Secretome analysis of Phanerochaete chrysosporium strain CIRM-BRFM41 grown on softwood

Proteomic analysis was performed to determine and differentiate the composition of the secretomes of Phanerochaete chrysosporium CIRM-BRFM41, a peroxidase hypersecretory strain grown under ligninolytic conditions and on softwood chips under biopulping conditions. Extracellular proteins from both cultures were analyzed by bidimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. A total of 37 spots were identified. The secretome in liquid synthetic medium comprised mainly peroxidases, while several wood-degrading enzymes and enzymes involved in fungal metabolism were detected in biopulping cultures on softwood. This prompted an analysis of the impact of secretome modulation in the presence of softwood chips. Biotreated wood was submitted to kraft cooking and chemical bleaching using chlorine dioxide. The fungal pre-treatment led to a significant increase in pulp yield and a better bleachability of the pulp. This bleachability improvement could be explained by the production of specific lignocellulose-degrading enzymes.     

培养于天然冷杉木片的黄孢原毛平革菌木质素过氧化物酶基因表达的RT-PCR分析

利用RT-PCR方法分析了生长于冷杉木片上的黄孢原毛平革菌木质素过氧化物酶基因lipA2(GLG3)、lipC1(GLG2)、lipC2(GLG5)、lipD2(GLG1)、lipE(LPO811)的表达。结果发现在不同的培养时间里仅有特定的基因表达,在第2周时仅有lipA2(GLG3)基因表达,在第4周时未检测到任何基因的表达,在第6周时lipD2(GLG1)和lipC1(GLG2)基因表达,在第8周时仅有lipA2(GLG3)基因表达。这些结果说明,在冷杉木片上培养的黄孢原毛平革菌的lip基因表达具有明显的时间特异性,并且与限定培养基中得到的结果明显不同。     

Laccase and Its Role in Production of Extracellular Reactive Oxygen Species during Wood Decay by the Brown Rot Basidiomycete Postia placenta

Brown rot basidiomycetes initiate wood decay by producing extracellular reactive oxygen species that depolymerize the structural polysaccharides of lignocellulose. Secreted fungal hydroquinones are considered one contributor because they have been shown to reduce Fe³⁺, thus generating perhydroxyl radicals and Fe²⁺, which subsequently react further to produce biodegradative hydroxyl radicals. However, many brown rot fungi also secrete high levels of oxalate, which chelates Fe³⁺ tightly, making it unreactive with hydroquinones. For hydroquinone-driven hydroxyl radical production to contribute in this environment, an alternative mechanism to oxidize hydroquinones is required. We show here that aspen wood undergoing decay by the oxalate producer Postia placenta contained both 2,5-dimethoxyhydroquinone and laccase activity. Mass spectrometric analysis of proteins extracted from the wood identified a putative laccase (Joint Genome Institute P. placenta protein identification number 111314), and heterologous expression of the corresponding gene confirmed this assignment. Ultrafiltration experiments with liquid pressed from the biodegrading wood showed that a high-molecular-weight component was required for it to oxidize 2,5-dimethoxyhydroquinone rapidly and that this component was replaceable by P. placenta laccase. The purified laccase oxidized 2,5-dimethoxyhydroquinone with a second-order rate constant near 10⁴ M⁻¹ s⁻¹, and measurements of the H₂O₂ produced indicated that approximately one perhydroxyl radical was generated per hydroquinone supplied. Using these values and a previously developed computer model, we estimate that the quantity of reactive oxygen species produced by P. placenta laccase in wood is large enough that it likely contributes to incipient decay.Brown rot basidiomycetes are the principal recyclers of woody biomass in coniferous forest ecosystems and also the chief cause of decay in wooden structures (8, 41). Unlike the closely related white rot fungi, they degrade the cellulose and hemicellulose in wood while mineralizing little of the lignin that shields these structural polysaccharides from enzymatic attack. As a result, extensively brown-rotted wood consists primarily of an oxidized, partially cleaved residue derived from the original lignin (7, 16, 21, 22, 40). This failure to remove lignin efficiently suggests that brown rot systems contain fewer components than white rot systems and, in agreement, the recently published genome sequence of Postia placenta shows that this brown rot fungus lacks the ligninolytic peroxidases generally thought important for white rot (26). Instead, brown rot fungi appear to rely, at least during incipient decay, on small agents that can penetrate the lignin to access the polysaccharides (10, 14).A better understanding of the biodegradative agents produced by P. placenta may provide clues about what constitutes a minimally effective system for the microbial deconstruction of lignocellulose. One potential low-molecular-weight contributor is an extracellular metabolite, 2,5-dimethoxyhydroquinone (2,5-DMHQ), which has been found in cultures of P. placenta and other brown rot fungi and also shown to reduce Fe³⁺ with concomitant H₂O₂ production, thus producing hydroxyl radicals (^·OH) via the Fenton reaction (Fig. ​(Fig.1,1, reaction 6) (4, 17, 20, 29, 34, 36). Past work has shown that the chemical changes introduced by brown rot fungi into wood, cellulose, and other polymers are consistent with attack by reactive oxygen species (ROS) such as ^·OH (4, 7, 19, 21-23). A second small agent with a proposed role is extracellular oxalic acid, which P. placenta produces in sufficient quantity to acidify colonized wood to pH 2 to 4. Assays in vitro have shown that cellulose is slowly hydrolyzed at these acidities (11).Open in a separate windowFIG. 1.Chemical reactions discussed in the text. For simplicity, the HOO^·/O₂^·− acid/base pair is shown only as HOO^·. H₂Q, hydroquinone; HQ^·, semiquinone; Q, quinone.However, there is an apparent contradiction between these two mechanisms: oxalate is a strong chelator of Fe³⁺, and the resulting Fe³⁺ trioxalate complex has too negative a reduction potential to react readily with methoxyhydroquinones such as 2,5-DMHQ (28, 37). In considering this problem, we noted the surprising finding that the P. placenta genome encodes two putative laccases, enzymes that are considered atypical of brown rot fungi (26). Laccases oxidize methoxyhydroquinones to semiquinone radicals, which generally have more negative reduction potentials than their parent hydroquinones (38), and are therefore expected to be better reductants of Fe³⁺. In addition, methoxysemiquinones reduce O₂ to generate perhydroxyl radicals (HOO^·) and their conjugate base superoxide (O₂^·−), which dismutate to produce H₂O₂. Furthermore, HOO^·/O₂^·− can reduce some Fe³⁺ chelates to generate additional Fe²⁺ and can oxidize some Fe²⁺ chelates to generate additional H₂O₂ (9, 13, 33, 38). By these routes, a P. placenta laccase could bypass the requirement for the hydroquinone to react directly with Fe³⁺ and could thus generate a complete Fenton system (Fig. ​(Fig.1,1, reactions 1 to 7).Here we have expressed one of the P. placenta putative laccase genes heterologously and thus demonstrate that it encodes a typical laccase. In addition, we show that laccase activity and this particular enzyme are present in wood undergoing decay by P. placenta. Furthermore, we report that 2,5-DMHQ is present in the biodegrading wood, that it is a substrate for the P. placenta laccase, and that its oxidation during incipient wood decay requires a macromolecular component that is replaceable by P. placenta laccase. Finally, we show that the oxidation of 2,5-DMHQ by the P. placenta laccase results in significant H₂O₂ production, and we estimate that the quantity of ROS produced by this route is large enough that it probably contributes to incipient brown rot.     

Penetrability of White Rot-Degraded Pine Wood by the Lignin Peroxidase of Phanerochaete chrysosporium

The penetration of enzymes into wood cell walls during white rot decay is an open question. A postembedding immunoelectron microscopic technique was the method of choice to answer that question. Infiltration of pine wood specimens with a concentrated culture filtrate greatly improved the labeling density and, thereby, reproducibility. Characterization of the concentrated culture filtrate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) revealed three closely spaced proteins of molecular weights about 42,000 showing immunoreactivity against anti-lignin peroxidase serum. It was shown by immunogold labeling that lignin peroxidase of Phanerochaete chrysosporium is located on the surface of the wood cell wall or within areas of heavy attack. It did not diffuse into undecayed parts of the cell wall. The reasons for preventing lignin peroxidase from penetrating wood cell walls during white rot decay are discussed.     

Xylanase Activity of Phanerochaete chrysosporium

Xylan-degrading enzymes were induced when Phanerochaete chrysosporium was grown at 30°C in shake flask media containing xylan, Avicel PH 102, or ground corn stalks. The highest xylanase activity was produced in the corn stalk medium, while the xylan-based fermentation resulted in the lowest induction. Analytical and preparative isoelectric focusing were used to characterize xylanase multienzyme components. Preparative focusing was performed only with the cultures grown on Avicel and corn stalk. Of over 30 protein bands separated by analytical focusing from the Avicel and corn stalk media, three main groups (I, II, and III) of about five isoenzymes each showed xylanase activity when a zymogram technique with a xylan overlay was used. Enzyme assays revealed the presence of 1,4-β-endoxylanase and arabinofuranosidase activities in all three isoenzyme groups separated by preparative isoelectric focusing. β-Xylosidase activity appeared in the first peak and also as an independent peak between peaks II and III. Denatured molecular masses for the three isoenzyme groups were found to be between 18 and 90 kDa, and pI values were in the range of 4.2 to 6.0. β-Xylosidase has an apparent molecular mass of 20, 30, and 90 kDa (peak I) and 18 and 45 kDa (independent peak), indicating a trimer and dimer structure, respectively, with pI values of 4.2 and 5.78, respectively. Three more minor xylanase groups were produced on corn stalk medium: a double peak in the acidic range (pI 6.25 to 6.65 and 6.65 to 7.12) and two minor peaks in the alkaline range (pI 8.09 to 8.29 and 9.28 to 9.48, respectively). The profile of xylanases separated by isoelectric focusing (zymogram) of culture filtrate from cells grown on corn stalk media was more complex than that of culture supernatants from cells grown on cellulose. The pH optima of the three major xylanase groups are in the range of pH 4 to 5.5.     

Enzyme Immunoassay of Herbicide Decomposition by Soil and Wood Decay Fungi

The effect of herbicide atrazine was studied on the growth and development of a number of soil and wood decay fungi: white-rot basidiomycetes (Cerrena maxima, Coriolopsis fulvocenerea, and Coriolus hirsutus), thermophilic micromycetes from self-heating grass composts (cellulolytic fungus Penicilliumsp. 13 and noncellulolytic ones Humicola lanuginosaspp. 5 and 12), and mesophilic phenol oxidase-producing micromycete Mycelia sterilia INBI 2-26. Detection of atrazine in liquid fungal cultures was performed by using the enzyme immune assay technique. Both stimulation (Humicola lanuginosa 5) and suppression (Humicola lanuginosa 12 and Penicillium sp. 13) of fungal growth with atrazine were observed on solid agar media. HyphomyceteMycelia sterilia INBI 2-26 was almost insensitive to the presence of atrazine. Neither of the thermophilic strains was capable of atrazine consumption in three-week cultivation. In contrast with that, active laccase producers Cerrena maxima, Coriolopsis fulvocenerea, and Coriolus hirsutus consumed up to 50% atrazine in 5-day cultivation in the presence of the xenobiotic and at least 80–92% in 40 days. Mycelia steriliaINBI 2-26, which also forms extracellular laccase, also consumed up to 70% atrazine in 17 days. The degree of atrazine consumption depended on the term of its addition to the fungal culture medium.     

Chloromethane, Methyl Donor in Veratryl Alcohol Biosynthesis in Phanerochaete chrysosporium and Other Lignin-Degrading Fungi

Chloromethane, a gaseous natural product implicated in methylation processes in Phellinus pomaceus, has been shown to act as methyl donor in veratryl alcohol biosynthesis in the lignin-degrading fungi Phanerochaete chrysosporium, Phlebia radiata, and Coriolus versicolor, none of which released detectable amounts of CH(3)Cl during growth. When P. chrysosporium was grown in a medium containing CH(3)Cl, levels of CH(3) incorporation into the 3- and 4-O-methyl groups of veratryl alcohol were very high and initially similar to those observed when the medium was supplemented with l-[methyl-H(3)]methionine. When CH(3)Cl was added to cultures actively synthesizing veratryl alcohol, incorporation of CH(3) was very rapid, with 81% of veratryl alcohol labeled after 12 h. By contrast, incorporation of CH(3) from l-[methyl-H(3)]methionine was comparatively slow, attaining 10% after 12 h. It is proposed that these lignin-degrading fungi possess a tightly channeled multienzyme system in which CH(3)Cl biosynthesis is closely coupled to CH(3)Cl utilization for methylation of veratryl alcohol precursors.     

Involvement of an Extracellular Glucan Sheath during Degradation of Populus Wood by Phanerochaete chrysosporium

Observations by transmission electron microscopy of wood samples of Populus tremula inoculated with the white rot fungus Phanerochaete chrysosporium showed that, at certain stages of their growth cycle, hyphae were encapsulated by a sheath which seems to play an active role in the wood cell wall degradation. Chemical and immunochemical techniques and C nuclear magnetic resonance spectroscopy were applied to demonstrate the beta-1,3-1,6-d-glucan nature of the sheath. Double-staining methods revealed the interaction between the extracellular peroxidases involved in lignin degradation and the glucan mucilage. The glucan was also shown to establish a material junction between the fungus and the wood cell wall. It was concluded that, by means of these interactions, the sheath provides a transient junction between the hyphae and the wood, thus establishing a point of attachment to the site of the degradation. The association of peroxidases to the glucan matrix is in favor of the role of the sheath as a supporting structure. Furthermore, that the sheath was hydrolyzed during the attack demonstrated its active role both in providing the H(2)O(2) necessary to the action of peroxidases and in providing a mode of transport of the fungal enzymes to their substrates at the surface of the wood cell wall.     

Impact of Phanerochaete chrysosporium on the Functional Diversity of Bacterial Communities Associated with Decaying Wood

Bacteria and fungi naturally coexist in various environments including forest ecosystems. While the role of saprotrophic basidiomycetes in wood decomposition is well established, the influence of these fungi on the functional diversity of the wood-associated bacterial communities has received much less attention. Based on a microcosm experiment, we tested the hypothesis that both the presence of the white-rot fungus Phanerochaete chrysosporium and the wood, as a growth substrate, impacted the functional diversity of these bacterial communities. Microcosms containing sterile sawdust were inoculated with a microbial inoculum extracted from a forest soil, in presence or in absence of P. chrysosporium and subsequently, three enrichment steps were performed. First, bacterial strains were isolated from different microcosms previously analyzed by 16S rRNA gene-based pyrosequencing. Strains isolated from P. chrysosporium mycosphere showed less antagonism against this fungus compared to the strains isolated from the initial forest soil inoculum, suggesting a selection by the fungus of less inhibitory bacterial communities. Moreover, the presence of the fungus in wood resulted in a selection of cellulolytic and xylanolytic bacterial strains, highlighting the role of mycospheric bacteria in wood decomposition. Additionally, the proportion of siderophore-producing bacteria increased along the enrichment steps, suggesting an important role of bacteria in iron mobilization in decaying-wood. Finally, taxonomic identification of 311 bacterial isolates revealed, at the family level, strong similarities with the high-throughput sequencing data as well as with other studies in terms of taxonomic composition of the wood-associated bacterial community, highlighting that the isolated strains are representative of the wood-associated bacterial communities.     

Production of Phanerochaete chrysosporium lignin peroxidase

Liginin peroxidase (ligninase) of the white rot fungus Phanerochaete chrysosporium Burdsall was discovered in 1982 as a secondary metabolite. Today multiple isoenzymes are known, which are often collectively called as lignin peroxidase. Lignin peroxidase has been characterized as a veratryl alcohol oxidizing enzyme, but it is a relatively unspecific enzyme catalyzing a variety of reactions with hydrogen peroxide as the electron acceptor. P. chrysosporium ligninases are heme glycoproteins. At least a number of isoenzymes are also phosphorylated. Two of the major isoenzymes have been crystallized. Until recently lignin peroxidase could only be produced in low yields in very small scale stationary cultures owing to shear sensitivity. Most strains produce the enzyme only after grown under nitrogen or carbon limitation, although strains producing lignin peroxidase under nutrient sufficiency have also been isolated. Activities over 2000 U dm(-3) (as determined at 30 degrees to 37 degrees C) have been reported in small scale Erlenmeyer cultures with the strain INA-12 grown on glycerol in the presence of soybean phospholipids under nitrogen sufficiency. In about 8 dm(3) liquid volume pilot scale higher than 100 U dm(-3) (as determined at 23 degrees C) have been obtained under agitation with immobilized P. chrysosporium strains ATCC 24725 or TKK 20512. Good results have been obtained for example with nylon web, polyurethane foam, sintered glass or silicon tubing as the carrier. The immobilized biocatalyst systems have also made large scale repeated batch and semicontinuous production possible. With nylon web as the carrier, lignin peroxidase production has recently been scaled up to 800 dm(3) liquid volume semicontinuous industrial production process.     

Metabolism of cyanide by Phanerochaete chrysosporium

The oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) by lignin peroxidase H2 (LiP H2) from the white rot fungus Phanerochaete chrysosporium was strongly inhibited by sodium cyanide. The I50 was estimated to be about 2-3 microM. In contrast, sodium cyanide binds to the native enzyme with an apparent sodium cyanide dissociation constant Kd of about 10 microM. Inhibition of the veratryl alcohol oxidase activity of LiP H2 by cyanide was reversible. Ligninolytic cultures of P. chrysosporium mineralized cyanide at a rate that was proportional to the concentration of cyanide to 2 mM. The N-tert-butyl-alpha-phenylnitrone-cyanyl radical adduct was observed by ESR spin trapping upon incubation of LiP H2 with H2O2 and sodium cyanide. The identity of the spin adduct was confirmed using 13C-labeled cyanide. Six-day-old cultures of the fungus were more tolerant to sodium cyanide toxicity than spores. Toxicity measurements were based on the effect of sodium cyanide on respiration of the fungus as determined by the metabolism of [14C]glucose to [14C]CO2. We propose that this tolerance of the mature fungus was due to its ability to mineralize cyanide and that this fungus might be effective in treating environmental pollution sites contaminated with cyanide.     

Effects of Nitrogen Supplements on Degradation of Aspen Wood Lignin and Carbohydrate Components by Phanerochaete chrysosporium

A supplement of KH₂PO₄, MgSO₄, CaCl₂, trace elements, and thiamine accelerated the initial rate of aspen wood decay by Phanerochaete chrysosporium but did not increase the extent of lignin degradation. Asparagine, casein hydrolysate, and urea supplements (1% added N) strongly inhibited lignin degradation and weight loss. The complex nitrogen sources peptone and yeast extract stimulated lignin degradation and weight loss. Albumen and NH₄Cl had intermediate effects. Conversion of [¹⁴C]lignin to ¹⁴CO₂ and water-soluble materials underestimated lignin degradation in the presence of the complex N sources. The highest ratio of lignin degradation to total weight loss and the largest increase in cellulase digestibility occurred during the decay of unsupplemented wood. Rotting of aspen wood by P. chrysosporium gives smaller digestibility increases than have been found with some other white-rot fungi.     

Improved Colorimetric Determination of Cell Wall Chitin in Wood Decay Fungi

The Svennerholm modification of the Elson-Morgan method for glucosamine analysis was evaluated for its applicability to the rapid determination of chitin in wood decay fungi. The evaluation included extent of chromogen interference, sensitivity, color stability, and hydrolysis conditions for maximum release of glucosamine from fungal cell walls. With our further modification, the Svennerholm method was shown to be suitable for rapid quantitative determination of fungal chitin without chromatographic separation of hydrolysate chromogens.     

Biocatalytic decolourisation of molasses by Phanerochaete chrysosporium

Bioremediation potential of Phanerochaete chrysosporium strains NCIM 1073, NCIM 1106 and NCIM 1197 to decolourise molasses in solid and liquid molasses media was studied. Strains varied in the pattern of molasses decolourisation on solid medium by Giant colony method. Under submerged cultivation conditions, strain NCIM 1073 did not decolourise molasses while, strains NCIM 1106 and NCIM 1197 could decolourise molasses up to 82% and 76%, respectively. Under stationary cultivation conditions, none of the strains could decolourise molasses. This was overcome by increasing the surface area of the culture in flat bottom glass bottles under stationary cultivation conditions. Under submerged cultivation conditions, growth was more or less same in all strains. However, the lignin peroxidase and manganese peroxidase activities were significantly less in the strain NCIM 1073. Under stationary cultivation conditions, none of the strains could produce enzymes lignin peroxidase, manganese peroxidase and laccase. However, all of them could produce lignin peroxidase and manganese peroxidase when cultivated in flat bottom glass bottles under stationary cultivation conditions.