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.

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.

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.

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.

Medium optimization for enhanced production of recombinant lignin peroxidase in Pichia pastoris.

OBJECTIVES: Different cultivation conditions and parameters were evaluated to improve the production and secretion of a recombinant Phanerochaete chrysosporium lipH8 gene in Komagataella phaffii (Pichia pastoris). RESULTS: The recombinant lipH8 gene with its native secretion signal was successfully cloned and expressed in Komagataella phaffii (Pichia pastoris) under the control of the alcohol oxidase 1 promoter (PAOX1). The results revealed that co-feeding with sorbitol and methanol increased rLiP secretion by 5.9-fold compared to the control conditions. The addition of 1 mM FeSO4 increased LiP activity a further 6.0-fold during the induction phase. Moreover, the combination of several optimal conditions and parameters yielded an extracellular rLiP activity of 20.05 U l-1, which is more than ten-fold higher relative to standard growth conditions (BMM10 medium, pH 6 and 30 °C). CONCLUSION: Extracellular activity of a recombinant LiP expressed in P. pastoris increased more than ten-fold when co-feeding sorbitol and methanol as carbon sources, together with urea as nitrogen source, FeSO4 supplementation, lower pH and lower cultivation temperature.

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.

Latent potentials of the white-rot basidiomycete Phanerochaete chrysosporium responsible for sesquiterpene metabolism: CYP5158A1 and CYP5144C8 decorate (E)-α-bisabolene.

Basidiomycetes produce various sesquiterpenoids and their relevance for pharmaceutical and agricultural applications and understanding their biosynthetic machinery to produce these secondary metabolites have attracted significant interest. Because sesquiterpene synthases (STSs) and cytochrome P450 monooxygenases (P450s) play pivotal roles in the production of sesquiterpenoids, functional characterization of these enzymes is fundamentally essential. In this study, we found 11 possible STSs from the white-rot basidiomycete Phanerochaete chrysosporium (PcSTSs) and isolated nine of these as full-length cDNAs encoding a mature open reading frame. Using the isolated cDNAs, we performed heterologous expression of PcSTSs in Saccharomyces cerevisiae. Metabolic studies revealed that seven of the PcSTSs produce a series of sesquiterpene scaffolds, including (E)-α-bisabolene. Furthermore, we constructed a co-expression system of (E)-α-bisabolene synthase and P450 from P. chrysosporium (PcCYP). Semi-comprehensive screening using 120 isoforms of PcCYPs resulted in the identification of CYP5158A1 and CYP5144C8, two P450s capable of decorating (E)-α-bisabolene.

Expression and characterization of a family 45 glycosyl hydrolase from Fomitopsis pinicola and comparison to Phanerochaete chrysosporium Cel45A.

To efficiently decompose biomass, fungi have developed various enzymatic and non-enzymatic strategies and are a source of versatile biocatalysts. The endoglucanases in glycosyl hydrolase CAZy family 45 (GH45) are known for their small size, a high thermostability and a broad substrate specificity that has been employed in textile and detergent industries. Here we report the heterologous expression and characterisation of an GH45 endoglucanase from the brown rot Fomitopsis pinicola and its direct comparison to an already characterised GH45 from the white rot Phanerochaete chrysosporium. Both enzymes were recombinantly expressed in Pichia pastoris and purified by two chromatographic steps. The biochemical characterisation highlighted the acidophilic character, with an optimal pH of 4, and a preference for amorphous substrates as carboxymethyl cellulose (CMC) and substrates containing ß-1,4-glucans rather than the previously reported ß-1,3/1,4-glucans lichenan and ß-glucan. The dominating products from ß-1,4-glucans were C3-C6 oligosaccharides, whereas from ß-1,3/1,4-glucans glucose was the main reaction product. From the characterisation no differences between the brown rot and the white rot GH45 was evident.

Comparison of glycoside hydrolase family 3 ß-xylosidases from basidiomycetes and ascomycetes reveals evolutionarily distinct xylan degradation systems.

Xylan is the most common hemicellulose in plant cell walls, though the structure of xylan polymers differs between plant species. Here, to gain a better understanding of fungal xylan degradation systems, which can enhance enzymatic saccharification of plant cell walls in industrial processes, we conducted a comparative study of two glycoside hydrolase family 3 (GH3) ß-xylosidases (Bxls), one from the basidiomycete Phanerochaete chrysosporium (PcBxl3), and the other from the ascomycete Trichoderma reesei (TrXyl3A). A comparison of the crystal structures of the two enzymes, both with saccharide bound at the catalytic center, provided insight into the basis of substrate binding at each subsite. PcBxl3 has a substrate-binding pocket at subsite -1, while TrXyl3A has an extra loop that contains additional binding subsites. Furthermore, kinetic experiments revealed that PcBxl3 degraded xylooligosaccharides faster than TrXyl3A, while the KM values of TrXyl3A were lower than those of PcBxl3. The relationship between substrate specificity and degree of polymerization of substrates suggested that PcBxl3 preferentially degrades xylobiose (X2), while TrXyl3A degrades longer xylooligosaccharides. Moreover, docking simulation supported the existence of extended positive subsites of TrXyl3A in the extra loop located at the N-terminus of the protein. Finally, phylogenetic analysis suggests that wood-decaying basidiomycetes use Bxls such as PcBxl3 that act efficiently on xylan structures from woody plants, whereas molds use instead Bxls that efficiently degrade xylan from grass. Our results provide added insights into fungal efficient xylan degradation systems.

Ethanol fermentation by saprotrophic white-rot fungus Phanerochaete sordida YK-624 during wood decay as a system for short-term resistance to hypoxic conditions.

In this study, major factors involved in regulating ethanol production from wood by the saprotrophic white-rot fungus Phanerochaete sordida YK-624 were investigated. P. sordida YK-624 produced ethanol from wood meal culture without the addition of any nutrients, and ethanol was produced from wood culture only when the oxygen concentration in headspace was reduced to ≤5%; thereafter, ethanol production ceased within a few days. Analyses of gene expression during aerobic incubation indicated that P. sordida simultaneously upregulates the glycolytic pathway from sugar uptake to pyruvate conversion during ethanol fermentation and suppresses pyruvate influx into the TCA cycle. Upon termination of ethanol fermentation, the expression of all tested genes was repressed, and the fungus ceased to grow. In contrast, the fungus could utilize ethanol for aerobic growth. These results suggest that ethanol fermentation by P. sordida functions as a short-term stress response system under anaerobic conditions during wood decay, enabling the fungus to rapidly resume growing when oxygen is supplied (e.g., following breakdown of plant cell walls or removal of the fungus from water immersion). This is the first report to describe the physiologic significance of ethanol fermentation in saprotrophic white-rot fungi.

Transcriptomic analysis reveals ligninolytic enzymes of white-rot fungus Phanerochaete sordida YK-624 participating in bisphenol F biodegradation under ligninolytic conditions.

Bisphenol F (BPF) is widely used in the plastic manufacturing industry as a replacement for bisphenol A (BPA) because BPF and BPA have similar structures and comparable properties. However, BPF is ubiquitously present in the environment and has higher toxicity to humans. This study is the first to report BPF degradation using the white-rot fungus Phanerochaete sordida YK-624 under ligninolytic conditions (pH=4.5, 30 °C). P. sordida YK-624 almost completely degraded BPF within 4 days. Moreover, functional genes involved in BPF degradation were detected by RNA-Seq. Metabolic processes and peroxidases were enriched by GO analysis, and the metabolic pathway was enriched according to the KEGG pathway analysis. These results suggested that P. sordida YK-624 could secrete higher levels of ligninolytic enzymes lignin peroxidase (LiP) and manganese peroxidase (MnP) for BPF degradation. The results indicated that LiPs and MnPs are important for BPF degradation and cytochrome P450s play a small role. Furthermore, reliability of the RNA-Seq results was validated by qRT-PCR.

Transcriptomics analysis reveals the high biodegradation efficiency of white-rot fungus Phanerochaete sordida YK-624 on native lignin.

Lignocellulosic biomass is an organic matrix composed of cellulose, hemicellulose, and lignin. In nature, lignin degradation by basidiomycetes is the key step in lignocellulose decay. The white-rot fungus Phanerochaete sordida YK-624 (YK-624) has been extensively studied due to its high lignin degradation ability. It was demonstrated that YK-624 can secrete lignin peroxidase and manganese peroxidase for lignin degradation. However, the underlying mechanism for lignin degradation by YK-624 remains unknown. Here, we analyzed YK-624 gene expression following growth under ligninolytic and nonligninolytic conditions and compared the differentially expressed genes in YK-624 to those in the model white-rot fungus Phanerochaete chrysosporium by next-generation sequencing. More ligninolytic enzymes and lignin-degrading auxiliary enzymes were upregulated in YK-624. This might explain the high degradation efficiency of YK-624. In addition, the genes involved in energy metabolism pathways such as the TCA cycle, lipid metabolism, carbon metabolism and glycolysis were upregulated under ligninolytic conditions in YK-624. The first differential gene expression analysis of YK-624 under ligninolytic and nonligninolytic conditions was reported in this study. The results obtained in this study indicated that YK-624 produces more enzymes involved in lignin degradation and energy metabolism.

Isolation and modification of nano-scale cellulose from organosolv-treated birch through the synergistic activity of LPMO and endoglucanases.

Nanocellulose isolation from lignocellulose is a tedious and expensive process with high energy and harsh chemical requirements, primarily due to the recalcitrance of the substrate, which otherwise would have been cost-effective due to its abundance. Replacing the chemical steps with biocatalytic processes offers opportunities to solve this bottleneck to a certain extent due to the enzymes substrate specificity and mild reaction chemistry. In this work, we demonstrate the isolation of sulphate-free nanocellulose from organosolv pretreated birch biomass using different glycosyl-hydrolases, along with accessory oxidative enzymes including a lytic polysaccharide monooxygenase (LPMO). The suggested process produced colloidal nanocellulose suspensions (ζ-potential -19.4 mV) with particles of 7-20 nm diameter, high carboxylate content and improved thermostability (To = 301 °C, Tmax = 337 °C). Nanocelluloses were subjected to post-modification using LPMOs of different regioselectivity. The sample from chemical route was the least favorable for LPMO to enhance the carboxylate content, while that from the C1-specific LPMO treatment showed the highest increase in carboxylate content.

Comparative characterization of glyoxal oxidase from Phanerochaete chrysosporium expressed at high levels in Pichia pastoris and Trichoderma reesei.

In the secretome of Phanerochaete chrysosporium, a white-rot fungus serving as a model organism to elucidate lignocellulose deconstruction, the copper containing metalloprotein glyoxal oxidase (GLOX) is potentially involved in the crucial production of hydrogen peroxide to fuel and initiate oxidative biomass degradation by lignin-degrading peroxidases. Its ability to oxidize a variety of aldehydes and α-hydroxy carbonyls with the concomitant reduction of dioxygen to hydrogen peroxide has attracted attention for its application as green biocatalyst in different industrial fields. Here we report and compare two efficient processes for the heterologous production of GLOX from P. chrysosporium using the well-established methanolytic yeast Pichia pastoris and the filamentous fungus Trichoderma reesei as expression hosts with subsequent purification by anion exchange and hydrophobic interaction chromatography. Both processes were shown to be suitable for the production of the target protein at high levels. GLOX produced in T. reesei carries mainly Man5 glycosylation while the enzyme produced in P. pastoris exhibits the typical high-mannose type N-glycosylation. The enzyme expressed in P. pastoris showed slightly higher specific activities which correlates with the higher copper loading of 65.5 % compared to 51.9 % for the protein from T. reesei. The pH optimum for both recombinant proteins was 6.0, however, GLOX activity was found to be highly affected by different buffer species. Both enzymes showed very similar substrate affinities and turnover numbers with the highest catalytic efficiency observed for methylglyoxal. GLOX from both expression hosts is therefore a suitable enzyme for further mechanistic characterization and application studies.

Oxidized glutathione promotes association between eukaryotic translation elongation factor 1Bγ and Ure2p glutathione transferase from Phanerochaete chrysosporium.

The eukaryotic translation elongation factor 1Bγ (eEF1Bγ) is an atypical member of the glutathione transferase (GST) superfamily. Contrary to more classical GSTs having a role in toxic compound detoxification, eEF1Bγ is suggested to act as a scaffold protein, anchoring the elongation factor complex EF1B to the endoplasmic reticulum. In this study, we show that eEF1Bγ from the basidiomycete Phanerochaete chrysosporium is fully active as a glutathione transferase in vitro and undergoes conformational changes upon binding of oxidized glutathione. Using real-time analyses of biomolecular interactions, we show that GSSG allows eEF1Bγ to physically interact with other GSTs from the Ure2p class, opening new perspectives for a better understanding of the role of eEF1Bγ in cellular oxidative stress response.

White-rot fungus Phanerochaete chrysosporium metabolizes chloropyridinyl-type neonicotinoid insecticides by an N-dealkylation reaction catalyzed by two cytochrome P450s.

We previously identified a cytochrome P450 (CYP) derived from the white-rot fungus Phanerochaete chrysosporium as involved in degradation of acetamiprid, a neonicotinoid (NEO) insecticide. In the present study, we investigated biodegradation of other NEOs by P. chrysosporium, and attempted to identify the CYP enzyme responsible for NEO degradation. P. chrysosporium was able to degrade some NEOs (acetamiprid, clothianidin, imidacloprid, and thiacloprid) in nutrient-rich medium. Two CYPs in P. chrysosporium (PcCYPs), CYP5037B3 and CYP5147A3, were identified as major isozymes involved in metabolism of three neonicotinoids that have in common a chloropyridinyl moiety (acetamiprid, imidacloprid, and thiacloprid) by screening yeast that heterologously express PcCYPs. Both PcCYPs catalyzed cleavage of the chloropyridinyl moiety and side chain of the three NEOs by N-dealkylation, resulting in 6-chloro-3-pyridinemethanol and respective side chain fragments. In a culture of P. chrysosporium, 97 % and 74 % of imidacloprid and thiacloprid were modified to form degradation products, and one of these, 6-chloro-3-pyridinemethanol, was further degraded. These two PcCYPs catalyzed almost the same reaction but their substrate specificity and expression pattern are slightly different. Altogether, we found that P. chrysosporium degrades NEOs via the activity of at least two different CYP isozymes.

Biodegradation mechanisms of sulfonamides by Phanerochaete chrysosporium - Luffa fiber system revealed at the transcriptome level.

The overuse of antibiotics and subsequent enrichment of antibiotic resistant microbes in the natural and built environments is a severe threat to global public health. In this study, a Phanerochaete chrysosporium fungal-luffa fiber system was found to efficiently biodegrade two sulfonamides, sulfadimethoxine (SDM) and sulfadizine (SDZ), in cow urine wastewater. Biodegradation pathways were proposed on the basis of key metabolites identified using high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (HPLC-QqTOF-MS). Transcriptomic, metabolomic, and free radical analyses were performed to explore the functional groups and detailed molecular mechanisms of SDM and SDZ degradation. A total of 27 UniGene clusters showed significant differences between luffa fiber and luffa fiber-free systems, which were significantly correlated to cellulose catabolism, carbohydrate metabolism, and oxidoreductase activity. Carbohydrate-active enzymes and oxidoreductases appear to play particularly important roles in SDM and SDZ degradation. Electron paramagnetic resonance (EPR) spectroscopy revealed the generation and evolution of OH and R during the biodegradation of SDM and SDZ, suggesting that beyond enzymatic degradation, SDM and SDZ were also transformed through a free radical pathway. Luffa fiber also acts as a co-substrate to improve the activity of enzymes for the degradation of SDM and SDZ. This research provides a potential strategy for removing SDM and SDZ from agricultural and industrial wastewater using fungal-luffa fiber systems.

Algicidal Molecular Mechanism and Toxicological Degradation of Microcystis aeruginosa by White-Rot Fungi.

Current research on the inhibition of Microcystis aeruginosa growth is primarily focused on algae-lysing bacteria, and few studies have investigated the inhibitory mechanisms by which fungi affect it at the molecular level. A comparative analysis of the effects of Phanerochaete chrysosporium on the expression of the algal cell antioxidant protease synthesis gene prx, the biological macromolecule damage and repair genes recA, grpE, and fabZ, and the photosynthesis system-related genes psaB, psbD1 and rbcL, as well as genes for algal toxin synthesis mcyB, were performed to elucidate the molecular mechanism of Phanerochaete chrysosporium against Microcystis aeruginosa cells. RT-qPCR technology was used to study the molecular mechanism of algal cell inhibition by Phanerochaete chrysosporium liquid containing metabolites of Phanerochaete chrysosporium, Phanerochaete chrysosporium supernatant and Phanerochaete chrysosporium inactivated via high temperature sterilization at the gene expression level. Compared with the control, the chlorophyll-a contents dropped, and the recA, grpE, fabZ, and prx increased, but the psaB, psbD1, rbcL and mcyB showed that they were significantly reduced, which indicated that Phanerochaete chrysosporium can not only effectively destroy algal cells, but they may also reduce the expression of the Microcystis aeruginosa toxin gene and significantly block the metabolic system underlying the growth of algal cells and the synthesis of microcystins.

The 206 kbp mitochondrial genome of Phanerochaete carnosa reveals dynamics of introns, accumulation of repeat sequences and plasmid-derived genes.

In this study, the mitogenome of Phanerochaete carnosa was sequenced and assembled by the next-generation sequencing. The P. carnosa mitogenome was composed of circular DNA molecules, with a total size of 206,437 bp. Intron sequence, repeat sequence and plasmid-derived genes together promoted the P. carnosa mitogenome to become the second largest mitogenome in Basidiomycota. Gene arrangement analysis revealed large-scale gene rearrangements between Polyporales mitogenomes, and P. carnosa contained a unique gene order. The number and position classes of introns varied between 14 Polyporales species tested, indicated numerous intron loss/gain events occurred in the evolution of Polyporales. Most core PCGs in the 14 Polyporales species we tested were found subjected to purifying selection. However, the Ka/Ks values of rps3 gene were found >1 between some Polyporales species, indicating pressure of positive selection may exist. Phylogenetic analysis based on the combined mitochondrial gene set obtained well-supported tree topologies, and P. carnosa was identified as a sister species to Phlebia radiata. This study served as the first report on the mitogenome in the family Phanerochaetaceae, which will promote the understanding of the phylogeny, population genetics, and evolution of this white-rot fungus and related fungi.

Flow cytometry-based system for screening of lignin peroxidase mutants with higher oxidative stability.

Lignin peroxidase (LiP) is a heme-containing oxidoreductase that oxidizes structurally diverse substrates in an H2O2-dependent manner. Its ability to oxidize many pollutants makes it suitable for bioremediation applications and an ideal candidate for optimization by mutagenesis and selection. In order to increase oxidative stability of LiP we generated a random mutagenesis library comprising 106 mutated LiP genes and screened for expressed enzymes with higher than wild-type activity after incubation in 30 mM H2O2 by flow cytometry with fluorescein-tyramide as a substrate. To preserve the genotype-phenotype connection, the LiP mutants were displayed on the yeast cell surface. Two rounds of sorting were performed, recovered colonies were then screened in microtiter plates, and activity analysis revealed a significant increase in the percentage of cells expressing LiP variants with higher oxidative stability than wtLiP. Two rounds of sorting increased the proportion of more-stable variants from 1.4% in the original library to 52.3%. The most stable variants after two rounds of sorting featured between two and four mutations and retained up to 80% of initial activity after 1 h incubation in 30 mM H2O2. We for the first-time applied flow cytometry for screening of any ligninolytic peroxidase library. Obtained results suggest that developed system may be applied for improvement of industrially important characteristics of lignin peroxidase.