Evidence for ligninolytic activity of the ascomycete fungus Podospora anserina

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 13 C 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 H 2 O 2 -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.

Lignin Degradation and Its Use in Signaling Development by the Coprophilous Ascomycete Podospora anserina

“…Through Py-GCMS, we confirm the data of van Erven et al [29] showing that the β-O-4 linkages are likely the preferred target for breakdown also on a natural substrate. Lignin is a complex polymer originating from the polymerization of three monolignols: coniferyl, sinapyl and p-coumaryl alcohols (Figure S3).…”

“…3.4. Spectroscopic Analyses Indicate That P. anserina Efficiently Breaks Down Lignin P. anserina is very efficient at retrieving nutrients from natural lignocellulose, likely because it is able to break down lignin [29]. We confirm here using alternative methods that it is able to alter the lignin components of the biomass, using first a color plate assay.…”

“…The reasons for this are likely multifactorial and include the secretion of different enzymatic cocktails, as previously demonstrated [29,70,71]. It was shown that the presence of lignin reduces the secretion of cellulases and galactomannanases and increases that of laccases and H 2 O 2 -producing enzymes, which should favor ligninolysis [29]. Crucial roles of laccases and catalases in the ability of the fungus to grow and fructify on wood shavings were previously demonstrated [27,72,73].…”

“…Even on its preferred natural growth substrate, dung, we detect large variations in the amounts of mycelium and fruiting bodies that the fungus produces. The reasons for this are likely multifactorial and include the secretion of different enzymatic cocktails, as previously demonstrated [29,70,71]. It was shown that the presence of lignin reduces the secretion of cellulases and galactomannanases and increases that of laccases and H 2 O 2 -producing enzymes, which should favor ligninolysis [29].…”

“…This indicates that it can extract enough nutrients from the complex lignocellulosic sources to build the multicellular fruiting bodies involved in ascospore production, or perithecia, that it uses for dispersal. A recent study demonstrated that it is able to mineralize lignin [29]. In this study, the starting lignocellulose source, i.e., pre-isolated lignin from wheat straw re-associated with the hemicellulose fraction of the straw, while enabling fine analyses of the lignin hydrolysis, was not a natural food source for the fungus.…”