{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T15:01:39Z","timestamp":1780498899817,"version":"3.54.1"},"reference-count":51,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2025,12,17]],"date-time":"2025-12-17T00:00:00Z","timestamp":1765929600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"European Commission\u2014NextGenerationEU","award":["IR0000005"],"award-info":[{"award-number":["IR0000005"]}]},{"DOI":"10.13039\/501100005389","name":"University of Insubria","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100005389","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["JoF"],"abstract":"<jats:p>The white-rot fungus Coriolopsis trogii MUT3379 produces Lac3379-1 laccase at high yields due to the previous development of a robust fermentation process. Throughout the extended use of this strain, we observed the occurrence of substrate-specific guaiacol and ABTS (2,2\u2019-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) oxidizing enzymes other than Lac3379-1 Since we did not succeed in producing these enzymes in significant amounts by conventional strain selection and fermentation tools, we developed an approach based on protoplast preparation and regeneration to isolate stable producers of these alternative oxidative enzymes from the complex multinucleate mycelium of C. trogii MUT3379. A cost-effective and efficient protocol for protoplast preparation was developed by using the enzymatic cocktail VinoTaste Pro by Novozymes. A total of 100 protoplast-derived clones were selected and screened to produce laccases and other oxidative enzymes. A variable spectrum of oxidative activity levels, including both high and low producers, was revealed. Notably, a subset of clones exhibited diverse guaiacol\/ABTS positive enzymatic patterns. These findings suggest that it is possible to isolate different lineages from the mycelium of C. trogii MUT337, each producing a distinct pattern of oxidative enzymes. This highlights the potential of protoplast-mediated genome separation to uncover novel metabolic traits that would otherwise remain cryptic. These data hold outstanding significance for accessing and producing novel oxidative enzymes from native fungal populations.<\/jats:p>","DOI":"10.3390\/jof11120890","type":"journal-article","created":{"date-parts":[[2025,12,17]],"date-time":"2025-12-17T10:41:23Z","timestamp":1765968083000},"page":"890","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Unravelling the Potential of Fungal Division of Labour in the Laccase Producer Coriolopsis trogii MUT3379 Through Protoplast Formation and Regeneration"],"prefix":"10.3390","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3987-3955","authenticated-orcid":false,"given":"Luca","family":"Mellere","sequence":"first","affiliation":[{"name":"Department of Biotechnology and Life Sciences, University of Insubria, 21100 Varese, Italy"},{"name":"BioC-CheM Solutions S.r.l., Insubrias BioPark, 21040 Gerenzano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Adriana","family":"Bava","sequence":"additional","affiliation":[{"name":"BioC-CheM Solutions S.r.l., Insubrias BioPark, 21040 Gerenzano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1589-445X","authenticated-orcid":false,"given":"Jean","family":"Armengaud","sequence":"additional","affiliation":[{"name":"D\u00e9partement M\u00e9dicaments et Technologies pour la Sant\u00e9 (DMTS), CEA, INRAE, SPI, Universit\u00e9 Paris-Saclay, 30200 Bagnols-sur-C\u00e8ze, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1913-9929","authenticated-orcid":false,"given":"Francesca","family":"Berini","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Life Sciences, University of Insubria, 21100 Varese, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9195-6777","authenticated-orcid":false,"given":"Flavia","family":"Marinelli","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Life Sciences, University of Insubria, 21100 Varese, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1455-6208","authenticated-orcid":false,"given":"Giovanna Cristina","family":"Varese","sequence":"additional","affiliation":[{"name":"Mycotheca Universitatis Taurinensis (MUT), Department of Life Sciences and Systems Biology, University of Turin, Viale Mattioli, 25, 10125 Turin, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2736-0570","authenticated-orcid":false,"given":"Federica","family":"Spina","sequence":"additional","affiliation":[{"name":"Mycotheca Universitatis Taurinensis (MUT), Department of Life Sciences and Systems Biology, University of Turin, Viale Mattioli, 25, 10125 Turin, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4464-3222","authenticated-orcid":false,"given":"Fabrizio","family":"Beltrametti","sequence":"additional","affiliation":[{"name":"BioC-CheM Solutions S.r.l., Insubrias BioPark, 21040 Gerenzano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,17]]},"reference":[{"key":"ref_1","first-page":"e00717","article-title":"Biocatalytic Potential of Basidiomycetes: Relevance, Challenges and Research Interventions in Industrial Processes","volume":"11","author":"Bentil","year":"2021","journal-title":"Sci. Afr."},{"key":"ref_2","first-page":"109","article-title":"Forest Microbiome: Diversity, Complexity and Dynamics","volume":"41","author":"Baldrian","year":"2017","journal-title":"FEMS Microbiol. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Skovgaard, N. (2002). The Mycota. A Comprehensive Treatise on Fungi as Experimental Systems for Basic and Applied Research, Springer.","DOI":"10.1016\/S0168-1605(02)00042-9"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Zhang, Z., Claessen, D., and Rozen, D.E. (2016). Understanding Microbial Divisions of Labor. Front. Microbiol., 7.","DOI":"10.3389\/fmicb.2016.02070"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"102148","DOI":"10.1016\/j.mib.2022.102148","article-title":"Ecological Drivers of Division of Labour in Streptomyces","volume":"67","author":"Traxler","year":"2022","journal-title":"Curr. Opin. Microbiol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"eaay5781","DOI":"10.1126\/sciadv.aay5781","article-title":"Antibiotic Production in Streptomyces Is Organized by a Division of Labor through Terminal Genomic Differentiation","volume":"6","author":"Zhang","year":"2020","journal-title":"Sci. Adv."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Mellere, L., Bava, A., Capozzoli, C., Branduardi, P., Berini, F., and Beltrametti, F. (2022). Strain Improvement and Strain Maintenance Revisited. The Use of Actinoplanes teichomyceticus ATCC 31121 Protoplasts in the Identification of Candidates for Enhanced Teicoplanin Production. Antibiotics, 11.","DOI":"10.3390\/antibiotics11010024"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2138","DOI":"10.1016\/j.biotechadv.2018.10.002","article-title":"Multiscale Heterogeneity in Filamentous Microbes","volume":"36","author":"Zacchetti","year":"2018","journal-title":"Biotechnol. Adv."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1345","DOI":"10.1128\/EC.00213-12","article-title":"Physiological Significance of Network Organization in Fungi","volume":"11","author":"Simonin","year":"2012","journal-title":"Eukaryot. Cell"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1808","DOI":"10.1021\/pr301154w","article-title":"Deletion of FlbA Results in Increased Secretome Complexity and Reduced Secretion Heterogeneity in Colonies of Aspergillus niger","volume":"12","author":"Krijgsheld","year":"2013","journal-title":"J. Proteome Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"969","DOI":"10.1099\/00221287-139-5-969","article-title":"In Situ Localization of the Secretion of Lignin Peroxidases in Colonies of Phanerochaete chrysosporium Using a Sandwiched Mode of Culture","volume":"139","author":"Moukha","year":"1993","journal-title":"J. Gen. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"3121","DOI":"10.1099\/mic.0.26451-0","article-title":"In Situ Localization of Manganese Peroxidase Production in Mycelial Pallets of Phanerochaete chrysosporium","volume":"149","author":"Kurzatkowski","year":"2003","journal-title":"Microbiology"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1186\/s40694-016-0022-x","article-title":"Two Genomes Are Better than One: History, Genetics, and Biotechnological Applications of Fungal Heterokaryons","volume":"3","author":"Strom","year":"2016","journal-title":"Fungal Biol. Biotechnol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1128\/9781555819583.ch10","article-title":"Molecular Mechanisms Regulating Cell Fusion and Heterokaryon Formation in Filamentous Fungi","volume":"5","author":"Daskalov","year":"2017","journal-title":"Fungal Kingd"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Brown, A.J.P. (2006). The Mycota XIII: Fungal Genomics, Springer.","DOI":"10.1007\/3-540-30809-1"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1146\/annurev-micro-041320-112443","article-title":"The Dynamic Fungal Genome: Polyploidy, Aneuploidy and Copy Number Variation in Response to Stress","volume":"77","author":"Zhou","year":"2023","journal-title":"Annu. Rev. Microbiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s13068-021-02043-4","article-title":"The Phenomenon of Degeneration of Industrial Trichoderma reesei Strains","volume":"14","author":"Martzy","year":"2021","journal-title":"Biotechnol. Biofuels"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Wang, X., Hong, S., Tang, G., and Wang, C. (2024). Accumulation of the Spontaneous and Random Mutations Is Causative of Fungal Culture Degeneration. Fundam. Res.","DOI":"10.1016\/j.fmre.2024.02.003"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Mellere, L., Bellasio, M., Berini, F., Marinelli, F., Armengaud, J., and Beltrametti, F. (2024). Coriolopsis Trogii MUT3379: A Novel Cell Factory for High-Yield Laccase Production. Fermentation, 10.","DOI":"10.3390\/fermentation10070376"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Savinova, O.S., Moiseenko, K.V., Vavilova, E.A., Chulkin, A.M., Fedorova, T.V., Tyazhelova, T.V., and Vasina, D.V. (2019). Evolutionary Relationships between the Laccase Genes of Polyporales: Orthology-Based Classification of Laccase Isozymes and Functional Insight from Trametes hirsuta. Front. Microbiol., 10.","DOI":"10.3389\/fmicb.2019.00152"},{"key":"ref_21","first-page":"1271","article-title":"A Modified Regeneration Method for Streptomycete Protoplasts","volume":"45","author":"Shirahama","year":"1981","journal-title":"Agric. Biol. Chem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1186\/s40168-020-00797-x","article-title":"Estimating Relative Biomasses of Organisms in Microbiota Using \u201cPhylopeptidomics\u201d","volume":"8","author":"Pible","year":"2020","journal-title":"Microbiome"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"de Groot, A., Dulermo, R., Ortet, P., Blanchard, L., Gu\u00e9rin, P., Fernandez, B., Vacherie, B., Dossat, C., Jolivet, E., and Siguier, P. (2009). Alliance of Proteomics and Genomics to Unravel the Specificities of Sahara Bacterium Deinococcus deserti. PLoS Genet., 5.","DOI":"10.1371\/journal.pgen.1000434"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1002\/jobm.200310365","article-title":"Production of Laccase Isoforms by Pleurotus pulmonarius in Response to Presence of Phenolic and Aromatic Compounds","volume":"44","author":"Tychanowicz","year":"2004","journal-title":"J. Basic. Microbiol."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Yang, J., Wang, G., Ng, T.B., Lin, J., and Ye, X. (2016). Laccase Production and Differential Transcription of Laccase Genes in Cerrena Sp. in Response to Metal Ions, Aromatic Compounds, and Nutrients. Front. Microbiol., 6.","DOI":"10.3389\/fmicb.2015.01558"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"716","DOI":"10.1038\/nrmicro.2016.111","article-title":"Division of Labour in Microorganisms: An Evolutionary Perspective","volume":"14","author":"West","year":"2016","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1241","DOI":"10.1139\/b95-384","article-title":"Origins and Significance of Genetic and Epigenetic Instability in Mycelial Systems","volume":"73","author":"Rayner","year":"1995","journal-title":"Can. J. Bot."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Zacharia, V.M., Ra, Y., Sue, C., Alcala, E., Reaso, J.N., Ruzin, S.E., and Traxler, M.F. (2021). Genetic Network Architecture and Environmental Cues Drive Spatial Organization of Phenotypic Division of Labor in Streptomyces coelicolor. mBio, 12.","DOI":"10.1128\/mBio.00794-21"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"R786","DOI":"10.1016\/j.cub.2011.06.042","article-title":"Nuclear and Genome Dynamics in Multinucleate Ascomycete Fungi","volume":"21","author":"Roper","year":"2011","journal-title":"Curr. Biol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"100729","DOI":"10.1016\/j.coche.2021.100729","article-title":"Fungal Morphology: A Challenge in Bioprocess Engineering Industries for Product Development","volume":"35","year":"2022","journal-title":"Curr. Opin. Chem. Eng."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2997","DOI":"10.1007\/s00253-018-8818-7","article-title":"The Filamentous Fungal Pellet\u2014Relationship between Morphology and Productivity","volume":"102","author":"Veiter","year":"2018","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1029","DOI":"10.1007\/s00449-018-1932-1","article-title":"Morphological Regulation of Aspergillus niger to Improve Citric Acid Production by ChsC Gene Silencing","volume":"41","author":"Sun","year":"2018","journal-title":"Bioprocess Biosyst. Eng."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s40694-020-00101-4","article-title":"The Mechanisms of Hyphal Pellet Formation Mediated by Polysaccharides, \u03b1-1,3-Glucan and Galactosaminogalactan, in Aspergillus Species","volume":"7","author":"Miyazawa","year":"2020","journal-title":"Fungal Biol. Biotechnol."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Pombeiro-Sponchiado, S.R., Sousa, G.S., Andrade, J.C.R., Lisboa, H.F., and Gon\u00e7alves, R.C.R. (2017). Production of Melanin Pigment by Fungi and Its Biotechnological Applications. Melanin, IntechOpen.","DOI":"10.5772\/67375"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1186\/s43008-021-00082-y","article-title":"Melanin Production and Laccase Mediated Oxidative Stress Alleviation during Fungal-Fungal Interaction among Basidiomycete Fungi","volume":"12","author":"Dullah","year":"2021","journal-title":"IMA Fungus"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Suthar, M., Dufoss\u00e9, L., and Singh, S.K. (2023). The Enigmatic World of Fungal Melanin: A Comprehensive Review. J. Fungi, 9.","DOI":"10.3390\/jof9090891"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"94","DOI":"10.1186\/1475-2859-11-94","article-title":"Physiological Heterogeneities in Microbial Populations and Implications for Physical Stress Tolerance","volume":"11","author":"Carlquist","year":"2012","journal-title":"Microb. Cell Fact."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1111\/j.1574-6968.2000.tb08975.x","article-title":"Selection of Pycnoporus cinnabarinus Strains for Laccase Production","volume":"183","author":"Moukha","year":"2000","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"a019679","DOI":"10.1101\/cshperspect.a019679","article-title":"Fungal Morphogenesis","volume":"5","author":"Lin","year":"2015","journal-title":"Cold Spring Harb. Perspect. Med."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"e0008722","DOI":"10.1128\/msphere.00087-22","article-title":"Evolutionary Significance of Fungal Hypermutators: Lessons Learned from Clinical Strains and Implications for Fungal Plant Pathogens","volume":"7","author":"Gambhir","year":"2022","journal-title":"mSphere"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"2311","DOI":"10.1128\/EC.00244-07","article-title":"Spatial Differentiation in the Vegetative Mycelium of Aspergillus niger","volume":"6","author":"Levin","year":"2007","journal-title":"Eukaryot. Cell"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Hao, H., Zhang, J., Wang, Q., Huang, J., Juan, J., Kuai, B., Feng, Z., and Chen, H. (2022). Transcriptome and Differentially Expressed Gene Profiles in Mycelium, Primordium and Fruiting Body Development in Stropharia rugosoannulata. Genes, 13.","DOI":"10.3390\/genes13061080"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"853","DOI":"10.1111\/j.1469-8137.2012.04278.x","article-title":"Significant Genetic and Phenotypic Changes Arising from Clonal Growth of a Single Spore of an Arbuscular Mycorrhizal Fungus over Multiple Generations","volume":"196","author":"Ehinger","year":"2012","journal-title":"New Phytol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"2514","DOI":"10.1038\/ismej.2016.29","article-title":"Population Genomics Reveals That Within-Fungus Polymorphism Is Common and Maintained in Populations of the Mycorrhizal Fungus Rhizophagus irregularis","volume":"10","author":"Wyss","year":"2016","journal-title":"ISME J."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"L\u00fcbeck, M., and L\u00fcbeck, P.S. (2022). Fungal Cell Factories for Efficient and Sustainable Production of Proteins and Peptides. Microorganisms, 10.","DOI":"10.3390\/microorganisms10040753"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1016\/0167-7799(94)90100-7","article-title":"Protein Secretion in Filamentous Fungi\u2014Trying to Understand a Highly Productive Black Box","volume":"12","author":"Peberdy","year":"1994","journal-title":"Trends Biotechnol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"386","DOI":"10.1002\/bit.10681","article-title":"Combined Sequence and Structure Analysis of the Fungal Laccase Family","volume":"83","author":"Kumar","year":"2003","journal-title":"Biotechnol. Bioeng."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"920","DOI":"10.1128\/AEM.66.3.920-924.2000","article-title":"Copper Induction of Laccase Isoenzymes in the Ligninolytic Fungus Pleurotus ostreatus","volume":"66","author":"Palmieri","year":"2000","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"104","DOI":"10.1186\/s13568-023-01608-w","article-title":"Genome-Wide Analysis of the Pleurotus eryngii Laccase Gene (PeLac) Family and Functional Identification of PeLac5","volume":"13","author":"Li","year":"2023","journal-title":"AMB Express"},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Yang, J., Xu, X., Ng, T.B., Lin, J., and Ye, X. (2016). Laccase Gene Family in Cerrena Sp. HYB07: Sequences, Heterologous Expression and Transcriptional Analysis. Molecules, 21.","DOI":"10.3390\/molecules21081017"},{"key":"ref_51","doi-asserted-by":"crossref","unstructured":"Vasina, D.V., Moiseenko, K.V., Fedorova, T.V., and Tyazhelova, T.V. (2017). Lignin-Degrading Peroxidases in White-Rot Fungus Trametes hirsuta 072. Absolute Expression Quantification of Full Multigene Family. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0173813"}],"container-title":["Journal of Fungi"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2309-608X\/11\/12\/890\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,19]],"date-time":"2025-12-19T05:10:50Z","timestamp":1766121050000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2309-608X\/11\/12\/890"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,17]]},"references-count":51,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["jof11120890"],"URL":"https:\/\/doi.org\/10.3390\/jof11120890","relation":{},"ISSN":["2309-608X"],"issn-type":[{"value":"2309-608X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,17]]}}}