A non-canonical melanin biosynthesis pathway protects Aspergillus terreus conidia from environmental stress

Pro- and eukaryotes produce melanin for protection from environmental stress or as virulence determinant. The human pathogenic fungus Aspergillus fumigatus and related Ascomycetes produce dihydroxynaphthalene (DHN) melanin in conidia, which is essential for inhibiting phagolysosome acidification. In contrast, Aspergillus terreus lacks genes for biosynthesis of DHN-melanin. Therefore, the origin of the pigment in A. terreus conidia was elucidated. Expression analyses from conidiation conditions identified genes coding for an unusual NRPS-like enzyme (MelA) and a tyrosinase. MelA produces aspulvinone E as precursor, which is activated for polymerisation by the tyrosinase TyrP as shown by heterologous in vivo and in vitro reconstitution of pigment formation. Functional studies revealed that the pigment confers resistance against UV-light and hampers phagocytosis by soil amoeba, but does not inhibit acidification of phagolysosomes. Since A. terreus conidia prefer persistence at acidic pH, this uncommon type of melanin, termed Asp-melanin, might specifically contribute to survival in the environment

Fungi Unearthed: Transcripts Encoding Lignocellulolytic and Chitinolytic Enzymes in Forest Soil

BACKGROUND: Fungi are the main organisms responsible for the degradation of biopolymers such as lignin, cellulose, hemicellulose, and chitin in forest ecosystems. Soil surveys largely target fungal diversity, paying less attention to fungal activity. METHODOLOGY/PRINCIPAL FINDINGS: Here we have focused on the organic horizon of a hardwood forest dominated by sugar maple that spreads widely across Eastern North America. The sampling site included three plots receiving normal atmospheric nitrogen deposition and three that received an extra 3 g nitrogen m(2) y(1) in form of sodium nitrate pellets since 1994, which led to increased accumulation of organic matter in the soil. Our aim was to assess, in samples taken from all six plots, transcript-level expression of fungal genes encoding lignocellulolytic and chitinolytic enzymes. For this we collected RNA from the forest soil, reverse-transcribed it, and amplified cDNAs of interest, using both published primer pairs as well as 23 newly developed ones. We thus detected transcript-level expression of 234 genes putatively encoding 26 different groups of fungal enzymes, notably major ligninolytic and diverse aromatic-oxidizing enzymes, various cellulose- and hemicellulose-degrading glycoside hydrolases and carbohydrate esterases, enzymes involved in chitin breakdown, N-acetylglucosamine metabolism, and cell wall degradation. Among the genes identified, 125 are homologous to known ascomycete genes and 105 to basidiomycete genes. Transcripts corresponding to all 26 enzyme groups were detected in both control and nitrogen-supplemented plots. CONCLUSIONS/SIGNIFICANCE: Many of these enzyme groups are known to be important in soil turnover processes, but the contribution of some is probably underestimated. Our data highlight the importance of ascomycetes, as well as basidiomycetes, in important biogeochemical cycles. In the nitrogen-supplemented plots, we have detected no transcript-level gap likely to explain the observed increased carbon storage, which is more likely due to community changes and perhaps transcriptional and/or post-transcriptional down-regulation of relevant genes

Exploration du genre Pycnoporus pour la production d'une nouvelle tyrosinase (de l'expression du gène aux applications biotechnologiques)

Ce travail a eu pour objectif l'exploration des potentialités du genre Pycnoporus à synthétiser une nouvelle tyrosinase d'intérêt biotechnologique dans le domaine agro-alimentaire. Une nouvelle tyrosinase a été isolée, purifiée et caractérisée à partir de la souche P. sanguineus BRFM49. Cette enzyme s'est révélée d'un grand intérêt pour la synthèse de molécules à haute valeur ajoutée comme des anti-oxydants ou des polymères de nouvelle génération. Deux voies complémentaires ont été explorées afin d'améliorer le niveau de production de la tyrosinase. La première, qui a consisté à sélectionner, par génétique classique, des lignées monocaryotiques génétiquement stables, n'a pas permis d'améliorer, de façon significative, l'activité tyrosinase chez P. sanguineus BRFM49. La seconde a consisté à réaliser la production de la tyrosinase dans un système d'expression hétérologue. Pour cela, le gène de la tyrosinase (2204 pb) et l'ADNc correspondant (1857 pb) ont été clonés et caractérisés. La production hétérologue de la tyrosinase de P. sanguineus a été réalisée chez un champignon hyper-producteur de protéines, Aspergillus niger. Pour cela, l'ADNc correspondant a été fusionné à la séquence d'adressage de la glucoamylase d'A. niger et placé sous le contrôle d'un promoteur constitutif fort. Cette construction a permis de sécréter la protéine active dans le milieu extracellulaire. La souche A. niger D15#26-e, présentant le meilleur niveau de production (50 mg/L), a été sélectionnée pour purifier et caractériser la tyrosinase recombinante et comparer celle-ci à l'enzyme native. Cette étude a montré que l'enzyme recombinante est aussi efficace que la tyrosinase native pour la réticulation de protéines modèlesAIX-MARSEILLE1-BU Sci.St Charles (130552104) / SudocSudocFranceF

Decolourization of black oxidized olive-mill wastewater by a new tannase-producing Aspergillus flavus strain isolated from soil

International audienceBy contaminating a Tunisian soil with black oxidized and sterilized olive-mill wastewaters (OMW), 30 new indigenous fungal soil strains able to overcome the OMW toxicity could be directly selected. Ten of the fungal strains previously isolated were screened for their capability to grow in a liquid culture medium containing oxidized OMW as the only source of carbon and energy. According to these preliminary tests, strain F2 showed the best capability of removing black colour and COD (chemical oxygen demand) and was further identified as Aspergillus flavus. After optimization of batch-liquid culture conditions in the presence of oxidized OMW, the time course of biomass and enzyme production by A. flavus F2 was followed in relation to colour and COD removal. A. flavus F2 could efficiently decolourize and detoxify the black oxidized OMW (58 and 46% of colour and COD removal, respectively, after 6 days of cultivation), concomitantly with the production of tannase (8000 UI/l on day 3)

Fungal tyrosinases: new prospects in molecular characteristics, bioengineering and biotechnological applications

International audienc

Cloning and characterization of a tyrosinase gene from the white-rot fungus Pycnoporus sanguineus, and overproduction of the recombinant protein in Aspergillus niger

A new tyrosinase-encoding gene (2,204 bp) and the corresponding cDNA (1,857 nucleotides) from the white-rot fungus Pycnoporus sanguineus BRFM49 were cloned. This gene consisted of seven exons and six introns and encoded a predicted protein of 68 kDa, exceeding the mature tyrosinase by 23 kDa. P. sanguineus tyrosinase cDNA was over-expressed in Aspergillus niger, a particularly suitable fungus for heterologous expression of proteins of biotechnological interest, under the control of the glyceraldehyde-3-phosphate-dehydrogenase promoter as strong and constitutive promoter. The glucoamylase preprosequence of A. niger was used to target the secretion. This construction enabled the production of recombinant tyrosinase in the extracellular medium of A. niger. The identity of the purified recombinant protein was confirmed by N-terminal amino acid sequencing. The maturation process was shown to be effective in A. niger, and the recombinant enzyme was fully active, with a molecular mass of 45 kDa. The best transformant obtained, A. niger D15#26-e, produced extracellular tyrosinase activities of 534 and 1,668 U l(-1) for monophenolase and diphenolase, respectively, which corresponded to a protein yield of ca. 20 mg l(-1)

Characterization of a new tyrosinase from <i>Pycnoporus</i> species with high potential for food technological applications

International audienceAims: Tyrosinase production by Pycnoporus cinnabarinus and Pycnoporus sanguineus was screened among 20 strains originating from various geographical areas, particularly from tropical environments. The tyrosinase from the most efficient strain was purified and characterized and tested for food additive applications. Methods and Results: Monophenolase and diphenolase activities of tyrosinase were measured from cell lysate from the 20 Pycnoporus strains, for 8-10 days of cultivation. The strain P. sanguineus CBS 614.73 showed the highest productivity (45.4 and 163.6 U g(-1) protein per day for monophenolase and diphenolase respectively). P. sanguineus CBS 614.73 tyrosinase was purified from concentrated cell lysate, anion-exchange, size-exclusion and hydroxyapatite chromatography, with a final yield of 2% and a purification factor of 35-38. The pure enzyme was a monomere with a molecular mass of 45 kDa and it showed four isoforms or isoenzymes with pI between 4.5-5. No N-glycosylation was found. The N-terminal amino acid sequence was IVTGPVGGQTEGAPAPNR. The enzyme was shown to be almost fully active in a pH range of 6-7, in a large temperature range (30-70degreesC), and was stable below 60degreesC. The main kinetic constants were determined. The tyrosinase was able to convert p-tyrosol and p-coumaric acid into hydroxytyrosol and caffeic acid, respectively, and it could also catalyse the cross-linking formation of a model protein. Conclusions: Among the genus Pycnoporus, known for the production of laccase, the strain P. sanguineus CBS 614.73 was shown to produce one other phenoloxidase, a new monomeric tyrosinase with a specific activity of 30 and 84 U mg(-1) protein for monophenolase and diphenolase respectively. Significance and Impact of the Study: This study identified P. sanguineus CBS 614.73 as a potential producer of a tyrosinase which demonstrated effectiveness in the synthesis of antioxidant molecules and in protein cross-linking

Comparison of the characteristics of fungal and plant tyrosinases

International audienceEnzymatic crosslinking provides valuable means for modifying functionality and structural properties of different polymers. Tyrosinases catalyze the hydroxylation of various monophenols to the corresponding o-diphenols, and the subsequent oxidation of o-diphenols to the corresponding quinones, which are highly reactive and can further undergo non-enzymatic reactions to produce mixed melanins and heterogeneous polymers. Tyrosinases are also capable of oxidizing protein- and peptide-bound tyrosyl residues, resulting in the formation of inter- and intra-molecular crosslinks. Tyrosinases from apple (AT), potato (PT), the white rot fungus Pycnoporus sanguineus (PsT), the filamentous fungus Trichoderma reesei (TrT) and the edible mushroom Agaricus bisporus (AbT) were compared for their biochemical characteristics. The enzymes showed different features in terms of substrate specificity, stereo-specificity, inhibition, and ability to crosslink the model protein, greek small letter alpha-casein. All enzymes were found to produce identical semiquinone radicals from the substrates as analyzed by electron spin resonance spectroscopy. The result suggests similar reaction mechanism between the tyrosinases. PsT enzyme had the highest monophenolase/diphenolase ratio for the oxidation of monophenolic l-tyrosine and diphenolic l-dopa, although the tyrosinases generally had noticeably lower activity on monophenols than on di- or triphenols. The activity of AT and PT on tyrosine was particularly low, which largely explains the poor crosslinking ability of the model protein greek small letter alpha-casein by these enzymes. AbT oxidized peptide-bound tyrosine, but was not able to crosslink greek small letter alpha-casein. Conversely, the activity of PsT on model peptides was relatively low, although the enzyme could crosslink greek small letter alpha-casein. In the reaction conditions studied, TrT showed the best ability to crosslink greek small letter alpha-casein. TrT also had the highest activity on most of the tested monophenols, and showed noticeable short lag periods prior to the oxidation