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The unique ability of basidiomycete white rot fungi to degrade all components of plant cell walls makes them indispensable organisms in the global carbon cycle. In this study, we analyzed the proteomes of two closely related white rot fungi, Obba rivulosa and Gelatoporia subvermispora , during eight-week cultivation on solid spruce wood. Plant cell wall degrading carbohydrate-active enzymes (CAZymes) represented approximately 5% of the total proteins in both species. A core set of orthologous plant cell wall degrading CAZymes was shared between these species on spruce suggesting a conserved plant biomass degradation approach in this clade of basidiomycete fungi. However, differences in time-dependent production of plant cell wall degrading enzymes may be due to differences among initial growth rates of these species on solid spruce wood. The obtained results provide insight into specific enzymes and enzyme sets that are produced during the degradation of solid spruce wood in these fungi. These findings expand the knowledge on enzyme production in nature-mimicking conditions and may contribute to the exploitation of white rot fungi and their enzymes for biotechnological applications.

Lignocellulosic biomass is an abundant and renewable resource, and its utilization has become the focus of research and biotechnology applications as a very promising raw material for the production of value-added compounds. Filamentous fungi play an important role in the production of various lignocellulolytic enzymes, while some of them have also been used for the production of important metabolites. However, wild type strains have limited efficiency in enzyme production or metabolic conversion, and therefore many efforts have been made to engineer improved strains. Examples of this are the manipulation of transcriptional regulators and/or promoters of enzyme-encoding genes to increase gene expression, and protein engineering to improve the biochemical characteristics of specific enzymes. This review provides and overview of the applications of filamentous fungi in lignocellulosic biomass based processes and the development and current status of various molecular engineering strategies to improve these processes., (Copyright © 2021 Elsevier Inc. All rights reserved.)

White-rot (WR) fungi are pivotal decomposers of dead organic matter in forest ecosystems and typically use a large array of hydrolytic and oxidative enzymes to deconstruct lignocellulose. However, the extent of lignin and cellulose degradation may vary between species and wood type. Here, we combined comparative genomics, transcriptomics and secretome proteomics to identify conserved enzymatic signatures at the onset of wood-decaying activity within the Basidiomycota genus Pycnoporus. We observed a strong conservation in the genome structures and the repertoires of protein-coding genes across the four Pycnoporus species described to date, despite the species having distinct geographic distributions. We further analysed the early response of P. cinnabarinus, P. coccineus and P. sanguineus to diverse (ligno)-cellulosic substrates. We identified a conserved set of enzymes mobilized by the three species for breaking down cellulose, hemicellulose and pectin. The co-occurrence in the exo-proteomes of H2O2-producing enzymes with H2O2-consuming enzymes was a common feature of the three species, although each enzymatic partner displayed independent transcriptional regulation. Finally, cellobiose dehydrogenase-coding genes were systematically co-regulated with at least one AA9 lytic polysaccharide monooxygenase gene, indicative of enzymatic synergy in vivo. This study highlights a conserved core white-rot fungal enzymatic mechanism behind the wood-decaying process., (© The Author(s) 2020. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

Fungi can decompose plant biomass into small oligo- and monosaccharides to be used as carbon sources. Some of these small molecules may induce metabolic pathways and the production of extracellular enzymes targeted for degradation of plant cell wall polymers. Despite extensive studies in ascomycete fungi, little is known about the nature of inducers for the lignocellulolytic systems of basidiomycetes. In this study, we analyzed six sugars known to induce the expression of lignocellulolytic genes in ascomycetes for their role as inducers in the basidiomycete white-rot fungus Dichomitus squalens using a transcriptomic approach. This identified cellobiose and l-rhamnose as the main inducers of cellulolytic and pectinolytic genes, respectively, of D. squalens Our results also identified differences in gene expression patterns between dikaryotic and monokaryotic strains of D. squalens cultivated on plant biomass-derived monosaccharides and the disaccharide cellobiose. This suggests that despite conservation of the induction between these two genetic forms of D. squalens , the fine-tuning in the gene regulation of lignocellulose conversion is differently organized in these strains. IMPORTANCE Wood-decomposing basidiomycete fungi have a major role in the global carbon cycle and are promising candidates for lignocellulosic biorefinery applications. However, information on which components trigger enzyme production is currently lacking, which is crucial for the efficient use of these fungi in biotechnology. In this study, transcriptomes of the white-rot fungus Dichomitus squalens from plant biomass-derived monosaccharide and cellobiose cultures were studied to identify compounds that induce the expression of genes involved in plant biomass degradation., (Copyright © 2018 American Society for Microbiology.)

Basidiomycete fungi can degrade a wide range of plant biomass, including living and dead trees, forest litter, crops, and plant matter in soils. Understanding the process of plant biomass decay by basidiomycetes could facilitate their application in various industrial sectors such as food & feed, detergents and biofuels, and also provide new insights into their essential biological role in the global carbon cycle. The fast expansion of basidiomycete genomic and functional genomics data (e.g. transcriptomics, proteomics) has facilitated exploration of key genes and regulatory mechanisms of plant biomass degradation. In this study, we comparatively analyzed 22 transcriptome datasets from basidiomycetes related to plant biomass degradation, and identified 328 commonly induced genes and 318 repressed genes, and defined a core set of carbohydrate active enzymes (CAZymes), which was shared by most of the basidiomycete species. High conservation of these CAZymes in genomes and similar regulation pattern in transcriptomics data from lignocellulosic substrates indicate their key role in plant biomass degradation and need for their further biochemical investigation., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The white button mushroom Agaricus bisporus is economically the most important commercially produced edible fungus. It is grown on carbon- and nitrogen-rich substrates, such as composted cereal straw and animal manure. The commercial mushroom production process is usually performed in buildings or tunnels under highly controlled environmental conditions. In nature, the basidiomycete A. bisporus has a significant impact on the carbon cycle in terrestrial ecosystems as a saprotrophic decayer of leaf litter. In this mini-review, the fate of the compost plant cell wall structures, xylan, cellulose and lignin, is discussed. A comparison is made from the structural changes observed to the occurrence and function of enzymes for lignocellulose degradation present, with a special focus on the extracellular enzymes produced by A. bisporus. In addition, recent advancements in whole genome level molecular studies in various growth stages of A. bisporus in compost are reviewed.

The global push toward an efficient and economical biobased economy has driven research to develop more cost-effective applications for the entirety of plant biomass, including lignocellulosic crops. As discussed elsewhere (Karlsson M, Atanasova L, Funck Jensen D, Zeilinger S, in Heitman J et al. [ed], Tuberculosis and the Tubercle Bacillus, 2nd ed, in press), significant progress has been made in the use of polysaccharide fractions from lignocellulose, cellulose, and various hemicellulose types. However, developing processes for use of the lignin fraction has been more challenging. In this chapter, we discuss characteristics of lignolytic enzymes and the fungi that produce them as well as potential and current uses of lignin-derived products.

White rot fungus Dichomitus squalens is an efficient lignocellulose degrading basidiomycete and a promising source for new plant cell wall polysaccharides depolymerizing enzymes. In this work, we focused on cellobiohydrolases (CBHs) of D. squalens. The native CBHI fraction of the fungus, consisting three isoenzymes, was purified and it maintained the activity for 60 min at 50°C, and was stable in acidic pH. Due to the lack of enzyme activity assay for detecting only CBHII activity, CBHII of D. squalens was produced recombinantly in an industrially important ascomycete host, Trichoderma reesei. CBH enzymes of D. squalens showed potential in hydrolysis of complex lignocellulose substrates sugar beet pulp and wheat bran, and microcrystalline cellulose, Avicel. Recombinant CBHII (rCel6A) of D. squalens hydrolysed all the studied plant biomasses. Compared to individual activities, synergistic effect between rCel6A and native CBHI fraction of D. squalens was significant in the hydrolysis of Avicel. Furthermore, the addition of laccase to the mixture of CBHI fraction and rCel6A significantly enhanced the amount of released reducing sugars from sugar beet pulp. Especially, synergy between individual enzymes is a crucial factor in the tailor-made enzyme mixtures needed for hydrolysis of different plant biomass feedstocks. Our data supports the importance of oxidoreductases in improved enzyme cocktails for lignocellulose saccharification.

Background: The fungal genus Phlebia consists of a number of species that are significant in wood decay. Biotechnological potential of a few species for enzyme production and degradation of lignin and pollutants has been previously studied, when most of the species of this genus are unknown. Therefore, we carried out a wider study on biochemistry and systematics of Phlebia species., Methods: Isolates belonging to the genus Phlebia were subjected to four-gene sequence analysis in order to clarify their phylogenetic placement at species level and evolutionary relationships of the genus among phlebioid Polyporales. rRNA-encoding (5.8S, partial LSU) and two protein-encoding gene (gapdh, rpb2) sequences were adopted for the evolutionary analysis, and ITS sequences (ITS1+5.8S+ITS2) were aligned for in-depth species-level phylogeny. The 49 fungal isolates were cultivated on semi-solid milled spruce wood medium for 21 days in order to follow their production of extracellular lignocellulose-converting oxidoreductases and carbohydrate active enzymes., Results: Four-gene phylogenetic analysis confirmed the polyphyletic nature of the genus Phlebia. Ten species-level subgroups were formed, and their lignocellulose-converting enzyme activity profiles coincided with the phylogenetic grouping. The highest enzyme activities for lignin modification (manganese peroxidase activity) were obtained for Phlebia radiata group, which supports our previous studies on the enzymology and gene expression of this species on lignocellulosic substrates., Conclusions: Our study implies that there is a species-level connection of molecular systematics (genotype) to the efficiency in production of both lignocellulose-converting carbohydrate active enzymes and oxidoreductases (enzyme phenotype) on spruce wood. Thus, we may propose a similar phylogrouping approach for prediction of lignocellulose-converting enzyme phenotypes in new fungal species or genetically and biochemically less-studied isolates of the wood-decay Polyporales.

The economically important edible basidiomycete mushroom Agaricus bisporus thrives on decaying plant material in forests and grasslands of North America and Europe. It degrades forest litter and contributes to global carbon recycling, depolymerizing (hemi-)cellulose and lignin in plant biomass. Relatively little is known about how A. bisporus grows in the controlled environment in commercial production facilities and utilizes its substrate. Using transcriptomics and proteomics, we showed that changes in plant biomass degradation by A. bisporus occur throughout its life cycle. Ligninolytic genes were only highly expressed during the spawning stage day 16. In contrast, (hemi-)cellulolytic genes were highly expressed at the first flush, whereas low expression was observed at the second flush. The essential role for many highly expressed plant biomass degrading genes was supported by exo-proteome analysis. Our data also support a model of sequential lignocellulose degradation by wood-decaying fungi proposed in previous studies, concluding that lignin is degraded at the initial stage of growth in compost and is not modified after the spawning stage. The observed differences in gene expression involved in (hemi-)cellulose degradation between the first and second flushes could partially explain the reduction in the number of mushrooms during the second flush., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The biological conversion of plant lignocellulose plays an essential role not only in carbon cycling in terrestrial ecosystems but also is an important part of the production of second generation biofuels and biochemicals. The presence of the recalcitrant aromatic polymer lignin is one of the major obstacles in the biofuel/biochemical production process and therefore microbial degradation of lignin is receiving a great deal of attention. Fungi are the main degraders of plant biomass, and in particular the basidiomycete white rot fungi are of major importance in converting plant aromatics due to their ability to degrade lignin. However, the aromatic monomers that are released from lignin and other aromatic compounds of plant biomass are toxic for most fungi already at low levels, and therefore conversion of these compounds to less toxic metabolites is essential for fungi. Although the release of aromatic compounds from plant biomass by fungi has been studied extensively, relatively little attention has been given to the metabolic pathways that convert the resulting aromatic monomers. In this review we provide an overview of the aromatic components of plant biomass, and their release and conversion by fungi. Finally, we will summarize the applications of fungal systems related to plant aromatics., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Biotechnological, microbiological, and genetic studies of Agaricus species other than A. bisporus, the white button mushroom, have been limited so far. To expand the knowledge in the genus Agaricus, six novel wild-type isolates of Agaricus spp. were studied on their nutritional demands for enzyme production and mycelial growth. All the selected Agaricus species produced extracellular manganese peroxidase (MnP) and laccase activities in semi-solid rye bran cultures. Moderate MnP activities were measured for A. bisporus, A. bernardii and A. campestris. The highest laccase activities were obtained for A. bisporus and A. campestris. On soy medium, the highest mycelial tyrosinase activity was determined for A. bernardii. For A. bisporus, addition of copper caused no increase in laccase or tyrosinase activities on soy or malt extract media. Hyphal growth rate of the isolates was studied on lignocellulose amended agar plates. Fastest growth was obtained for A. bisporus on wheat bran and birch leaf litter agar. Except for A. bernardii, hyphal growth rates correlated well with MnP and laccase production levels between Agaricus species. Molecular taxonomy of the novel Agaricus spp. positioned them to distinct phylogenetic clusters with species-level identity. In conclusion, our data point to the importance of both MnP and multicopper enzymes in Agaricus spp. while growing on lignocelluloses., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Production of the oxidoreductive lignin-modifying enzymes - lignin and manganese peroxidases (MnPs), and laccase - of the white-rot basidiomycete Phlebia radiata was investigated in semi-solid cultures supplemented with milled grey alder or Norway spruce and charcoal. Concentrations of nutrient nitrogen and Cu-supplement varied also in the cultures. According to extracellular activities, production of both lignin peroxidase (LiP) and MnP was significantly promoted with wood as carbon source, with milled alder (MA) and low nitrogen (LN) resulting with the maximal LiP activities (550 nkat l(-1)) and noticeable levels of MnP (3 μkat l(-1)). Activities of LiP and MnP were also elevated on high nitrogen (HN) complex medium when supplemented with spruce and charcoal. Maximal laccase activities (22 and 29 μkat l(-1)) were obtained in extra high nitrogen (eHN) containing defined and complex media supplemented with 1.5 mM Cu(2+). However, the nitrogen source, either peptone or ammonium nitrate and asparagine, caused no stimulation on laccase production without Cu-supplement. This is also the first report to demonstrate a new, on high Cu(2+) amended medium produced extracellular laccase of P. radiata with pI value of 4.9, thereby complementing our previous findings on gene expression, and cloning of a second laccase of this fungus., (Copyright © 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Filamentous fungi owe powerful abilities for decomposition of the extensive plant material, lignocellulose, and thereby are indispensable for the Earth's carbon cycle, generation of soil humic matter and formation of soil fine structure. The filamentous wood-decaying fungi belong to the phyla Basidiomycota and Ascomycota, and are unique organisms specified to degradation of the xylem cell wall components (cellulose, hemicelluloses, lignins and extractives). The basidiomycetous wood-decaying fungi form brackets, caps or resupinaceous (corticioid) fruiting bodies when growing on wood for dissemination of their sexual basidiospores. In particular, the ability to decompose the aromatic lignin polymers in wood is mostly restricted to the white rot basidiomycetes. The white-rot decay of wood is possible due to secretion of organic acids, secondary metabolites, and oxidoreductive metalloenzymes, heme peroxidases and laccases, encoded by divergent gene families in these fungi. The brown rot basidiomycetes obviously depend more on a non-enzymatic strategy for decomposition of wood cellulose and modification of lignin. This review gives a current ecological, genomic, and protein functional and phylogenetic perspective of the wood and lignocellulose-decaying basidiomycetous fungi.