Evidence for ligninolytic activity of the ascomycete fungus Podospora anserina

Gijs van Erven; Anne F Kleijn; Aleksandrina Patyshakuliyeva; Marcos Di Falco; Adrian Tsang; Ronald P de Vries; Willem J H van Berkel; Mirjam A Kabel

doi:10.1186/s13068-020-01713-z

Evidence for ligninolytic activity of the ascomycete fungus Podospora anserina

Biotechnol Biofuels. 2020 Apr 16:13:75. doi: 10.1186/s13068-020-01713-z. eCollection 2020.

Authors

Gijs van Erven¹, Anne F Kleijn¹, Aleksandrina Patyshakuliyeva², Marcos Di Falco³, Adrian Tsang³, Ronald P de Vries², Willem J H van Berkel¹, Mirjam A Kabel¹

Affiliations

¹ 1Laboratory of Food Chemistry, Wageningen University and Research, Bornse Weilanden 9, 6708 WG Wageningen, The Netherlands.
² 2Fungal Physiology, Westerdijk Fungal Biodiversity Institute and Fungal Molecular Physiology, Utrecht University, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
³ 3Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke Street West, Montréal, Québec H4B 1R6 Canada.

Abstract

Background: The ascomycete fungus Podospora anserina has been appreciated for its targeted carbohydrate-active enzymatic arsenal. As a late colonizer of herbivorous dung, the fungus acts specifically on the more recalcitrant fraction of lignocellulose and this lignin-rich biotope might have resulted in the evolution of ligninolytic activities. However, the lignin-degrading abilities of the fungus have not been demonstrated by chemical analyses at the molecular level and are, thus far, solely based on genome and secretome predictions. To evaluate whether P. anserina might provide a novel source of lignin-active enzymes to tap into for potential biotechnological applications, we comprehensively mapped wheat straw lignin during fungal growth and characterized the fungal secretome.

Results: Quantitative ¹³C lignin internal standard py-GC-MS analysis showed substantial lignin removal during the 7 days of fungal growth (24% w/w), though carbohydrates were preferably targeted (58% w/w removal). Structural characterization of residual lignin by using py-GC-MS and HSQC NMR analyses demonstrated that C_α-oxidized substructures significantly increased through fungal action, while intact β-O-4' aryl ether linkages, p-coumarate and ferulate moieties decreased, albeit to lesser extents than observed for the action of basidiomycetes. Proteomic analysis indicated that the presence of lignin induced considerable changes in the secretome of P. anserina. This was particularly reflected in a strong reduction of cellulases and galactomannanases, while H₂O₂-producing enzymes clearly increased. The latter enzymes, together with laccases, were likely involved in the observed ligninolysis.

Conclusions: For the first time, we provide unambiguous evidence for the ligninolytic activity of the ascomycete fungus P. anserina and expand the view on its enzymatic repertoire beyond carbohydrate degradation. Our results can be of significance for the development of biological lignin conversion technologies by contributing to the quest for novel lignin-active enzymes and organisms.

Keywords: Biomass; Enzymes; Laccase; Lignin; NMR spectroscopy; Proteomics; Secretomics; py-GC–MS.