Applied Microbiology and Biotechnology

, Volume 103, Issue 1, pp 411–425 | Cite as

Gongronella sp. w5 elevates Coprinopsis cinerea laccase production by carbon source syntrophism and secondary metabolite induction

  • Jun Hu
  • Yinliang Zhang
  • Yong Xu
  • Qiuying Sun
  • Juanjuan Liu
  • Wei Fang
  • Yazhong Xiao
  • Ursula KüesEmail author
  • Zemin FangEmail author
Applied microbial and cell physiology


When sucrose was used as the carbon source, the Basidiomycete Coprinopsis cinerea showed poor growth and low laccase activity in pure culture, but greatly enhanced the level of laccase activity (>1800 U/L) during coculture with the Mucoromycete Gongronella sp. w5. As a result, the mechanism of laccase overproduction in coculture was investigated by starting from clarifying the function of sucrose. Results demonstrated that Gongronella sp. w5 in the coculture system hydrolyzed sucrose to glucose and fructose by an intracellular invertase. Fructose rather than glucose was supplied by Gongronella sp. w5  as the readily available carbon source for C. cinerea, and contributed to an alteration of its growth behavior and a basal laccase secretion of 110.6 ± 3.3 U/L. On the other hand, separating Gongronella sp. w5 of C. cinerea by transfer into dialysis tubes yielded the same level of laccase activity as without separation, indicating that enhanced laccase production probably resulted from the metabolites in the fermentation broth. Further investigation showed that the ethyl acetate–extracted metabolites generated by Gongronella sp. w5 induced C. cinerea laccase production. One of the laccase-inducing compounds namely p-hydroxybenzoic acid (HBA) was purified and identified from the extract. When using HBA as the inducer and fructose as the carbon source in monoculture, C. cinerea observed similar high laccase activity to that in coculture, and zymograms revealed the same expression of laccase Lcc9 as the main and Lcc1 and Lcc5 as the minor enzymes. Overall, our experiments verified that Gongronella sp. w5 elevates Coprinopsis cinerea laccase production by carbon source syntrophism and secondary metabolite induction.


Carbon source syntrophism Coculture Coprinopsis cinerea Laccase Secondary metabolites Gongronella sp. w5 



The authors are grateful to Prof. Patricia J. Pukkila (University of North Carolina, Chapel Hill, USA) for providing the strain C. cinerea strain Okayama 7 (#130), and to Jingjing Wang, Nannan Zhao, and Prof. Xiaotang Wang for suggestions.

Funding information

This work was supported by grants from the Natural Sciences Foundation of China (31870098, 31300044), the Chinese Scholarship Council (201706505019) for a research stay of ZF in Goettingen, and the National Natural Science Foundation of Anhui Province (1308085QC46).

Compliance with ethical standards

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Competing interests

The authors declare that they have no competing interests.

Supplementary material

253_2018_9469_MOESM1_ESM.pdf (317 kb)
ESM 1 (PDF 317 kb)


  1. Afri Y, Levasseur A, Record E (2013) Differential gene expression in Pycnoporus coccineus during interspecific mycelial interactions with different competitors. Appl Environ Microbiol 79:6626–6636CrossRefGoogle Scholar
  2. Alexandre G, Zhulin LB (2000) Laccases are widespread in bacteria. Trends Biotechnol 18:41–42CrossRefGoogle Scholar
  3. Armand D, Thivend S (1965) The production of phenolic acids by mycelia of hymenomycetes on a glucose medium. C R Acad Sci III 260:1472–1473Google Scholar
  4. Badalyan SM, Rapior S, Doko L, Lequang J, Jacob M, Serrano JJ, Andary C (1996) Chemical and pharmacological study of higher fungi. II. Comparative investigation of carpophores of some Nematoloma species: chemical composition and cultural characteristics. Mikol Fitopatol 30:79–86Google Scholar
  5. Bader J, Mast-Gerlach E, Popović MK, Bajpai R, Stahl U (2010) Relevance of microbial coculture fermentations in biotechnology. J Appl Microbiol 109:371–387CrossRefGoogle Scholar
  6. Bahn YS, Xue C, Idnurm A, Rutherford JC, Heitman J, Cardenas ME (2007) Sensing the environment: lessons from fungi. Nat Rev Microbiol 5:57–69CrossRefGoogle Scholar
  7. Baldrian P (2004) Increase of laccase activities during interspecific interactions of white-rot fungi. FEMS Microbiol Ecol 50:245–253CrossRefGoogle Scholar
  8. Baldrian P (2006) Fungal laccases – occurrence and properties. FEMS Microbiol Rev 30:215–242CrossRefGoogle Scholar
  9. Bertrand S, Bohni N, Schnee S, Schumpp O, Gindro K, Wolfender JL (2014) Metabolite induction via microorganism co-culture: a potential way to enhance chemical diversity for drug discovery. Biotechnol Adv 32:1180–1204CrossRefGoogle Scholar
  10. Bourbonnais R, Paice MG (1990) Oxidation of non-phenolic substrates. FEBS Lett 267(1):99–102CrossRefGoogle Scholar
  11. Cannatelli MD, Ragauskas AJ (2017) Two decades of laccases: advancing sustainability in the chemical industry. Chem Rec 17(1):122–140CrossRefGoogle Scholar
  12. Chen S, Ge W, Buswell JA (2004) Biochemical and molecular characterization of a laccase from the edible straw mushroom, Volvariella volvacea. Eur J Biochem 271(2):318–328CrossRefGoogle Scholar
  13. Chi Y, Hatakka A, Maijala P (2007) Can co-culturing of two white-rot fungi increase lignin degradation and the production of lignin-degrading enzymes? Int Biodeterior Biodegrad 59:32–39CrossRefGoogle Scholar
  14. Crowe JD, Olsson S (2001) Induction of laccase activity in Rhizoctonia solani by antagonistic Pseudomonas fluorescens strains and a range of chemical treatments. Appl Environ Microbiol 67:2088–2094CrossRefGoogle Scholar
  15. Dong YQ, Sun QY, Wang XJ, Zhang YL, Liu P, Xiao YZ, Fang ZM (2018) Complete genome of Gongronella sp. w5 provides insight into its relationship with plant. J Biotechnol 286:1–4.
  16. Flores C, Vidal C, Trejo-Hernández MR, Galindo E, Serrano-Carreón L (2009) Selection of Trichoderma strains capable of increasing laccase production by Pleurotus ostreatus and Agaricus bisporus in dual cultures. J Appl Microbiol 106:249–257CrossRefGoogle Scholar
  17. Floudas D, Binder M, Riley R, Barry K, Blanchette RA, Henrissat B, Martínez AT, Otillar R, Spatafora JW, Yadav JS, Aerts A, Benoit I, Boyd A, Carlson A, Copeland A, Coutinho PM, de Vries RP, Ferreira P, Findley K, Foster B, Gaskell J, Glotzer D, Górecki P, Heitman J, Hesse C, Hori C, Igarashi K, Jurgens JA, Kallen N, Kersten P, Kohler A, Kües U, Kumar TKA, Kuo A, LaButti K, Larrondo LF, Lindquist E, Ling A, Lombard V, Lucas S, Lundell T, Martin R, McLaughlin DJ, Morgenstern I, Morin E, Murat C, Nagy LG, Nolan M, Ohm RA, Patyshakuliyeva A, Rokas A, Ruiz-Dueñas FJ, Sabat G, Salamov A, Samejima M, Schmutz J, Slot JC, St. John F, Stenlid J, Sun H, Sun S, Syed K, Tsang A, Wiebenga A, Young D, Pisabarro A, Eastwood DC, Martin F, Cullen D, Grigoriev IV, Hibbett DS (2012) The paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes. Science 336(6089):1715–1719CrossRefGoogle Scholar
  18. Galhaup C, Wagner H, Barbara H, Haltrich D (2002) Increased production of laccase by the wood-degrading basidiomycete Trametes pubescens. Enzyme Microb Technol 30:529–536CrossRefGoogle Scholar
  19. Guo C, Zhao L, Wang F, Lu J, Ding Z, Shi G (2017) β-Carotene from yeasts enhances laccase production of Pleurotus eryngii var. ferulae in co-culture. Front Microbiol 8:1101CrossRefGoogle Scholar
  20. Hiscox J, Boddy L (2017) Armed and dangerous – chemical warfare in wood decay communities. Fungal Biol Rev 31(4):169–184CrossRefGoogle Scholar
  21. Hoegger PJ, Kilaru S, James TY, Thacker JR, Kües U (2006) Phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequences. FEBS J 273:2308–2326CrossRefGoogle Scholar
  22. Iakovlev A, Stenlid J (2000) Spatiotemporal patterns of laccase activity in interacting mycelia of wood-decaying basidiomycete fungi. Microb Ecol 39:236–245PubMedGoogle Scholar
  23. Jiang H, Ma Y, Chi Z, Liu GL, Chi ZM (2016) Production, purification, and gene cloning of a β-fructofuranosidase with a high inulin-hydrolyzing activity produced by a novel yeast Aureobasidium sp. P6 isolated from a mangrove ecosystem. Mar Biotechnol (NY) 18(4):500–510CrossRefGoogle Scholar
  24. Johannes C, Majcherczyk A (2000) Natural mediators in the oxidation of polycyclic aromatic hydrocarbons by laccase mediator systems. Appl Environ Microbiol 66:524–528CrossRefGoogle Scholar
  25. Kilaru S, Hoegger PJ, Kües U (2006) The laccase multi-gene family in Coprinopsis cinerea has seventeen different members that divide into two distinct subfamilies. Curr Genet 50(1):45–60Google Scholar
  26. Klonowska A, Petit JL, Tron T (2001) Enhancement of minor laccases production in the basidiomycete Marasmius quercophilus C30. FEMS Microbiol Lett 200:25–30CrossRefGoogle Scholar
  27. Kobayashi DY, Crouch JA (2009) Bacterial/fungal interaction: from pathogens to mutualistic endosymbionts. Annu Rev Phytopathol 47:63–82CrossRefGoogle Scholar
  28. Kudanga T, Nemadziva B, Le Roes-Hill M (2017) Laccase catalysis for the synthesis of bioactive compounds. Appl Microbiol Biotechnol 101(1):13–33CrossRefGoogle Scholar
  29. Kües U (2000) Life history and developmental processes in the basidiomycete Coprinus cinereus. Microbiol Mol Biol Rev 64:316–353CrossRefGoogle Scholar
  30. Kües U, Rühl M (2011) Multiple multi-copper oxidase gene families in basidiomycetes - what for? Curr Genomics 12:72–94CrossRefGoogle Scholar
  31. Lakshmanan D, Sadasivan C (2016) Trichoderma viride laccase plays a crucial role in defense mechanism against antagonistic organisms. Front Microbiol 7:741.
  32. Lammens W, Le Roy K, Schroeven L, Van Laere A, Rabijns A, Van den Ende W (2009) Structural insights into glycoside hydrolase family 32 and 68 enzymes: functional implications. J Exp Bot 60(3):727–740CrossRefGoogle Scholar
  33. Li P, Wang H, Liu G, Li X, Yao J (2011) The effect of carbon source succession on laccase activity in the co-culture process of Ganoderma lucidum and a yeast. Enzym Microb Technol 48:1–6CrossRefGoogle Scholar
  34. Marques WL, Raghavendran V, Stambuk BU, Gombert AK (2016) Sucrose and Saccharomyces cerevisiae: a relationship most sweet. FEMS Yeast Res 16(1):fov107CrossRefGoogle Scholar
  35. Mata G, Murrieta Hernández DM, Iglesias Andreu LG (2005) Changes in lignocellulolytic enzyme activites in six Pleurotus spp. strains cultivated on coffee pulp in confrontation with Trichoderma spp. World J Microbiol Biotechnol 21:143–150CrossRefGoogle Scholar
  36. Moore D (1969) Sources of carbon and energy used by Coprinus lagopus sensu Buller. J Gen Microbiol 58:49–56CrossRefGoogle Scholar
  37. Pan K, Zhao N, Yin Q, Zhang T, Xu X, Fang W, Hong Y, Fang Z, Xiao Y (2014) Induction of laccase Lcc9 from Coprinopsis cinerea by fungal coculture and its application on indigo dye decolorization. Bioresour Technol 162:45–52CrossRefGoogle Scholar
  38. Periasamy R, Palvannan T (2010) Optimization of laccase production by Pleurotus ostreatus IMI 395545 using the Taguchi DOE methodology. J Basic Microbiol 50(6):548–556CrossRefGoogle Scholar
  39. Petersen TN, Brunak S, Heijne GV, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8(10):785–786CrossRefGoogle Scholar
  40. Piscitelli A, Giardina P, Lettera V, Pezzella C, Sannia G, Faraco V (2011) Induction and transcriptional regulation of laccases in fungi. Curr Genomics 12:104–112CrossRefGoogle Scholar
  41. Rühl M, Majcherczyk A, Kües U (2013) Lcc1 and Lcc5 are the main laccases secreted in liquid cultures of Coprinopsis cinerea strains. Antonie van Leeuwenhoek 103(5):1029–1039Google Scholar
  42. Savoie JM, Mata G, Billette C (1998) Extracellular laccase production during hyphal interactions between Trichoderma sp. and Shiitake, Lentinula edodes. Appl Microbiol Biotechnol 49:589–593CrossRefGoogle Scholar
  43. Savoie JM, Mata G, Mamoun M (2001) Variability in brown line formation and extracellular laccase production during interaction between white-rot basidiomycetes and Trichoderma harzianum biotype Th2. Mycologia 93:243–248CrossRefGoogle Scholar
  44. Schroeckh V, Scherlach K, Nützmann HW, Shelest E, Schmidt-Heck W, Schuemann J, Martin K, Hertweck C, Brakhage AA (2009) Intimate bacterial–fungal interaction triggers biosynthesis of archetypal polyketides in Aspergillus nidulans. Proc Natl Acad Sci U S A 106:14558–14563CrossRefGoogle Scholar
  45. Sjaarda CP, Abubaker KS, Castle AJ (2015) Induction of lcc2 expression and activity by Agaricus bisporus provides defence against Trichoderma aggressivum toxic extracts. Microbe Biotechnol 8:918–929. CrossRefGoogle Scholar
  46. Stajich JE, Wilke SK, Ahren D, Au CH, Birren BW, Borodovsky M, Burns C, Canbäck B, Casselton LA, Cheng CK, Deng J, Dietrich FS, Fargo DC, Farman ML, Gathman AC, Goldberg J, Guigo R, Hoegger PJ, Hooker JB, Huggins A, James TY, Kamada T, Kilaru S, Kodira C, Kües U, Kupfer D, Kwan HS, Lomsadze A, Li W, Lilly WW, Ma LJ, Mackey AJ, Manning G, Martin F, Muraguchi H, Natvig DO, Palmerini H, Ramesh MA, Rehmeyer CJ, Roe BA, Shenoy N, Stanke M, Ter-Hovhannisyan V, Tunlid A, Velagapudi R, Vision TJ, Zeng Q, Zolan ME, Pukkila PJ (2010) Insights into evolution of multicellular fungi from the assembled chromosomes of the mushroom Coprinopsis cinerea (Coprinus cinereus). Proc Natl Acad Sci U S A 107(26):11889–11894Google Scholar
  47. Tavares APM, Coelho MAZ, Coutinho JAP, Xavier A (2005) Laccase improvement in submerged cultivation: induced production and kinetic modelling. J Chem Technol Biotechnol 80(6):669–676CrossRefGoogle Scholar
  48. Ujor VC, Adukwu EC, Okonkwo CC (2018) Fungal wars: the underlying molecular repertoires of combating mycelia. Fungal Biol 122(4):191–202CrossRefGoogle Scholar
  49. Van der Nest MA, Steenkamp ET, McTaggart AR, Trollip C, Godlonton T, Sauerman E, Roodt D, Naidoo K, Coetzee MP, Wilken PM, Wingfield MJ, Wingfield BD (2015) Saprophytic and pathogenic fungi in the Ceratocystidaceae differ in their ability to metabolize plant-derived sucrose. BMC Evol Biol 15:273CrossRefGoogle Scholar
  50. Vargas WA, Mandawe JC, Kenerley CM (2009) Plant-derived sucrose is a key element in the symbiotic association between Trichoderma virens and maize plants. Plant Physiol 151:792–808CrossRefGoogle Scholar
  51. Vargas WA, Crutcher FK, Kenerley CM (2011) Functional characterization of a plant-like sucrose transporter from the beneficial fungus Trichoderma virens. Regulation of the symbiotic association with plants by sucrose metabolism inside the fungal cells. New Phytol 189:777–789CrossRefGoogle Scholar
  52. Velázquez-Cedeño M, Farnet AM, Mata G, Savoie JM (2008) Role of Bacillus spp. in antagonism between Pleurotus ostreatus and Trichoderma harzianum in heat-treated wheat-straw substrates. Bioresour Technol 99(15):6966–6973CrossRefGoogle Scholar
  53. Wahl R, Wippel K, Goos S, Kämper J, Sauer N (2010) A novel high-affinity sucrose transporter is required for virulence of the plant pathogen Ustilago maydis. PLoS Biol 8(2):e1000303Google Scholar
  54. Wang H, Yu G, Li P, Gu Y, Li J, Liu G, Yao J (2009) Overproduction of Trametes versicolor laccase by making glucose starvation using yeast. Enzym Microb Technol 45:146–149CrossRefGoogle Scholar
  55. Wang H, Peng L, Ding Z, Wu J, Shi G (2015) Stimulated laccase production of Pleurotus ferulae JM301 fungus by Rhodotorula mucilaginosa yeast in co-culture. Process Biochem 50(6):901–905CrossRefGoogle Scholar
  56. Wei F, Hong Y, Liu J, Yuan J, Fang W, Peng H, Xiao Y (2010) Gongronella sp. induces overproduction of laccase in Panus rudis. J Basic Microbiol l50:98–103CrossRefGoogle Scholar
  57. Xiao YZ, Tu XM, Wang J, Zhang M, Cheng Q, Zeng WY, Shi YY (2003) Purification, molecular characterization and reactivity with aromatic compounds of a laccase from basidiomycete Trametes sp. strain AH28-2. Appl Microbiol Biotechnol 60(6):700–707CrossRefGoogle Scholar
  58. Xiao YZ, Chen Q, Hang J, Shi YY, Wu J, Hong YZ, Wang YP (2004) Selective induction, purification and characterization of a laccase isozyme from the basidiomycete Trametes sp. AH28-2. Mycologia 96:26–35CrossRefGoogle Scholar
  59. Xie N, Chapeland-Leclerc F, Silar P, Ruprich-Robert G (2014) Systematic gene deletions evidences that laccases are involved in several stages of wood degradation in the filamentous fungus Podospora anserina. Environ Microbiol 16:141–161CrossRefGoogle Scholar
  60. Yang J, Li W, Ng TB, Deng X, Lin J, Ye X (2017) Laccases: production, expression regulation, and applications in pharmaceutical biodegradation. Front Microbiol 8:832. CrossRefPubMedPubMedCentralGoogle Scholar
  61. Yin Y, Mao X, Yang J, Chen X, Mao F, Xu Y (2012) dbCAN: a web resource for automated carbohydrate-active enzyme annotation. Nucleic Acids Res 40:W445–W451CrossRefGoogle Scholar
  62. Zhang H, Hong YZ, Xiao YZ, Yuan J, Tu XM, Zhang XQ (2006) Efficient production of laccases by Trametes sp. AH28-2 in cocultivation with a Trichoderma strain. Appl Microbiol Biotechnol 73:89–94CrossRefGoogle Scholar
  63. Zhang L, Feng G, Declerck S (2018) Signal beyond nutrient, fructose, exuded by an arbuscular mycorrhizal fungus triggers phytate mineralization by a phosphate solubilizing bacterium. ISME J 12:2339–2351. CrossRefPubMedPubMedCentralGoogle Scholar
  64. Zhong Z, Li L, Chang P, Xie H, Zhang H, Igarashi Y, Li N, Luo F (2017) Differential gene expression profiling analysis in Pleurotus ostreatus during interspecific antagonistic interactions with Dichomitus squalens and Trametes versicolor. Fungal Biol 121(12):1025–1036CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.School of Life SciencesAnhui UniversityHefeiChina
  2. 2.Anhui Key Laboratory of Modern BiomanufacturingHefeiChina
  3. 3.Anhui Provincial Engineering Technology Research Center of Microorganisms and BiocatalysisHefeiChina
  4. 4.Molecular Wood Biotechnology and Technical Mycology, Büsgen-InstituteUniversity of GoettingenGoettingenGermany
  5. 5.Goettingen Center for Molecular Biosciences (GZMB)University of GoettingenGoettingenGermany

Personalised recommendations