Biotransformation and detoxification of tetrabromobisphenol A by white-rot fungus Phanerochaete sordida YK-624.

The bulky phenolic compound tetrabromobisphenol A (TBBPA) is a brominated flame retardant used in a wide range of products; however, it diffuses into the environment, and has been reported to have toxic effects. Although it is well-known that white-rot fungi degrade TBBPA through ligninolytic enzymes, no other metabolic enzymes have yet been identified, and the toxicity of the reaction products and their risks have not yet been examined. We found that the white-rot fungus Phanerochaete sordida YK-624 converted TBBPA to TBBPA-O-ß-D-glucopyranoside when grown under non-ligninolytic-enzyme-producing conditions. The metabolite showed less cytotoxicity and mitochondrial toxicity than TBBPA in neuroblastoma cells. From molecular biological and genetic engineering experiments, two P. sordida glycosyltransferases (PsGT1c and PsGT1e) that catalyze the glycosylation of TBBPA were newly identified; these enzymes showed dramatically different glycosylation activities for TBBPA and bisphenol A. The results of computational analyses indicated that the difference in substrate specificity is likely due to differences in the structure of the substrate-binding pocket. It appears that P. sordida YK-624 takes up TBBPA, and reduces its cytotoxicity via these glycosyltransferases.

Pathway crosstalk between the central metabolic and heme biosynthetic pathways in Phanerochaete chrysosporium.

A comprehensive analysis to survey heme-binding proteins produced by the white-rot fungus Phanerochaete chrysosporium was achieved using a biotinylated heme-streptavidin beads system. Mitochondrial citrate synthase (PcCS), glyceraldehyde 3-phosphate dehydrogenase (PcGAPDH), and 2-Cys thioredoxin peroxidase (mammalian HBP23 homolog) were identified as putative heme-binding proteins. Among these, PcCS and PcGAPDH were further characterized using heterologously expressed recombinant proteins. Difference spectra of PcCS titrated with hemin exhibited an increase in the Soret absorbance at 414 nm, suggesting that the axial ligand of the heme is a His residue. The activity of PcCS was strongly inhibited by hemin with Ki oxaloacetate of 8.7 µM and Ki acetyl-CoA of 5.8 µM. Since the final step of heme biosynthesis occurred at the mitochondrial inner membrane, the inhibition of PcCS by heme is thought to be a physiological event. The inhibitory mode of the heme was similar to that of CoA analogues, suggesting that heme binds to PcCS at His347 at the AcCoA-CoA binding site, which was supported by the homology model of PcCS. PcGAPDH was also inhibited by heme, with a lower concentration than that for PcCS. This might be caused by the different location of these enzymes. From the integration of these phenomena, it was concluded that metabolic regulations by heme in the central metabolic and heme synthetic pathways occurred in the mitochondria and cytosol. This novel pathway crosstalk between the central metabolic and heme biosynthetic pathways, via a heme molecule, is important in regulating the metabolic balance (heme synthesis, ATP synthesis, flux balance of the tricarboxylic acid (TCA) cycle and cellular redox balance (NADPH production) during fungal aromatic degradation. KEY POINTS: • A comprehensive survey of heme-binding proteins in P. chrysosporium was achieved. • Several heme-binding proteins including CS and GAPDH were identified. • A novel metabolic regulation by heme in the central metabolic pathways was found.

Effect of Phanerochaete chrysosporium induced phosphate precipitation on bacterial diversity during the soil remediation process.

Biomineralization by phosphate minerals and phosphate solubilizing fungi (PSF) has attracted great interest as a novel remediation method for heavy metal(loid) co-contaminated soil. It was very essential to investigate the microenvironment response with the application of amendments. In this study, three grain sizes of hydroxyapatites (HAP) and Phanerochaete chrysosporium (P. chrysosporium) were used to investigate the change in heavy metal(loid) fractions, soil physicochemical properties, and bacterial community during the remediation of Mangchang and Dabaoshan acidic mine soils. The results showed that the residual fractions in the two soils increased significantly after 35 days of remediation, especially that of As and Zn in Dabaoshan soils were presented at over 87%. In addition, soil pH, organic matter (OM), and available phosphorous (AP) were almost improved. 16S rRNA sequencing analysis indicated that the introduction of culture medium and P. chrysosporium alone changed bacterial abundance, but the addition of HAP changed the bacterial diversity and community composition by altering environmental conditions. The amendments in the research showed good performance on immobilizing heavy metal(loid)s and reducing their bioavailability. Moreover, the research suggested that environmental factors and soil inherent properties could influence the microbial community structure and composition.

Identification and characterization of methoxy- and dimethoxyhydroquinone 1,2-dioxygenase from Phanerochaete chrysosporium.

White-rot fungi, such as Phanerochaete chrysosporium, are the most efficient degraders of lignin, a major component of plant biomass. Enzymes produced by these fungi, such as lignin peroxidases and manganese peroxidases, break down lignin polymers into various aromatic compounds based on guaiacyl, syringyl, and hydroxyphenyl units. These intermediates are further degraded, and the aromatic ring is cleaved by 1,2,4-trihydroxybenzene dioxygenases. This study aimed to characterize homogentisate dioxygenase (HGD)-like proteins from P. chrysosporium that are strongly induced by the G-unit fragment of vanillin. We overexpressed two homologous recombinant HGDs, PcHGD1 and PcHGD2, in Escherichia coli. Both PcHGD1 and PcHGD2 catalyzed the ring cleavage in methoxyhydroquinone (MHQ) and dimethoxyhydroquinone (DMHQ). The two enzymes had the highest catalytic efficiency (kcat/Km) for MHQ, and therefore, we named PcHGD1 and PcHGD2 as MHQ dioxygenases 1 and 2 (PcMHQD1 and PcMHQD2), respectively, from P. chrysosporium. This is the first study to identify and characterize MHQ and DMHQ dioxygenase activities in members of the HGD superfamily. These findings highlight the unique and broad substrate spectra of PcHGDs, rendering them attractive candidates for biotechnological applications.IMPORTANCEThis study aimed to elucidate the properties of enzymes responsible for degrading lignin, a dominant natural polymer in terrestrial lignocellulosic biomass. We focused on two homogentisate dioxygenase (HGD) homologs from the white-rot fungus, P. chrysosporium, and investigated their roles in the degradation of lignin-derived aromatic compounds. In the P. chrysosporium genome database, PcMHQD1 and PcMHQD2 were annotated as HGDs that could cleave the aromatic rings of methoxyhydroquinone (MHQ) and dimethoxyhydroquinone (DMHQ) with a preference for MHQ. These findings suggest that MHQD1 and/or MHQD2 play important roles in the degradation of lignin-derived aromatic compounds by P. chrysosporium. The preference of PcMHQDs for MHQ and DMHQ not only highlights their potential for biotechnological applications but also underscores their critical role in understanding lignin degradation by a representative of white-rot fungus, P. chrysosporium.

Effect of one or more microorganisms on the yield components of upland rice under greenhouse conditions.

The use of beneficial microorganisms is an important strategy to improve rice production in a sustainable way. The study was carried out to determine the effect of single and combined beneficial microorganism on the development of upland rice. The experiment was performed in greenhouse and arranged in a completely randomized design with 29 treatments and 4 replications. Treatments consisted of rice seeds cultivar BRS A501 CL treated with single and combined multifunctional microorganisms (1 (Serratia marcescens), 2 (Bacillus toyonensis), 3 (Phanerochaete australis), 4 (Trichoderma koningiopsis), 5 (Azospirillum brasilense), 6 (Azospirillum sp.), 7 (Bacillus sp.), 8 to 28 (combination of all these microorganisms in pairs) and 29 (control)). Inoculation of upland rice with sole and combined microorganism on upland rice increased the roots and shoots development, yield components and grain yield of upland rice. The combinations of Bacillus sp. (BRM 63573) and A. brasilense (AbV5), Azospirillum sp. (BRM 63574) + B. toyonensis (BRM 32110) and Phanerochaete australis (BRM 62389) + Serratia marcenscens (BRM 32114) led to improved roots and shoots development; increased number of panicles and grains per pot, 1000 grains weight and grain yield of rice plants. Besides, the combinations allow helped in increased accumulation of nutrients in roots, shoots and grains of rice plants.

Removal of perfluorooctanoic acid (PFOA) in the liquid culture of Phanerochaete chrysosporium.

Perfluorooctanoic acid (PFOA) is becoming a concern due to its persistence, bioaccumulation, and potential harmful effects on humans and the environment. In this study, the fungus Phanerochaete chrysosporium (P. chrysosporium) was used to remove the PFOA in liquid culture system. The results showed that the average activities of laccase (Lac), lignin peroxidase (LiP), and manganese peroxidase (MnP) enzymes secreted by P. chrysosporium were 0.0003 U/mL, 0.013 U/mL, and 0.0059 U/mL, respectively, during the incubation times of 0-75 days. The pH of 3 and incubation time of 45-55 days were the optimum parameters for the three enzymes activities. The enzyme activities in P. chrysosporium incubation system were firstly inhibited by adding PFOA and then they were enhanced after 14 days. The maximum removal efficiency of PFOA (69.23%) was achieved after 35 days in P. chrysosporium incubation system with an initial PFOA concentration of 0.002 mM and no veratryl alcohol (VA). Adsorption was not a main pathway for PFOA removal and the PFOA adsorbed in fungi mycelial mat accounted for merely 1.91%. The possible products of PFOA contained partially fluorinated aldehyde, alcohol, and aromatic ring. These partially fluorinated compounds might result from PFOA degradation via a combination of cross-coupling and rearrangement of free radicals.

Degradation of co-applied Atrazine and Fipronil in Phanerochaete Chrysosporium Augmented Biobeds.

White rot fungi possess an enzymatic system that is non-specific to any pesticide and can be used for pesticide detoxification in biobeds. The present study evaluated potential of Phanerochaete chrysosporium to degrade co-applied atrazine and fipronil in ash or biochar biomixtures. Five biomixtures were prepared by partially replacing compost in rice straw-compost biomixture (BM) with 10% rice husk ash (RHA), 10% sugarcane bagasse ash (SBA), and 1 and 5% wheat straw biochar (WBC). Results suggested that after 30 days P. chrysosporium augmented biobeds resulted in 60.52-72.72% atrazine and 69.57-72.52% fipronil degradation. Hydroxyatrazine and fipronil sulfone were detected as the only metabolite of atrazine and fipronil, respectively, and were further degraded. Although, SBA significantly enhanced atrazine degradation, RHA or SBA had no significant effect on fipronil degradation. WBC (5%) slowed down degradation of both pesticides.

Synergistic lignin degradation between Phanerochaete chrysosporium and Fenton chemistry is mediated through iron cycling and ligninolytic enzyme induction.

Removal of recalcitrant lignin from wastewater remains a critical bottleneck in multiple aspects relating to microbial carbon cycling ranging from incomplete treatment of biosolids during wastewater treatment to limited conversion of biomass feedstock to biofuels. Based on previous studies showing that the white rot fungus Phanerochaete chrysosporium and Fenton chemistry synergistically degrade lignin, we sought to determine optimum levels of Fenton addition and the mechanisms underlying this synergy. We tested the extent of degradation of lignin under different ratios of Fenton reagents and found that relatively low levels of H2O2 and Fe(II) enhanced fungal lignin degradation, achieving 80.4 ± 1.61 % lignin degradation at 1.5 mM H2O2 and 0.3 mM Fe(II). Using a combination of whole-transcriptome sequencing and iron speciation assays, we determined that at these concentrations, Fenton chemistry induced the upregulation of 80 differentially expressed genes in P. ch including several oxidative enzymes. This study underlines the importance of non-canonical, auxiliary lignin-degrading pathways in the synergy between white rot fungi and Fenton chemistry in lignin degradation. We also found that, relative to the abiotic control, P. ch. increases the availability of Fe(II) for the production of hydroxyl radicals in the Fenton reaction by recycling Fe(III) (p < 0.001), decreasing the Fe(II) inputs necessary for lignin degradation via the Fenton reaction.

Regulating pH and Phanerochaete chrysosporium inoculation improved the humification and succession of fungal community at the cooling stage of composting.

This study aimed to explore the effect of regulating pH and Phanerochaete chrysosporium inoculation at the cooling stage of composting on the lignocellulose degradation, humification process and related precursors as well as fungal community for secondary fermentation. Results showed that composting with P. chrysosporium inoculation and pH regulation (T4) had 58% cellulose decomposition, 73% lignin degradation and improved enzyme activities for lignin decomposition. There was 81.98% increase of humic substance content and more transformation of polyphenols and amino acids in T4 compared to control. Inoculating P. chrysosporium affected the fungal community diversity, and regulating pH helped to increase the colonization of P. chrysosporium. Network analysis showed that the network complexity and synergy between microorganisms was improved in T4. Correlation and Random Forest analysis suggested that enriched Phanerochaete and Thermomyces in the mature stage of T4 were key taxa for lignocellulose degradation, and humic acid formation by accumulating precursors.

Genomic and Secretomic Analyses of the Newly Isolated Fungus Perenniporia fraxinea SS3 Identified CAZymes Potentially Related to a Serious Pathogenesis of Hardwood Trees.

Perenniporia fraxinea can colonize living trees and cause severe damage to standing hardwoods by secreting a number of carbohydrate-activate enzymes (CAZymes), unlike other well-studied Polyporales. However, significant knowledge gaps exist in understanding the detailed mechanisms for this hardwood-pathogenic fungus. To address this issue, five monokaryotic P. fraxinea strains, SS1 to SS5, were isolated from the tree species Robinia pseudoacacia, and high polysaccharide-degrading activities and the fastest growth were found for P. fraxinea SS3 among the isolates. The whole genome of P. fraxinea SS3 was sequenced, and its unique CAZyme potential for tree pathogenicity was determined in comparison to the genomes of other nonpathogenic Polyporales. These CAZyme features are well conserved in a distantly related tree pathogen, Heterobasidion annosum. Furthermore, the carbon source-dependent CAZyme secretions of P. fraxinea SS3 and a nonpathogenic and strong white-rot Polyporales member, Phanerochaete chrysosporium RP78, were compared by activity measurements and proteomic analyses. As seen in the genome comparisons, P. fraxinea SS3 exhibited higher pectin-degrading activities and higher laccase activities than P. chrysosporium RP78, which were attributed to the secretion of abundant glycoside hydrolase family 28 (GH28) pectinases and auxiliary activity family 1_1 (AA1_1) laccases, respectively. These enzymes are possibly related to fungal invasion into the tree lumens and the detoxification of tree defense substances. Additionally, P. fraxinea SS3 showed secondary cell wall degradation capabilities at the same level as that of P. chrysosporium RP78. Overall, this study suggested mechanisms for how this fungus can attack the cell walls of living trees as a serious pathogen and differs from other nonpathogenic white-rot fungi. IMPORTANCE Many studies have been done to understand the mechanisms underlying the degradation of plant cell walls of dead trees by wood decay fungi. However, little is known about how some of these fungi weaken living trees as pathogens. P. fraxinea belongs to the Polyporales, a group of strong wood decayers, and is known to aggressively attack and fell standing hardwood trees all over the world. Here, we report CAZymes potentially related to plant cell wall degradation and pathogenesis factors in a newly isolated fungus, P. fraxinea SS3, by genome sequencing in conjunction with comparative genomic and secretomic analyses. The present study provides insights into the mechanisms of the degradation of standing hardwood trees by the tree pathogen, which will contribute to the prevention of this serious tree disease.

Recombinant Lignin Peroxidase with Superior Thermal Stability and Melanin Decolorization Efficiency in a Typical Human Skin-Mimicking Environment.

Recently, the desire for a safe and effective method for skin whitening has been growing in the cosmetics industry. Commonly used tyrosinase-inhibiting chemical reagents exhibit side effects. Thus, recent studies have focused on performing melanin decolorization with enzymes as an alternative due to the low toxicity of enzymes and their ability to decolorize melanin selectively. Herein, 10 different isozymes were expressed as recombinant lignin peroxidases (LiPs) from Phanerochaete chrysosporium (PcLiPs), and PcLiP isozyme 4 (PcLiP04) was selected due to its high stability and activity at pH 5.5 and 37 °C, which is close to human skin conditions. In vitro melanin decolorization results indicated that PcLiP04 exhibited at least 2.9-fold higher efficiency than that of well-known lignin peroxidase (PcLiP01) in a typical human skin-mimicking environment. The interaction force between melanin films measured by a surface forces apparatus (SFA) revealed that the decolorization of melanin by PcLiP04 harbors a disrupted structure, possibly interrupting π-π stacking and/or hydrogen bonds. In addition, a 3D reconstructed human pigmented epidermis skin model showed a decrease in melanin area to 59.8% using PcLiP04, which suggests that PcLiP04 exhibits a strong potential for skin whitening.

Roles of various enzymes in the biotransformation of 6:2 fluorotelomer alcohol (6:2 FTOH) by a white-rot fungus.

Six-carbon-chained polyfluoroalkyl substances, such as 6:2 fluorotelomer alcohol (6:2 FTOH), are being used to replace longer chained compounds in the manufacture of various commercial products. This study examined the effects of growth substrates and nutrients on specific intracellular and extracellular enzymes mediating 6:2 FTOH aerobic biotransformation by the white-rot fungus, Phanerochaete chrysosporium. Cellulolytic conditions with limited glucose were a suitable composition, resulting in high 5:3 FTCA yield (37 mol%), which is a key intermediate in 6:2 FTOH degradation without forming significant amounts of terminal perfluorocarboxylic acids (PFCAs). Sulfate and ethylenediaminetetraacetic acid (EDTA) were also essential for 5:3 FTCA production, but, at lower levels, resulted in the buildup of 5:2 sFTOH (52 mol%) and 6:2 FTUCA (20 mol%), respectively. In non-ligninolytic nutrient-rich medium, 45 mol% 6:2 FTOH was transformed but produced only 12.7 mol% 5:3 FTCA. Enzyme activity studies imply that cellulolytic conditions induce the intracellular cytochrome P450 system. In contrast, extracellular peroxidase synthesis is independent of 6:2 FTOH exposure. Gene expression studies further verified that peroxidases were relevant in catalyzing the downstream transformations from 5:3 FTCA. Collectively, the identification of nutrients and enzymatic systems will help elucidate underlying mechanisms and biogeochemical conditions favorable for fungal transformation of PFCA precursors in the environment.

Insights into characteristics of white rot fungus during environmental plastics adhesion and degradation mechanism of plastics.

Since the 1980s, plastic waste in the environment has been accumulating, and little is known about fungi biodegradation, especially in dry environments. Therefore, the research on plastic degradation technology is urgent. In this study, we demonstrated that Phanerochaete chrysosporium (P. chrysposporium), a typical species of white rot fungi, could react as a highly efficient biodegrader of polylactic acid (PLA), and 34.35 % of PLA degradation was obtained during 35-day incubation. A similar mass loss of 19.71 % could be achieved for polystyrene (PS) degradation. Here, we presented the visualization of the plastic deterioration process and their negative reciprocal on cell development, which may be caused by the challenge of using PS as a substrate. The RNA-seq analysis indicated that adaptations in energy metabolism and cellular defense were downregulated in the PS group, while lipid synthesis was upregulated in the PLA-treated group. Possible differentially expressed genes (DEG) of plastic degradation, such as hydrophobic proteins, lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Lac), Cytochrome P450 (CYP450), and genes involved in styrene or benzoic acid degradation pathways have been recorded, and we proposed a PS degradation pathway.

Exogenous microbial antagonism affects the bioaugmentation of humus formation under different inoculation using Trichoderma reesei and Phanerochaete chrysosporium.

This study was aimed at exploring the effect of antagonism of Trichoderma reesei (T.r) and Phanerochaete chrysosporium (P.c) on humification during fermentation of rice (RS) and canola straw (CS). Results showed that exogeneous fungi accelerated straw degradation and enzyme activities of CMCase, xylanase and LiP. P.c inhibited the activity of LiP when co-existing with T.r beginning, it promoted the degradation of lignin and further increased the production of humus-like substances (HLS) and humic-like acid (HLA) in later fermentation when nutrients were insufficient. The HLS of RTP was 54.9 g/kg RS, higher than the other treatments, and displayed more complex structure and higher thermostability. Brucella and Bacillus were the main HLA bacterial producers. P.c was the HLA fungal producer, while T.r assisted FLA and polyphenol transformation. Therefore, RTP was recommended to advance technologies converting crop straw into humus resources.

Loosenin-Like Proteins from Phanerochaete carnosa Impact Both Cellulose and Chitin Fiber Networks.

Microbial expansin-related proteins are ubiquitous across bacterial and fungal organisms and reportedly play a role in the modification and deconstruction of cell wall polysaccharides, including lignocellulose. So far, very few microbial expansin-related proteins, including loosenins and loosenin-like (LOOL) proteins, have been functionally characterized. Herein, four LOOLs encoded by Phanerochaete carnosa and belonging to different subfamilies (i.e., PcaLOOL7 and PcaLOOL9 from subfamily A and PcaLOOL2 and PcaLOOL12 from subfamily B) were recombinantly produced and the purified proteins were characterized using diverse cellulose and chitin substrates. The purified PcaLOOLs weakened cellulose filter paper and cellulose nanofibril networks (CNF); however, none significantly boosted cellulase activity on the selected cellulose substrates (Avicel and Whatman paper). Although fusing the family 63 carbohydrate-binding module (CBM63) of BsEXLX1 encoded by Bacillus subtilis to PcaLOOLs increased their binding to cellulose, the CBM63 fusion appeared to reduce the cellulose filter paper weakening observed using wild-type proteins. Binding of PcaLOOLs to alpha-chitin was considerably higher than that to cellulose (Avicel) and was pH dependent, with the highest binding at pH 5.0. Amendment of certain PcaLOOLs in fungal liquid cultivations also impacted the density of the cultivated mycelia. The present study reveals the potential of fungal expansin-related proteins to impact both cellulose and chitin networks and points to a possible biological role in fungal cell wall processing. IMPORTANCE The present study deepens investigations of microbial expansin-related proteins and their applied significance by (i) reporting a detailed comparison of diverse loosenins encoded by the same organism, (ii) considering both cellulosic and chitin-containing materials as targeted substrates, and (iii) investigating the impact of the C-terminal carbohydrate binding module (CBM) present in other expansin-related proteins on loosenin function. By revealing the potential of fungal loosenins to impact both cellulose and chitin-containing networks, our study reveals a possible biological and applied role of loosenins in fungal cell wall processing.

RNA-Seq analysis of Phanerochaete sordida YK-624 degrades neonicotinoid pesticide acetamiprid.

Acetamiprid (ACE) belongs to the group of neonicotinoid pesticides, which have become the most widely utilised pesticides around the world in the last two decades. The ability of Phanerochaete sordida YK-624 to degrade ACE under ligninolytic conditions has been demonstrated; however, the functional genes involved in ACE degradation have not been fully elucidated. In the present study, the differentially expressed genes of P. sordida YK-624 under ACE-degrading conditions and in the absence of ACE were elucidated by RNA sequencing (RNA-Seq). Based on the gene ontology enrichment results, the cell wall and cell membrane were significantly affected under ACE-degrading conditions. This result suggested that intracellular degradation of ACE might be mediated by this fungus. In addition, genes in metabolic pathways were the most enriched upregulated differentially expressed genes according to the KEGG pathway analysis. Eleven differentially expressed genes characterised as cytochrome P450s were upregulated, and these genes were determined to be particularly important for ACE degradation by P. sordida YK-624 under ligninolytic conditions.

Enhanced lignin degradation of paddy straw and pine needle biomass by combinatorial approach of chemical treatment and fungal enzymes for pulp making.

Paddy straw (PS) and pine needles (PN) are one of the challenging biomasses in terms of disposal and compost making due to their high silica and tannin contents. Particulate air pollution, loss of biodiversity and respiratory impairments are some of disastrous outcomes caused by burning. However, high percentage of cellulose and hemicellulose makes them potential substrate for paper and pulp industries. The main aim of work was to study and utilize a combinatorial approach of weak chemical treatment and lignin degrading fungal species as agents of effective production of lignin modifying enzymes (LME's) for lignin depolymerisation from the biomasses. Phanerochaete chrysosporium was found to be the best degrader of lignin (47.11 % in PS + PN in 28 days) with maximum LME's production between 10th-17th days. Efficient lignin degradation in the PS and PN biomass will aid further application in pulp production supporting the transition to a circular economy in a greener way.

Response of microbial community to the remediation of neonicotinoid insecticide imidacloprid contaminated wetland soil by Phanerochaete chrysosporium.

Imidacloprid (IMI), a typic neonicotinoid insecticide, is widely used and persist in soils with long half-time causing serious threat to ecosystem and human health. It is urgent to develop suitable and effective methods to accelerate it degradation and alleviate its negative impacts in soil. In this study, the introduction of functional microbe white-rot fungus Phanerochaete chrysosporium to remediate IMI contaminated wetland soil was carried out. The remediation performance and the response of the soil microbial community were examined. The results showed that P. chrysosporium could improve the degradation of IMI in soil no matter the soil was sterilized or not. The bioaugmentation was especially observed in non-sterilized soil under the inoculation patterns of FE and SP with the maximum IMI degradation rate of 91% and 93% in 7 days, respectively. The invertase activity in soil was also enhanced with P. chrysosporium inoculation. Microbial community analysis revealed that P. chrysosporium inoculation could increase the diversity and richness of bacterial community, and stimulate some IMI degraders genera including Ochrobactrum, Leifsonia, Achromobacter, and Bacillus. Moreover, the xenobiotic degradation and metabolism pathway was generally enhanced with P. chrysosporium inoculation based on PICRUSt analysis. These obtained results demonstrated that the introduction of white-rot fungus is of great potentially enabling the remediation of neonicotinoids contaminated soil.

Humification process enhancement through relative abundance promotion of Talaromyces and Coprinopsis by inoculated Phanerochaete chrysosporium during the secondary fermentation of composting.

To explore the mechanism of Phanerochaete chrysosporium (P. chrysosporium) inoculation driving the humification process of maize straw composting, the treatments without P. chrysosporium inoculation (T1) and that with P. chrysosporium inoculation (T2) were carried out separately during the secondary fermentation of the co-composting of maize straw and rapeseed cake. The key microorganisms were determined by evaluating the succession of the fungal community and its relationship with humification process parameters. The results showed that P. chrysosporium inoculation (T2) reduced fungal diversity but increased the relative abundance of Coprinopsis and Talaromyces. At the end of the composting (day 36), the relative abundance of Talaromyces and Coprinopsis in T2 increased by 1223.7% and 30.2%, respectively, compared with T1. Combined CCA and SEMs analyses demonstrated the microbially driven mechanisms that enhance the humification process of composting, that is, P. chrysosporium inoculation promoted lignin continuous degradation by promoting the relative abundance of Talaromyces and Coprinopsis during the secondary fermentation of composting; meanwhile, P. Chrysosporium inoculation further intensified the biological process of humification in composting.

Biochemical characterization of hydroquinone hydroxylase from Phanerochaete chrysosporium.

The white-rot fungus Phanerochaete chrysosporium can degrade lignin polymers using extracellular, non-specific, one-electron oxidizing enzymes. This results in the formation of guaiacyl (G), syringyl (S), and hydroxyphenyl (H) units, such as vanillic acid, syringic acid, and p-hydroxybenzoic acid (p-HBA) and the corresponding aldehydes, which are further metabolized intracellularly. Therefore, the aim of this study was to identify proteins involved in the hydroxylation of H-unit fragments such as p-HBA and its decarboxylated product hydroquinone (HQ) in P. chrysosporium. A flavoprotein monooxygenase (FPMO), PcFPMO2, was identified and its activity was characterized. Recombinant PcFPMO2 with an N-terminal polyhistidine tag was produced in Escherichia coli and purified. In the presence of NADPH, PcFPMO2 used six phenolic compounds as substrates. PcFPMO2 catalyzed the hydroxylation of the H-unit fragments such as p-HBA and HQ, and the G-unit derivative methoxyhydroquinone (MHQ). The highest catalytic efficiency (kcat/Km) was observed with HQ, indicating that PcFPMO2 could be involved in HQ hydroxylation in vivo. Additionally, PcFPMO2 converted MHQ to 3-, 5-, and 6-methoxy-1,2,4-trihydroxybenzene (3-, 5-, and 6-MTHB), respectively, suggesting that PcFPMO2 might partially be involved in MHQ degradation, following aromatic ring fission, via three MTHBs. FPMOs are divided into eight groups (groups A to H). This is the first study to show MHQ hydroxylase activity of a FPMO-group A superfamily member. These findings highlight the unique substrate spectrum of PcFPMO2, making it an attractive candidate for biotechnological applications.