imparts major impact on human health as well as environment. The white rot fungus Pycnoporus cinnabarinus Laccase is generally used to degrade these toxic dyes. In order to decipher the mechanism of process by which Laccase degrade dyes, it is essential to know its 3D structure. Homology modeling was performed in presented work, by satisfying Spatial restrains using Modeller Program, which is considered as standard in this field, to generate 3D structure of Laccase in unison, SWISSMODEL web server was also utilized to generate and verify the alternative models. We observed that models created using Modeller stands better on structure evaluation tests. This study can further be used in molecular docking techniques, to understand the interaction of enzyme with its mediators like 2, 2-azinobis (3-ethylbenzthiazoline-6-sulfonate) (ABTS) and Vanillin that are known to enhance the Laccase activity.
| S-EPMC3040475 | biostudies-literature
Project description:The genome of the white rot fungus Pycnoporus cinnabarinus includes a large number of genes encoding enzymes implicated in lignin degradation. Among these, three genes are predicted to encode glyoxal oxidase, an enzyme previously isolated from Phanerochaete chrysosporium The glyoxal oxidase of P. chrysosporium is physiologically coupled to lignin-oxidizing peroxidases via generation of extracellular H2O2 and utilizes an array of aldehydes and ?-hydroxycarbonyls as the substrates. Two of the predicted glyoxal oxidases of P. cinnabarinus, GLOX1 (PciGLOX1) and GLOX2 (PciGLOX2), were heterologously produced in Aspergillus niger strain D15#26 (pyrG negative) and purified using immobilized metal ion affinity chromatography, yielding 59 and 5 mg of protein for PciGLOX1 and PciGLOX2, respectively. Both proteins were approximately 60 kDa in size and N-glycosylated. The optimum temperature for the activity of these enzymes was 50°C, and the optimum pH was 6. The enzymes retained most of their activity after incubation at 50°C for 4 h. The highest relative activity and the highest catalytic efficiency of both enzymes occurred with glyoxylic acid as the substrate. The two P. cinnabarinus enzymes generally exhibited similar substrate preferences, but PciGLOX2 showed a broader substrate specificity and was significantly more active on 3-phenylpropionaldehyde.This study addresses the poorly understood role of how fungal peroxidases obtain an in situ supply of hydrogen peroxide to enable them to oxidize a variety of organic and inorganic compounds. This cooperative activity is intrinsic in the living organism to control the amount of toxic H2O2 in its environment, thus providing a feed-on-demand scenario, and can be used biotechnologically to supply a cheap source of peroxide for the peroxidase reaction. The secretion of multiple glyoxal oxidases by filamentous fungi as part of a lignocellulolytic mechanism suggests a controlled system, especially as these enzymes utilize fungal metabolites as the substrates. Two glyoxal oxidases have been isolated and characterized to date, and the differentiation of the substrate specificity of the two enzymes produced by Pycnoporus cinnabarinus illustrates the alternative mechanisms existing in a single fungus, together with the utilization of these enzymes to prepare platform chemicals for industry.
| S-EPMC4968546 | biostudies-literature
Project description:White-rot basidiomycete fungi are potent degraders of plant biomass with the ability to mineralize all lignocellulose components. Recent comparative genomics studies showed that these fungi use a wide diversity of enzymes for wood degradation. Deeper functional analyses are however necessary to understand the enzymatic mechanisms leading to lignocellulose breakdown. In the present study we analyzed the early response of the Polyporales fungi Pycnoporus coccineus CIRM-BRFM310, Pycnoporus cinnabarinus CIRM-BRFM137 and Pycnoporus sanguineus CIRM-BRFM 1264 to various carbon sources including lignocellulosic biomass.
2017-02-21 | GSE82427 | GEO
Project description:It was recently shown that the white rot basidiomycete Pycnoporus cinnabarinus secretes an unusual set of phenoloxidases when it is grown under conditions that stimulate ligninolysis (C. Eggert, U. Temp, and K.-E. L. Eriksson, Appl. Environ. Microbiol. 62:1151-1158, 1996). In this report we describe the results of a cloning and structural analysis of the laccase-encoding gene (lcc3-1) expressed by P. cinnabarinus during growth under xylidine-induced conditions. The coding region of the genomic laccase sequence, which is preceded by the eukaryotic promoter elements TATA and CAATA, spans more than 2,390 bp. The corresponding laccase cDNA was identical to the genomic sequence except for 10 introns that were 50 to 60 bp long. A sequence analysis indicated that the P. cinnabarinus lcc3-1 product has a Phe residue at a position likely to influence the reduction-oxidation potential of the enzyme's type 1 copper center. The P. cinnabarinus lcc3-1 sequence was most similar to the sequence encoding a laccase from Coriolus hirsutus (level of similarity, 84%).
| S-EPMC106228 | biostudies-literature
Project description:BACKGROUND: Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by lignocellulose-degrading fungi including Pycnoporus cinnabarinus. We investigated the cellulolytic system of P. cinnabarinus, focusing on the involvement of CDH in the deconstruction of lignocellulosic biomass. RESULTS: First, P. cinnabarinus growth conditions were optimized for CDH production. Following growth under cellulolytic conditions, the main components secreted were cellulases, xylanases and CDH. To investigate the contribution of P. cinnabarinus secretome in saccharification processes, the Trichoderma reesei enzymatic cocktail was supplemented with the P. cinnabarinus secretome. A significant enhancement of the degradation of wheat straw was observed with (i) the production of a large amount of gluconic acid, (ii) increased hemicellulose degradation, and (iii) increased overall degradation of the lignocellulosic material. P. cinnabarinus CDH was heterologously expressed in Pichia pastoris to obtain large amounts of pure enzyme. In a bioreactor, the recombinant CDH (rCDH) expression level reached 7800 U/L. rCDH exhibited values of biochemical parameters similar to those of the natural enzyme, and was able to bind cellulose despite the absence of a carbohydrate-binding module (CBM). Following supplementation of purified rCDH to T. reesei enzymatic cocktail, formation of gluconic acid and increased hemicellulose degradation were observed, thus confirming the previous results observed with P. cinnabarinus secretome. CONCLUSIONS: We demonstrate that CDH offers an attractive tool for saccharification process enhancement due to gluconic acid production from raw lignocellulosic material.
| S-EPMC3268779 | biostudies-literature
Project description:BackgroundThe demand for natural pigments in general, and for fungi-derived pigments in particular, is constantly rising. Wood-decomposing fungi represent a promising source for natural pigments and they are usually easy to cultivate in pure culture. One of them, i.e., Pycnoporus cinnabarinus, offers a highly interesting spectrum of bioactivity, partly due to the formation of the orange-red pigment cinnabarin. However, apart from a few studies addressing its diverse potential biotechnological applications, there is still a large gap of knowledge concerning the influence of light on the formation of cinnabarin. The aim of this work was to investigate the effect of different irradiations on the cinnabarin content, the growth, and the morphology of three different P. cinnabarinus strains. We used highly standardized irradiation conditions and cultivation techniques in combination with newly developed methods for the extraction and direct quantification of cinnabarin.ResultsRed, green, blue, and UV-A irradiation (mean irradiance Ee = 1.5 ± 0.18 W m-2) had considerable effects on the growth and colony appearance of all three P. cinnabarinus strains tested. The cinnabarin content determined was, thus, dependent on the irradiation wavelength applied, allowing strain-specific thresholds to be defined. Irradiation with wavelengths below this strain-specific threshold corresponded to a lower cinnabarin content, at least at the intensity applied. The orange-red pigment appeared by light microscopy as incrusted extracellular plaques present on the hyphal walls. Highly efficient vegetative propagation occurred by arthroconidia, and we observed the tendency that this asexual reproduction was (i) most frequent in the dark but (ii) never occurred under UV-A exposure.Conclusion This study highlights a differential photo-dependence of growth, morphology, and cinnabarin formation in P. cinnabarinus. This confirms that it is advisable to consider the wavelength of the light used in future biotechnological productions of natural pigments.
| S-EPMC10709268 | biostudies-literature