Fungus Phanerochaete Sordida YK-624 Research Articles

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-β-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.

Construction of white-rot fungal-bacterial consortia with improved ligninolytic properties and stable bacterial community structure

It is believed that wood-rot fungi change their wood decay activities due to influences from co-existing bacterial communities; however, it is difficult to elucidate experimentally the interaction mechanisms in fungal-bacterial consortia because the bacterial community structure is quite unstable and readily changes. Indeed, the wood decay properties of fungal-bacterial consortia consisting of a white-rot fungus Phanerochaete sordida YK-624 and a natural bacterial community changed dramatically during several sub-cultivations on wood. Therefore, development of a sub-cultivation method that imparts stability to the bacterial community structure and fungal phenotype was attempted. The adopted method using agar medium enabled maintenance of fungal phenotypes relating to wood decay and the bacterial community even through dozens of repetitive sub-cultures. Some bacterial metabolic pathways identified based on gene predictions were screened as candidates involved in P. sordida–bacterial interactions. In particular, pathways related to prenyl naphthoquinone biosynthesis appeared to be involved in an interaction that promotes higher lignin degradation selectivity by the consortia, as naphthoquinone derivatives induced phenol-oxidizing activity. Based on these results, it is expected that detailed analyses of the relationship between the wood-degrading properties of white-rot fungal-bacterial consortia and bacterial community structures will be feasible using the sub-cultivation method developed in this study.

Biotransformation of bisphenol F by white-rot fungus Phanerochaete sordida YK-624 under non-ligninolytic condition.

Environmental bisphenol F (BPF) has a cyclic endocrine disruption effect, seriously threatening animal and human health. It is frequently detected in environmental samples worldwide. For BPF remediation, biological methods are more environmentally friendly than physicochemical methods. White-rot fungi have been increasingly studied due to their potential capability to degrade environmental pollutants. Phanerochaete sordida YK-624 has been shown to degrade BPF by ligninolytic enzymes under ligninolytic conditions. In the present study, degradation of BPF under non-ligninolytic conditions (no production of ligninolytic enzymes) was investigated. Our results showed that BPF could be completely removed after 7-d incubation. A metabolite of BPF, 4,4'-dihydroxybenzophenone (DHBP) was identified by mass spectrometry and nuclear magnetic resonance, and DHBP was further degraded by this fungus to form 4-hydroxyphenyl 4-hydroxybenzoate (HPHB). DHBP and HPHB were the intermediate metabolites of BPF and would be further degraded by P. sordida YK-624. We also found that cytochrome P450s played an important role in BPF degradation. Additionally, transcriptomic analysis further supported the involvement of these enzymes in the action of BPF degradation. Therefore, BPF is transformed to DHBP and then to HPHB likely oxidized by cytochrome P450s in P. sordida YK-624. Furthermore, the toxicological studies demonstrated that the order of endocrine-disrupting activity for BPF and its metabolites was HPHB > BPF > DHBP. KEY POINTS: • White-rot fungus Phanerochaete sordida YK-624 could degrade BPF. • Cytochrome P450s were involved in the BPF degradation. • The order of endocrine disrupting activity was: HPHB > BPF > DHBP.

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.

Draft Genome Sequence of the White-Rot Fungus Phanerochaete sordida YK-624.

ABSTRACTHere, we report the draft genome sequence of the white-rot basidiomycete fungus Phanerochaete sordida YK-624 (ATCC 90872), which was isolated in Yakushima, Kagoshima, Japan. The genome of this strain was found to be 41.2 Mbp, with a G+C content of 58.7%, and to comprise 17,108 predicted protein-coding sequences.

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 Phanerochaetechrysosporium 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.

Self-fusion and fusion cell isolation of transformants derived from white rot fungus Phanerochaete sordida YK-624 by simple visual method

In order to develop a simple method for crossing two transformants, we first attempted to elucidate the fusion type (self-compatibility or -incompatible) of Phanerochaete sordida YK-624. Two transformants expressing green or red fluorescent protein derived from an auxotrophic mutant were constructed. Each recombinant protein fluoresced by expression as a fused protein with glyceraldehyde-3-phosphate dehydrogenase. On co-culture of both transformants, a number of sequential hyphal cells emitting dual fluorescence were formed at the contact areas of both hyphae. Some of the single cells isolated as protoplasts and chlamydospore from the co-cultures also expressed these fluorescent proteins. These results suggest that P.sordida YK-624 possesses a self-compatible fusion system. In addition, transformant strains with different fluorescence derived from this fungus can readily undergo self-fusion and nuclear interchange events by confrontational and mixed cultivation, and we developed a simple method that allows fused cells to be isolated as chlamydospores.

Biotransformation and detoxification of the neonicotinoid insecticides nitenpyram and dinotefuran by Phanerochaete sordida YK-624

Neonicotinoid insecticides have been widely used throughout the world over the last two decades. In the present study, we investigated the degradation of neonicotinoid insecticides nitenpyram (NIT) and dinotefuran (DIN) by the white-rot fungus Phanerochaete sordida YK-624. While NIT was completely degraded by P. sordida YK-624 under ligninolytic conditions, only a 20% decrease was observed under nonligninolytic conditions. On the other hand, P. sordida YK-624 degraded 31% of DIN under ligninolytic conditions after a 20-day incubation, while it did not degrade DIN under nonligninolytic conditions. We found that cytochromes P450 played a key role in the biotransformation of NIT and DIN by P. sordida YK-624. A novel NIT metabolite (E)-N-((6-chloropyridin-3-yl)methyl)-N-ethyl-N′-hydroxy acetimidamide (CPMHA) and a novel DIN metabolite N-((4aS,7aS,E)-1-methylhexahydrofuro[2,3-d]pyrimidin-2(1H)-ylidene)nitramide (PHPF) were identified in this study. In addition, to evaluate neurotoxicity, the effects of NIT, DIN and their metabolites on the viability of human neuroblastoma cells SH-SY5Y were determined. PHPF showed higher neurological toxicity than DIN, whereas the metabolite of NIT, CPMHA, showed no toxic effect. Our results indicated that the neurological toxicity of NIT could be effectively removed by P. sordida YK-624.

Effect on growth, sugar consumption, and aerobic ethanol fermentation of homologous expression of the sugar transporter gene Pshxt1 in the white rot fungus Phanerochaete sordida YK-624

Major facilitator superfamily (MFS) transporters are found in all organisms. Although numerous studies have examined the functions of yeast and mold MFS transporters in terms of sugar affinity and metabolic regulation, no functional analyses of MFS sugar transporters in white rot fungi have been reported. This study identified an MFS sugar transporter gene (Pshxt1) of the white rot fungus Phanerochaete sordida YK-624 expressed in liquid culture containing low concentrations of nitrogen source. Homologous expression of Pshxt1 dramatically increased the rates of glucose, fructose, mannose, and xylose consumption. Galactose consumption increased slightly but significantly. These data suggest that Pshxt1 has broad affinity for monosaccharides. In contrast, a transformant homologously expressing Pshxt1 consumed glucose in preference to xylose in wood enzymatic-digestion liquor and liquid culture. Additionally, homologous expression of Pshxt1 improved mycelial growth, aerobic ethanol production, and simultaneous aerobic saccharification and fermentation efficiency, whereas secretion of the ligninolytic enzyme manganese peroxidase was clearly decreased in the presence of glucose by Pshxt1 expression. These results suggest that Pshxt1 is involved in the repression of ligninolytic enzyme activity via carbon catabolite repression at sufficiently high glucose concentrations for activation of primary metabolism.

Effects of Glucose Concentration on Ethanol Fermentation of White-Rot Fungus Phanerochaete sordida YK-624 Under Aerobic Conditions.

White-rot fungi are microorganisms capable of ethanol fermentation; however, the specific conditions activating ethanol fermentation are unclear in contrast to fermentation by yeasts. In this study, we investigated the conditions favoring ethanol fermentation by the white-rot fungus Phanerochaete sordida YK-624, which is able to produce ethanol from woody material. In aerobic stationary cultivation with various concentrations of glucose (0.8-33g/l), the fungus produced ethanol in media containing an initial glucose concentration of 2.8g/l or higher. The amount of glucose consumption, mycelial weight, and ethanol production on the second day of culture increased in a concentration-dependent manner at low glucose concentrations; however, these were saturated at high concentrations. Biomass yields (growth/glucose consumption) were decreased until the initial glucose concentration increased to 6.0g/l, after which the biomass yields showed constant values at higher concentrations (12-33g/l). On the other hand, ethanol yields increased with decreasing biomass yields. In short shaking cultivation using mycelial suspension, trace amounts of instantaneous aerobic ethanol production were observed with 1.1 and 2.1g/l glucose, but the relative gene expression levels of key enzymes at the pyruvate branch point showed no significant differences between ethanol production and non-production conditions. From these experimental results, it appears that the white-rot fungus P. sordida YK-624 produces ethanol due to overflow in sugar metabolism under aerobic conditions, although P. sordida YK-624 prioritizes glucose utilization for respiratory growth.

Functional characterization of the manganese transporter smf2 homologue gene, PsMnt, of Phanerochaete sordida YK-624 via homologous overexpression.

A homologue gene of the yeast natural resistance-associated macrophage protein (Nramp) family transporter smf2 was identified in the white-rot fungus Phanerochaete sordida YK-624. Relative expression levels of the homologue, designated PsMnt, were roughly equivalent in cultures containing 0 to 1000 μM Mn(II), a concentration non-toxic to the fungus. In the PsMnt-overexpressing mutant, cellular Mn accumulation and manganese peroxidase (MnP) activity increased significantly in 4-day cultures containing 10 μM MnSO4. Compared with the wild-type strain, MnP activity in the overexpressing mutants was higher at lower Mn concentrations (specifically 10-15 times higher). These results suggest that PsMnt is a high-affinity Mn transporter involved in cellular Mn accumulation under Mn-deficient conditions. This is the first report of an smf2 homologue in wood rot fungi.

Direct lactic acid production from beech wood by transgenic white-rot fungus Phanerochaete sordida YK-624

A lactic acid (LA)-producing strain of the hyper-lignin-degrading fungus Phanerochaete sordida YK-624 with the lactate dehydrogenase-encoding gene from Bifidobacterium longum (Blldh) was constructed. When the endogenous pyruvate decarboxylase gene-knocked down and Blldh-expressing transformant was cultured with beech wood meal, the transformant was able to successively delignify and ferment the substrate. Supplementation of calcium carbonate into the culture medium, significantly increased the level of LA accumulation. Direct LA production (at 0.29g/l) from wood was confirmed, and additional inclusion of exogenous cellulase in this fermentation yielded significant further improvement in LA accumulation (up to 1.44g/l). This study provides the first report of direct production of LA by fermentation from woody biomass by a single microorganism, and indicates that transgenic white-rot fungi have a potential use for development of simple/easy applications for wood biorefinery.

Improvement of ethanol production by recombinant expression of pyruvate decarboxylase in the white-rot fungus Phanerochaete sordida YK-624

To improve ethanol production by Phanerochaete sordida YK-624, the pyruvate decarboxylase (PDC) gene was cloned from and reintroduced into this hyper lignin-degrading fungus; the gene encodes a key enzyme in alcoholic fermentation. We screened 16 transformant P.sordida YK-624 strains that each expressed a second, recombinant PDC gene (pdc) and then identified the transformant strain (designated GP7) with the highest ethanol production. Direct ethanol production from hardwood was 1.41 higher with GP7 than with wild-type P.sordida YK-624. RT-PCR analysis indicated that the increased PDC activity was caused by elevated recombinant pdc expression. Taken together, these results suggested that ethanol production by P.sordida YK-624 can be improved by the stable expression of an additional, recombinant pdc.

Improving xylitol production through recombinant expression of xylose reductase in the white-rot fungus Phanerochaete sordida YK-624

We generated an expression construct consisting of the xylose reductase (XR) gene (xr) from Phanerochaete chrysosporium. Transformant X7 exhibited increased xylitol production and markedly higher XR activities than the wild-type strain. RT-PCR analysis demonstrated that the increased XR activity was associated with constant expression of the recombinant xr gene.

Metabolism of bisphenol A by hyper lignin-degrading fungus Phanerochaete sordida YK-624 under non-ligninolytic condition

Recently, we reported the conversion of bisphenol A (BPA) to 4-(2-(4-hydroxyphenyl)propan-2-yl)benzene-1,2-diol (hydroxy-BPA) by hyper lignin-degrading fungus Phanerochaete sordida YK-624 under non-ligninolytic condition. In the present study, the metabolism of hydroxy-BPA by P. sordida YK-624 was demonstrated under non-ligninolytic condition. Under these conditions, approximately 66% of hydroxy-BPA was degraded after 7 d of incubation. High-resolution electrospray ionization mass spectra and nuclear magnetic resonance analyses of the metabolites isolated from the culture broth indicated that hydroxy-BPA was metabolized to 4-(2-(4-hydroxyphenyl)propan-2-yl)-2-methoxyphenol (methoxy-BPA) and to 4-(2-(3,4-dimethoxyphenyl)propan-2-yl)phenol (dimethoxy-BPA) by sequential methylation events. These metabolites showed reduced estrogenic activity compared to BPA. These results suggested that the hydroxy BPA is methylated to two low toxic-methylation metabolites.

Hydroxylation of bisphenol A by hyper lignin-degrading fungus Phanerochaete sordida YK-624 under non-ligninolytic condition

Bisphenol A (BPA) is one of the representative compounds of the endocrine disrupting compounds group and the highest volume chemicals produced worldwide. As a result, BPA is often detected in many soil and water environments. In this study, we demonstrated the transformation of BPA from liquid cultures inoculated with hyper lignin-degrading fungus Phanerochaete sordida YK-624. Under non-ligninolytic condition, approximately 80% of BPA was eliminated after 7d of incubation. High-resolution electrospray ionization mass spectra and nuclear magnetic resonance analyses of a metabolite isolated from the culture supernatant suggested that BPA was metabolized to hydroxy-BPA, 4-(2-(4-hydroxyphenyl)propan-2-yl)benzene-1,2-diol, which has a much lower estrogenic activity than BPA. In addition, we investigated the effect of the cytochrome P450 inhibitor piperonyl butoxide (PB) on the hydroxylation of BPA, markedly lower transformation activity of BPA was observed in cultures containing PB. These results suggest that cytochrome P450 plays an important role in the hydroxylation of BPA by P. sordida YK-624 under non-ligninolytic condition.

Improvement of Manganese Peroxidase Production by the Hyper Lignin-Degrading Fungus Phanerochaete sordida YK-624 by Recombinant Expression of the 5-Aminolevulinic Acid Synthase Gene

The manganese peroxidase (MnP) gene (mnp4) promoter of Phanerochaete sordida YK-624 was used to drive expression of 5-aminolevulinic acid synthase (als), which is a key heme biosynthesis enzyme. The expression plasmid pMnP4pro-als was transformed into P. sordida YK-624 uracil auxotrophic mutant UV-64, and 14 recombinant als expressing-transformants were generated. Average cumulative MnP activities in the transformants were 1.18-fold higher than that of control transformants. In particular, transformants A-14 and A-61 showed significantly higher MnP activity (approximately 2.8-fold) than wild type. RT-PCR analysis indicated that the increased MnP activity was caused by elevated recombinant als expression. These results suggest that the production of MnP is improved by high expression of als.

Dimerization of Bisphenol A by Hyper Lignin-Degrading Fungus Phanerochaete sordida YK-624 Under Ligninolytic Condition

Bisphenol A (BPA) was treated with hyper lignin-degrading fungus Phanerochaete sordida YK-624 under ligninolytic condition. After preculturing P. sordida YK-624 for 4days, BPA (final concentration, 1 and 0.1mM) was added to cultures. Both 1- and 0.1-mM BPA were effectively decreased within a 24-h treatment and two metabolites were detected. Two metabolites (5,5'-bis-[1-(4-hydroxy-phenyl)1-methyl-ethyl]-biphenyl-2,2'-diol and 4-(2-(4-hydroxy-phenyl) propan-2-yl)-2-(4-(2-(4-hydroxyphenyl) propan-2-yl) phenoxy)phenol) were identified by ESI-MS and NMR analysis. These results indicated that BPA was oxidized to BPA phenoxy radicals by ligninolytic enzymes and then dimerized at extracellular region.

Improvement of ligninolytic properties in the hyper lignin-degrading fungus Phanerochaete sordida YK-624 using a novel gene promoter

We identified a highly expressed protein (BUNA2) by two-dimensional gel electrophoresis from the hyper lignin-degrading fungus Phanerochaete sordida YK-624 under wood-rotting conditions. Partial amino acid sequences of BUNA2 were determined by LC-MS/MS analysis, and BUNA2 gene (bee2) and promoter region were PCR-cloned and sequenced. The bee2 promoter was used to drive the expression of the manganese peroxidase gene (mnp4) in P. sordida YK-624. Eighteen mnp4-expressing clones were obtained, with most showing higher ligninolytic activity and selectivity than wild-type YK-624. Examination of the ligninolytic properties of the most effective lignin-degrading transformant, BM-65, cultured on wood meal revealed that this strain exhibited higher lignin degradation and MnP activities than those of wild type. Transcriptional analysis confirmed the increased expression of recombinant mnp4 in the transformant. These results indicate that use of the bee2 promoter to drive the expression of ligninolytic enzymes may be an effective approach for improving the lignin-degrading properties of white-rot fungi.