The lignin-degrading abilities of Gelatoporia subvermispora gat1 and pex1 mutants generated via CRISPR/Cas9.

White-rot fungi efficiently degrade wood lignin; however, the mechanisms involved remain largely unknown. Recently, a forward genetics approach to identify several genes in Pleurotus ostreatus (Agaricales) in which mutations cause defects in wood lignin degradation was used. For example, pex1 encodes a peroxisome biogenesis factor and gat1 encodes a putative Agaricomycetes-specific DNA-binding transcription factor. In this study, we examined the effects of single-gene mutations in pex1 or gat1 on wood lignin degradation in another white-rot fungus, Gelatoporia (Ceriporiopsis) subvermispora (Polyporales), to investigate conserved and derived degradation mechanisms in white-rot fungi. G. subvermispora pex1 and gat1 single-gene mutant strains were generated from a monokaryotic wild-type strain, FP-90031-Sp/1, using plasmid-based CRISPR/Cas9. As in P. ostreatus, Gsgat1 mutants were nearly unable to degrade lignin sourced from beech wood sawdust medium (BWS), while Gspex1 mutants exhibited a delay in lignin degradation. We also found that the transcripts of lignin-modifying enzyme-encoding genes, mnp4, mnp5, mnp6, mnp7, and mnp11, which predominantly accumulate in FP-90031-Sp/1 cultured with BWS, were greatly downregulated in Gsgat1 mutants. Taken together, the results suggest that Gat1 may be a conserved regulator of the ligninolytic system of white-rot fungi and that the contribution of peroxisomes to the ligninolytic system may differ among species.

CRISPR/Cas9 using a transient transformation system in Ceriporiopsis subvermispora.

Ceriporiopsis subvermispora is a white-rot fungus with great potential for industrial and biotechnological applications, such as the pretreatment of lignocellulose in biorefineries, as it decomposes the lignin in the plant cell wall without causing severe cellulose degradation. A genetic transformation system was recently developed; however, gene-targeting experiments to disrupt or modify the gene(s) of interest remain challenging, and this is a bottleneck for further molecular genetic studies and breeding of C. subvermispora. Herein, we report efficient clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted gene mutagenesis in this fungus. Two plasmids expressing Cas9 together with a different pyrG-targeting single-guide RNA were separately introduced into the monokaryotic C. subvermispora strain FP-90031-Sp/1, which frequently generated strains that exhibited resistance to 5-fluoroorotic acid and uridine/uracil auxotrophy. Southern blot analyses and genomic polymerase chain reaction followed by DNA sequencing of some mutants revealed that they were pyrG mutants. We also observed that hygromycin resistance of the pyrG mutants was frequently lost after repeated subcultivations, indicating that a maker-free genome editing occurred successfully. It is also suggested that a gene mutation(s) can be introduced via a transient expression of Cas9 and a single-guide RNA; this feature, together with high-frequency gene targeting using the CRISPR/Cas9 system, would be helpful for studies on lignocellulose-degrading systems in C. subvermispora. KEY POINTS: • Efficient plasmid-based CRISPR/Cas9 was established in C. subvermispora. • The mutations can be introduced via a transient expression of Cas9 and sgRNA. • A maker-free CRISPR/Cas9 is established in this fungus.

Characterization of the effects of terminators and introns on recombinant gene expression in the basidiomycete Ceriporiopsis subvermispora.

Terminators and introns are vital regulators of gene expression in many eukaryotes; however, the functional importance of these elements for controlling gene expression in Agaricomycetes remains unclear. In this study, the effects of Ceriporiopsis subvermispora terminators and introns on the expression of a recombinant hygromycin B phosphotransferase gene (hph) were characterized. Using a transient transformation system, we proved that a highly active terminator (e.g., the gpd terminator) is required for the efficient expression of the hph gene. Mutational analyses of the C. subvermispora gpd terminator revealed that hph expression was dictated by an A-rich region, which included a putative positioning element, and polyadenylation sites. In contrast, our results indicated that introns are not required for the expression of hph directed by the Csß1-tub and Csgpd promoters in C. subvermispora. This study provides insights into the functions and cis-element requirements of transcriptional terminators in Agaricomycetes, which may be relevant for designing recombinant genes for this important fungal class.

The white-rot fungus pleurotus ostreatus transformant overproduced intracellular cAMP and laccase.

Transformation of Pleurotus ostreatus PC9 with the mutated heterotrimeric G protein alpha subunit (Gα) gene resulted in higher laccase (Lac) activity and intracellular cyclic adenosine monophosphate (cAMP) concentrations as compared to those in wild-type PC9. The transformant also exhibited higher Lac activity than the wild type when cultured in a medium containing known Lac inducers CuSO4 and ferulic acid.

A calmodulin inhibitor, W-7 influences the effect of cyclic adenosine 3', 5'-monophosphate signaling on ligninolytic enzyme gene expression in Phanerochaete chrysosporium.

The capacity of white-rot fungi to degrade wood lignin may be highly applicable to the development of novel bioreactor systems, but the mechanisms underlying this function are not yet fully understood. Lignin peroxidase (LiP) and manganese peroxidase (MnP), which are thought to be very important for the ligninolytic property, demonstrated increased activity in Phanerochaete chrysosporium RP-78 (FGSC #9002, ATCC MYA-4764™) cultures following exposure to 5 mM cyclic adenosine 3', 5'-monophosphate (cAMP) and 500 µM 3'-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that transcription of most LiP and MnP isozyme genes was statistically significantly upregulated in the presence of the cAMP and IBMX compared to the untreated condition. However, 100 µM calmodulin (CaM) inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which had insignificant effects on fungal growth and intracellular cAMP concentration, not only offset the increased activity and transcription induced by the drugs, but also decreased them to below basal levels. Like the isozyme genes, transcription of the CaM gene (cam) was also upregulated by cAMP and IBMX. These results suggest that cAMP signaling functions to increase the transcription of LiP and MnP through the induction of cam transcription.

Absolute configuration of ceriporic acids, the iron redox-silencing metabolites produced by a selective lignin-degrading fungus, Ceriporiopsis subvermispora.

Ceriporic acids are a class of alk(en)ylitaconic acids produced by a selective lignin-degrading fungus, Ceriporiopsis subvermispora. The unique function of alkylitaconic acid is the redox silencing of the Fenton reaction system by inhibiting reduction of Fe(3+). Ceriporic acids have an asymmetric centre at carbon-3, but absolute configuration has not been determined. We have isolated a series of ceriporic acids from the cultures of C. subvermispora, and measured their NMR spectra using a chiral shift reagent. In comparison with NMR spectra of (R)-(-)- and (S)-(+)-methylsuccinic acid and those of natural and chemically synthesized racemic mixtures of ceriporic acids, we have determined the absolute configuration of ceriporic acids as (R)-3-tetradecylitaconic acid (ceriporic acid A), (R)-3-hexadecylitaconic acid (ceriporic acid B) and (R,Z)-2-(hexadec-7-enyl)-3-itaconic acid (ceriporic acid C). We herein discuss their stereoselective biosynthetic pathway and the structural diversity of fungal secondary metabolites.

Redox silencing of the Fenton reaction system by an alkylitaconic acid, ceriporic acid B produced by a selective lignin-degrading fungus, Ceriporiopsis subvermispora.

The selective lignin-degrading fungus, Ceriporiopsis subvermispora secretes alkylitaconic acids (ceriporic acids) during wood decay. We reported that ceriporic acid B (hexadecylitaconic acid) was protective against the depolymerization of cellulose by the Fenton reaction. To understand the redox silencing effects, we analyzed the physicochemical and redox properties of itaconic, octylitaconic and hexadecylitaconic acids. The initial rate of HO production by the Fenton system with Fe(3+), H(2)O(2) and L-cysteine was suppressed by hexadecylitaconic and octylitaconic acids by 0.04 and 0.16 of the reaction rate without chelators. ESR, O(2) uptake and the assay of Fe(2+) with BPS demonstrated that Fe(3+) reduction by L-cysteine was suppressed by hexadecylitaconic and octylitaconic acids while the reaction of Fe(2+) with H(2)O(2) was not suppressed by the two alkylitaconic acids. Ligand exchange experiments with NTA demonstrated that Fe(3+) chelation by two carboxyl groups of alkylitaconic acids is a critical step in iron redox modulation. In stark contrast, the production of HO* and reduction of Fe(3+) were not suppressed by itaconic acid due to HO*--initiated degradation of the chelator. The strong redox silencing effects by a series of alkylitaconic acids have attracted interest in controlling microbial plant cell wall degradation and chemoprotection against cellular oxidative injury.

Microbial scission of sulfide linkages in vulcanized natural rubber by a white rot basidiomycete, Ceriporiopsis subvermispora.

A white rot basidiomycete, Ceriporiopsis subvermispora, degraded vulcanized natural rubber (NR) sheets on a wood medium. The fungus decreased the total sulfur content of the rubber by 29% in 200 days, accompanied by the cleavage of sulfide bonds between polyisoprene chains. X-ray photoelectron spectroscopy (XPS) demonstrated that C. subvermispora reduced the frequency of S-C bonds by 69% with a concomitant formation of S-O bonds during the culture period. Dipolar decoupling/magic angle spinning (DD/MAS) solid state 13C NMR revealed that the fungus preferentially decomposed monosulfide bonds linked to a cis- and trans-1,4-isoprene backbone but the cleavage of polysulfide bonds was also observed. In contrast, no decrease in weight or devulcanization of rubber was observed in cultures of a white rot fungus, Dichomitus squalens. The oxidative cleavage of sulfide bonds by C. subvermispora demonstrates that ligninolytic basidiomycetes are potential microbes for the biological devulcanization of rubber products.

Chemical synthesis, iron redox interactions and charge transfer complex formation of alkylitaconic acids from Ceriporiopsis subvermispora.

In 1999, we first reported that a white rot fungus, Ceriporiopsis subvermispora produced a series of novel alkylitaconic acids (ceriporic acids). In the present paper we synthesized the metabolite, 1-nonadecene-2,3-dicarboxylic acid (ceriporic acid B) by Grignard reaction to analyze chemical properties of the alkylitaconates. Mass spectrometer (MS) and nuclear magnetic resonance (NMR) spectra of the synthetic compound was identical to those of the fungal metabolite isolated. The dicarboxylic acid inhibited autoxidation of Fe(2+) to Fe(3+) as well as reduction of Fe(3+) to Fe(2+) by the strong natural reductants, cysteine, glutathione, and ascorbic acid. The formation of charge transfer complexes (CTCs) between 1-heptadecene-2,3-dicarboxylic acid and oxidized intermediates from phenolic substrates were also observed. Thus, we herein report that the new class of lipid-related metabolites produced by C. subvermispora are potential metabolites participating in the control of iron redox reactions and CTCs formation from oxidized lignin fragments.

A selective lignin-degrading fungus, Ceriporiopsis subvermispora, produces alkylitaconates that inhibit the production of a cellulolytic active oxygen species, hydroxyl radical in the presence of iron and H(2)O(2).

A cellulolytic active oxygen species, hydroxyl radicals (.OH), play a leading role in the erosion of wood cell walls by brown-rot and non-selective white-rot fungi. In contrast, selective white-rot fungi have been considered to possess unknown systems for the suppression of .OH production due to their wood decay pattern with a minimum loss of cellulose. In the present paper, we first report that 1-nonadecene-2,3-dicarboxylic acid, an alkylitaconic acid (ceriporic acid B) produced by the selective white-rot fungus Ceriporiopsis subvermispora intensively inhibited .OH production by the Fenton reaction by direct interaction with Fe ions, while non-substituted itaconic acid promoted the Fenton reaction. Suppression of the Fenton reaction by the alkylitaconic acid was observed even in the presence of the Fe(3+) reductants, cysteine and hydroquinone. The inhibition of .OH production by the diffusible fungal metabolite accounts for the extracellular system of the fungus that attenuates the formation of .OH in the presence of iron, molecular oxygen, and free radicals produced during lignin biodegradation.