White-rot fungi, such as Dichomitus squalens, degrade all wood components and inhabit mixed wood forests containing both soft- and hardwood species. In this study, we evaluated how D. squalens ...responded to the compositional differences in softwood (guaiacyl (G) lignin and higher mannan content) and hardwood (syringyl/guaiacyl (S/G) lignin and higher xylan content) using semi-natural solid cultures. Spruce (softwood) and birch (hardwood) sticks were degraded by D. squalens as measured by oxidation of the lignins using 2D-NMR. The fungal response as measured by transcriptomics, proteomics and enzyme activities showed a partial tailoring to wood composition. Mannanolytic transcripts and proteins were more abundant in spruce cultures, while a proportionally higher xylanolytic activity was detected in birch cultures. Both wood types induced manganese peroxidases to a much higher level than laccases, but higher transcript and protein levels of the manganese peroxidases were observed on the G-lignin rich spruce. Overall, the molecular responses demonstrated a stronger adaptation to the spruce rather than birch composition, possibly because D. squalens is mainly found degrading softwoods in nature, which supports the ability of the solid wood cultures to reflect the natural environment.
•Fluoroquinolone antibiotics are easily degradable by ligninolytic fungi.•Irpex lacteus and Trametes versicolor degraded fluoroquinolones most efficiently.•The compounds are attacked at the ...piperazinyl moiety: substitution or decomposition.•Only I. lacteus removed the antibiotic activity during the degradation.•PCA indicated that manganese peroxidase might participate in the degradation.
A group of white rot fungi (Irpex lacteus, Panus tigrinus, Dichomitus squalens, Trametes versicolor and Pleurotus ostreatus) was investigated for the biodegradation of norfloxacin (NOR), ofloxacin (OF) and ciprofloxacin (CIP). The selected fluoroquinolones were readily degraded almost completely by I. lacteus and T. versicolor within 10 and 14d of incubation in liquid medium, respectively. The biodegradation products were identified by liquid chromatography–mass spectrometry. The analyses indicated that the fungi use similar mechanisms to degrade structurally related antibiotics. The piperazine ring of the molecules is preferably attacked via either substitution or/and decomposition. In addition to the degradation efficiency, attention was devoted to the residual antibiotic activities estimated using Gram-positive and Gram-negative bacteria. Only I. lacteus was able to remove the antibiotic activity during the course of the degradation of NOR and OF. The product-effect correlations evaluated by Principal Component Analysis (PCA) enabled elucidation of the participation of the individual metabolites in the residual antibacterial activity. Most of the metabolites correlated with the antibacterial activity, explaining the rather high residual activity remaining after the biodegradation. PCA of ligninolytic enzyme activities indicated that manganese peroxidase might participate in the degradation.
Dichomitus squalens is an efficient white-rot fungus that generates a wide range of extracellular enzymes to degrade lignocellulose in nature. Although a protoplast-mediated transformation method for ...D. squalens has been developed, the transformation efficiency remains low. Here, we established a highly efficient Agrobacterium tumefaciens-mediated transformation (ATMT) procedure for D. squalens by transferring a binary vector harboring the neomycin phosphotransferase II (nptII) resistance gene fused with DsRed-Express2, under the control of the native glyceraldehyde-3-phosphate dehydrogenase (GPD) gene promoter. Key factors affecting the efficiency of transformation were tested. A. tumefaciens EHA105 strain with a cell density of 0.4 OD600nm and 96 h co-cultivation resulted in the highest transformation efficiency, with an average of 98 ± 11 transformants per co-cultivation plate. Besides, the strong expression of DsRed-Express2 indicates the effectiveness of the DsGPD promoter in driving gene expression in D. squalens. This ATMT system of D. squalens would be beneficial for its molecular genetic studies.
•Establishment and optimization of the Agrobacterium tumefaciens–mediated transformation method for Dichomitus squalens.•An average of 98 ± 11 transformants per co-cultivation plate was obtained.•Homologous promoter ofDsGPD efficiently drives the expression of the nptII-DsRed Express2 fusion gene.•Transgenes were expressed stably in D. squalens.
Production of value-added compounds from a renewable aromatic polymer, lignin, has proven to be challenging. Chemical procedures, involving harsh reaction conditions, are costly and often result in ...nonselective degradation of lignin linkages. Therefore, enzymatic catalysis with selective cleavage of lignin bonds provides a sustainable option for lignin valorization. In this study, we describe the first functionally characterized fungal intracellular β-etherase from the wood-degrading white-rot basidiomycete Dichomitus squalens. This enzyme, Ds-GST1, from the glutathione-S-transferase superfamily selectively cleaved the β-O-4 aryl ether bond of a dimeric lignin model compound in a glutathione-dependent reaction. Ds-GST1 also demonstrated activity on polymeric synthetic lignin fractions, shown by a decrease in molecular weight distribution of the laccase-oxidized guaiacyl dehydrogenation polymer. In addition to a possible role of Ds-GST1 in intracellular catabolism of lignin-derived aromatic compounds, the cleavage of the most abundant linkages in lignin under mild reaction conditions makes this biocatalyst an attractive green alternative in biotechnological applications.
Summary
The ability to obtain carbon and energy is a major requirement to exist in any environment. For several ascomycete fungi, (post‐)genomic analyses have shown that species that occupy a large ...variety of habitats possess a diverse enzymatic machinery, while species with a specific habitat have a more focused enzyme repertoire that is well‐adapted to the prevailing substrate. White‐rot basidiomycete fungi also live in a specific habitat, as they are found exclusively in wood. In this study, we evaluated how well the enzymatic machinery of the white‐rot fungus Dichomitus squalens is tailored to degrade its natural wood substrate. The transcriptome and exoproteome of D. squalens were analyzed after cultivation on two natural substrates, aspen and spruce wood, and two non‐woody substrates, wheat bran and cotton seed hulls. D. squalens produced ligninolytic enzymes mainly at the early time point of the wood cultures, indicating the need to degrade lignin to get access to wood polysaccharides. Surprisingly, the response of the fungus to the non‐woody polysaccharides was nearly as good a match to the substrate composition as observed for the wood polysaccharides. This indicates that D. squalens has preserved its ability to efficiently degrade plant biomass types not present in its natural habitat.
•Decomposers show species-specific responses to climate variables and substrate quality.•Wood quality takes relative predominance over climate on fungal decomposition.•Climate and wood quality ...interactions modify the decomposer species response.
Any process that affects wood decomposition and decomposers in boreal forests may also affect the role that dead wood has on global carbon storages. We investigated under controlled laboratory conditions the impact of three major variables – temperature, humidity and wood quality – on Scots pine wood decomposition by four different fungal species. To reveal these effects, we conducted a nine-month factorial experiment. Wood quality was found to have a much more pronounced effect on fungal wood decay than climate variables. Furthermore, the fast-grown pine wood from managed forests decayed much faster than centuries old ‘kelo’ pine trees from natural forests as well as the slow-grown wood from managed forests. We found an overall increase in decomposition with temperature and humidity in Gloeophyllum protractum, except that the decay rate of the fast-grown wood declined with increasing temperature at higher humidity levels. The overall decomposition rates varied greatly with decayer species and wood type, and several interactions between temperature, humidity and wood quality effects were documented. In particular, we found that the fast decayers, Dichomitus squalens and Fomitopsis pinicola did not show any response to climate variables, but responded to wood quality only. The slow decayers Antrodia xantha and G. protractum responded to wood quality and interaction effects of climate and wood quality. Our results demonstrated species-specific effects of climate and wood quality when tested simultaneously, and show that it is critical to understand the different and complex mechanisms that affect wood decomposition and, consequently, carbon storages in forests, in order to increase the reliability of the climate-carbon prediction models.
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•Mechanisms of white-rot fungal plant polysaccharide degradation are mostly unknown.•White-rot fungus Dichomitus squalens shows cellobiose-based response to ...cellulose.•Pectin-polysaccharides trigger a broad targeted pectinolytic response in D. squalens.•Co-expression analysis reveals candidate regulators of polysaccharide degradation.•Plant polysaccharides induce complete transcriptional response in white-rot fungi.
Wood-degrading white-rot fungi can efficiently degrade all plant biomass components, but the molecular mechanisms behind the degradation of plant polysaccharides remain poorly understood. For example, the gene sets and expression levels induced by the plant polysaccharide-derived monosaccharides in white-rot fungi do not reflect those induced by crude plant biomass substrates. To explore the molecular response of the white-rot fungus Dichomitus squalens to plant-derived oligo- and polysaccharides, we investigated the transcriptomes from mono- and dikaryotic strains of the fungus on 10 substrates and compared the expression of carbohydrate-active enzyme-encoding genes to that previously reported for different monosaccharides and cellobiose. Our results revealed that in D. squalens, a robust response to cellulose leads to its effective depolymerization, with an orthologue of the ascomycete Trichoderma reesei ACE3 likely acting as a central transcriptional regulator. The conserved response between cellulose and cellobiose further confirms cellobiose as the main cellulase inducer in D. squalens. Surprisingly, despite low abundance of pectin in the natural wood substrate of D. squalens, we identified polygalacturonic acid as a major inducer of a broad-targeted pectinolytic response including pectinase, pectin-related sugar transporter and catabolism genes, and four fungal specific transcription factors. This indicates that D. squalens has not only maintained its ability to degrade minor polysaccharide components in its biotope, but also a regulatory system spanning from extracellular degradation to metabolic conversion. Our study contributes to a deeper understanding of the molecular mechanisms behind white-rot fungal plant polysaccharide degradation and provides leads for functional studies of potential transcriptional regulators in basidiomycetes.
The sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden or Tiger milk mushroom (Polyporales, Basidiomycota) is a valuable folk medicine for indigenous peoples in Southeast Asia. Despite the ...increasing interest in this ethnobotanical mushroom, very little is known about the molecular and genetic basis of its medicinal and nutraceutical properties.
The de novo assembled 34.3 Mb L. rhinocerotis genome encodes 10,742 putative genes with 84.30% of them having detectable sequence similarities to others available in public databases. Phylogenetic analysis revealed a close evolutionary relationship of L. rhinocerotis to Ganoderma lucidum, Dichomitus squalens, and Trametes versicolor in the core polyporoid clade. The L. rhinocerotis genome encodes a repertoire of enzymes engaged in carbohydrate and glycoconjugate metabolism, along with cytochrome P450s, putative bioactive proteins (lectins and fungal immunomodulatory proteins) and laccases. Other genes annotated include those encoding key enzymes for secondary metabolite biosynthesis, including those from polyketide, nonribosomal peptide, and triterpenoid pathways. Among them, the L. rhinocerotis genome is particularly enriched with sesquiterpenoid biosynthesis genes.
The genome content of L. rhinocerotis provides insights into the genetic basis of its reported medicinal properties as well as serving as a platform to further characterize putative bioactive proteins and secondary metabolite pathway enzymes and as a reference for comparative genomics of polyporoid fungi.
Endocrine-disrupting compounds (EDCs) represent a large group of substances of natural and anthropogenic origin. They are widely distributed in the environment and can pose serious risks to aquatic ...organisms and to public health. In this study, 4-n-nonylphenol, technical 4-nonylphenol, bisphenol A, 17α-ethinylestradiol, and triclosan were biodegraded by eight ligninolytic fungal strains (
Irpex lacteus 617/93,
Bjerkandera adusta 606/93,
Phanerochaete chrysosporium ME 446,
Phanerochaete magnoliae CCBAS 134/I,
Pleurotus ostreatus 3004 CCBAS 278,
Trametes versicolor 167/93,
Pycnoporus cinnabarinus CCBAS 595,
Dichomitus squalens CCBAS 750). The results show that under the used conditions the fungi were able to degrade the EDCs within 14
d of cultivation with exception of
B. adusta and
P. chrysosporium in the case of triclosane and bisphenol A, respectively.
I. lacteus and
P. ostreatus were found to be most efficient EDC degraders with their degradation efficiency exceeding 90% or 80%, respectively, in 7
d. Both fungi degraded technical 4-nonylphenol, bisphenol-A, and 17α-ethinylestradiol below the detection limit within first 3
d of cultivation. In general, estrogenic activities assayed with a recombinant yeast test decreased with advanced degradation. However, in case of
I. lacteus,
P. ostreatus, and
P. chrysosporium the yeast assay showed a residual estrogenic activity (28-85% of initial) in 17α-ethinylestradiol cultures. Estrogenic activity in
B. adusta cultures temporally increased during degradation of technical 4-nonylphenol, suggesting a production of endocrine-active intermediates. Attention was paid also to the effects of EDCs on the ligninolytic enzyme activities of the different fungi strains to evaluate their possible stimulation or suppression of activities during the biodegradation processes.
Lignocellulosic plant biomass is an important feedstock for bio-based economy. In particular, it is an abundant renewable source of aromatic compounds, which are present as part of lignin, as ...side-groups of xylan and pectin, and in other forms, such as tannins. As filamentous fungi are the main organisms that modify and degrade lignocellulose, they have developed a versatile metabolism to convert the aromatic compounds that are toxic at relatively low concentrations to less toxic ones. During this process, fungi form metabolites some of which represent high-value platform chemicals or important chemical building blocks, such as benzoic, vanillic, and protocatechuic acid. Especially basidiomycete white-rot fungi with unique ability to degrade the recalcitrant lignin polymer are expected to perform highly efficient enzymatic conversions of aromatic compounds, thus having huge potential for biotechnological exploitation. However, the aromatic metabolism of basidiomycete fungi is poorly studied and knowledge on them is based on the combined results of studies in variety of species, leaving the overall picture in each organism unclear. Dichomitus squalens is an efficiently wood-degrading white-rot basidiomycete that produces a diverse set of extracellular enzymes targeted for lignocellulose degradation, including oxidative enzymes that act on lignin. Our recent study showed that several intra- and extracellular aromatic compounds were produced when D. squalens was cultivated on spruce wood, indicating also versatile aromatic metabolic abilities for this species. In order to provide the first molecular level systematic insight into the conversion of plant biomass derived aromatic compounds by basidiomycete fungi, we analyzed the transcriptomes of D. squalens when grown with 10 different lignocellulose-related aromatic monomers. Significant differences for example with respect to the expression of lignocellulose degradation related genes, but also putative genes encoding transporters and catabolic pathway genes were observed between the cultivations supplemented with the different aromatic compounds. The results demonstrate that the transcriptional response of D. squalens is highly dependent on the specific aromatic compounds present suggesting that instead of a common regulatory system, fine-tuned regulation is needed for aromatic metabolism.
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