Identification of two mutations that cause defects in the ligninolytic system through an efficient forward genetics in the white‐rot agaricomycete Pleurotus ostreatus

White‐rot fungi play an important role in the global carbon cycle because they are the species that almost exclusively biodegrade wood lignin in nature. Lignin peroxidases (LiPs), manganese peroxidases (MnPs) and versatile peroxidases (VPs) are considered key players in the ligninolytic system. Apart from LiPs, MnPs and VPs, however, only few other factors involved in the ligninolytic system have been investigated using molecular genetics, implying the existence of unidentified elements. By combining classical genetic techniques with next‐generation sequencing technology, they successfully showed an efficient forward genetics approach to identify mutations causing defects in the ligninolytic system of the white‐rot fungus Pleurotus ostreatus. In this study, they identified two genes – chd1 and wtr1 – mutations in which cause an almost complete loss of Mn²⁺‐dependent peroxidase activity. The chd1 gene encodes a putative chromatin modifier, and wtr1 encodes an agaricomycete‐specific protein with a putative DNA‐binding domain. The chd1‐1 mutation and targeted disruption of wtr1 hamper the ability of P. ostreatus to biodegrade wood lignin. Examination of the effects of the aforementioned mutation and disruption on the expression of certain MnP/VP genes suggests that a complex mechanism underlies the ligninolytic system in P. ostreatus.     

Predominance of a versatile-peroxidase-encoding gene, mnp4, as demonstrated by gene replacement via a gene targeting system for Pleurotus ostreatus

Pleurotus ostreatus (the oyster mushroom) and other white rot filamentous basidiomycetes are key players in the global carbon cycle. P. ostreatus is also a commercially important edible fungus with medicinal properties and is important for biotechnological and environmental applications. Efficient gene targeting via homologous recombination (HR) is a fundamental tool for facilitating comprehensive gene function studies. Since the natural HR frequency in Pleurotus transformations is low (2.3%), transformed DNA is predominantly integrated ectopically. To overcome this limitation, a general gene targeting system was developed by producing a P. ostreatus PC9 homokaryon Δku80 strain, using carboxin resistance complemented by the development of a protocol for hygromycin B resistance protoplast-based DNA transformation and homokaryon isolation. The Δku80 strain exhibited exclusive (100%) HR in the integration of transforming DNA, providing a high efficiency of gene targeting. Furthermore, the Δku80 strains produced showed a phenotype similar to that of the wild-type PC9 strain, with similar growth fitness, ligninolytic functionality, and capability of mating with the incompatible strain PC15 to produce a dikaryon which retained its resistance to the corresponding selection and was capable of producing typical fruiting bodies. The applicability of this system is demonstrated by inactivation of the versatile peroxidase (VP) encoded by mnp4. This enzyme is part of the ligninolytic system of P. ostreatus, being one of the nine members of the manganese-peroxidase (MnP) gene family, and is the predominantly expressed VP in Mn(2+)-deficient media. mnp4 inactivation provided a direct proof that mnp4 encodes a key VP responsible for the Mn(2+)-dependent and Mn(2+)-independent peroxidase activity under Mn(2+)-deficient culture conditions.     

Experimental evidence that lignin-modifying enzymes are essential for degrading plant cell wall lignin by Pleurotus ostreatus using CRISPR/Cas9

Lignin-modifying enzymes (LMEs), which include laccases (Lacs), manganese peroxidases (MnPs), versatile peroxidases (VPs), and lignin peroxidases (LiPs), have been considered key factors in lignin degradation by white-rot fungi because they oxidize lignin model compounds and depolymerize synthetic lignin in vitro. However, it remains unclear whether these enzymes are essential/important in the actual degradation of natural lignin in plant cell walls. To address this long-standing issue, we examined the lignin-degrading abilities of multiple mnp/vp/lac mutants of Pleurotus ostreatus. One vp2/vp3/mnp3/mnp6 quadruple-gene mutant was generated from a monokaryotic wild-type strain PC9 using plasmid-based CRISPR/Cas9. Also, two vp2/vp3/mnp2/mnp3/mnp6, two vp2/vp3/mnp3/mnp6/lac2 quintuple-gene mutants, and two vp2/vp3/mnp2/mnp3/mnp6/lac2 sextuple-gene mutants were generated. The lignin-degrading abilities of the sextuple and vp2/vp3/mnp2/mnp3/mnp6 quintuple-gene mutants on the Beech wood sawdust medium reduced drastically, but not so much for those of the vp2/vp3/mnp3/mnp6/lac2 mutants and the quadruple mutant strain. The sextuple-gene mutants also barely degraded lignin in Japanese Cedar wood sawdust and milled rice straw. Thus, this study presented evidence that the LMEs, especially MnPs and VPs, play a crucial role in the degradation of natural lignin by P. ostreatus for the first time.     

Involvement of ligninolytic enzymes and Fenton-like reaction in humic acid degradation by Trametes sp

Trametes sp. M23, isolated from biosolids compost was found to decompose humic acids (HA). A low N (LN) medium (C/N, 53) provided suitable conditions for HA degradation, whereas in a high N (HN) medium (C/N, 10), HA was not degraded. In the absence of Mn²⁺, HA degradation was similar to that in Mn²⁺-containing medium. In contrast, MnP activity was significantly affected by Mn²⁺. Laccase activity exhibited a negative correlation to HA degradation, while LiP activity was not detected. Thus, ligninolytic enzymes activity could provide only a partial explanation for the HA-degradation mechanism. The decolorization of two dyes, Orange II and Brilliant Blue R250, was also determined. Similar to HA degradation, under LN conditions, decolorization occurred independently of the presence of Mn²⁺. We investigated the possible involvement of a Fenton-like reaction in HA degradation. The addition of DMSO, an OH-radical scavenger, to LN media resulted in a significant decrease in HA bleaching. The rate of extracellular Fe³⁺ reduction was much higher in the LN vs. HN medium. In addition, the rate of reduction was even higher in the presence of HA in the medium. In vitro HA bleaching in non-inoculated media was observed with H₂O₂ amendment to a final concentration of 200 mM (obtained by 50 mM amendments for 4 days) and Fe²⁺ (36 mM). After 4 days of incubation, HA decolorization was similar to the biological treatment. These results support our hypothesis that a Fenton-like reaction is involved in HA degradation by Trametes sp. M23.     

Lignin-degrading peroxidases from genome of selective ligninolytic fungus Ceriporiopsis subvermispora

The white-rot fungus Ceriporiopsis subvermispora delignifies lignocellulose with high selectivity, but until now it has appeared to lack the specialized peroxidases, termed lignin peroxidases (LiPs) and versatile peroxidases (VPs), that are generally thought important for ligninolysis. We screened the recently sequenced C. subvermispora genome for genes that encode peroxidases with a potential ligninolytic role. A total of 26 peroxidase genes was apparent after a structural-functional classification based on homology modeling and a search for diagnostic catalytic amino acid residues. In addition to revealing the presence of nine heme-thiolate peroxidase superfamily members and the unexpected absence of the dye-decolorizing peroxidase superfamily, the search showed that the C. subvermispora genome encodes 16 class II enzymes in the plant-fungal-bacterial peroxidase superfamily, where LiPs and VPs are classified. The 16 encoded enzymes include 13 putative manganese peroxidases and one generic peroxidase but most notably two peroxidases containing the catalytic tryptophan characteristic of LiPs and VPs. We expressed these two enzymes in Escherichia coli and determined their substrate specificities on typical LiP/VP substrates, including nonphenolic lignin model monomers and dimers, as well as synthetic lignin. The results show that the two newly discovered C. subvermispora peroxidases are functionally competent LiPs and also suggest that they are phylogenetically and catalytically intermediate between classical LiPs and VPs. These results offer new insight into selective lignin degradation by C. subvermispora.     

Production of versatile peroxidase from Pleurotus eryngii by solid-state fermentation using agricultural residues and evaluation of its catalytic properties

Interest in production of ligninolytic enzymes has been growing over recent years for their use in various applications such as recalcitrant pollutants bioremediation; specifically, versatile peroxidase (VP) presents a great potential due to its catalytic versatility. The proper selection of the fermentation mode and the culture medium should be an imperative to ensure a successful production by an economic and available medium that favors the process viability. VP was produced by solid-state fermentation (SSF) of Pleurotus eryngii, using the agricultural residue banana peel as growth medium; an enzymatic activity of 10,800 U L^?1 (36 U g^?1 of substrate) was detected after 18 days, whereas only 1800 U L^?1 was reached by conventional submerged fermentation (SF) with glucose-based medium. The kinetic parameters were determined by evaluating the H₂O₂ and Mn²⁺ concentration effects on the Mn³⁺-tartrate complex formation. The results indicated that although the H₂O₂ inhibitory effect was observed for the enzyme produced by both media, the reaction rates for VP obtained by SSF were less impacted. This outcome suggests the presence of substances released from banana peel during the fermentation, which might exhibit a protective effect resulting in an improved kinetic behavior of the enzyme.     

Cloning and Functional Analysis of <Emphasis Type="Italic">SaCLCc1</Emphasis>, a Gene Belonging to the Chloride Channel Family (<Emphasis Type="Italic">CLC</Emphasis>), from the Halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis> (L.) Pall.

One of the genes of the CLC (Chloride Channel) family, SaCLCc1, from the halophyte Suaeda altissima (L.) Pall. was cloned. To investigate the function of SaCLCc1, it was expressed in the S. cerevisiae deletion mutant Δgef1::LEU2 for the only gene of the CLC family in this organism. The growth of the transformed SaCLCc1-expressing mutant Δgef1 was restored when cells were grown in Fe²⁺-deficient YPEG medium, in minimal synthetic media SD and SR (pH 7.0), and in rich YPD medium containing Mn²⁺. The complementation of the Δgef1 mutant phenotype with the SaClCc1 gene indicates the involvement of the SaClCc1 protein in the transport of Cl^– ions.     

Rapid decolorization of azo dyes by crude manganese peroxidase from Schizophyllum sp. F17 in solid-state fermentation

The production of ligninolytic enzymes by the fungus Schizophyllum sp. F17 using a cost-effective medium comprised of agro-industrial residues in solid-state fermentation (SSF) was optimized. The maximum activities of the enzymes manganese peroxidase (MnP), laccase (Lac), and lignin peroxidases (LiP) were 1,200, 586, and 109 U/L, respectively, on day 5 of SSF. In vitro decolorization of three structurally different azo dyes by the extracellular enzymes was monitored to determine its decolorization capability. The results indicated that crude MnP, but not LiP and Lac, played a crucial role in the decolorization of azo dyes. After optimization of the dye decolorization system with crude MnP, the decolorization rates of Orange IV and Orange G, at an initial dye concentration of 50 mg/L, were enhanced to 76 and 57%, respectively, after 20 min of reaction at pH 4 and 35°C. However, only 8% decolorization of Congo red was observed. This enzymatic reaction system revealed a rapid decolorization of azo dyes with a low MnP activity of 24 U/L. Thus, this study could be the basis for the production and application of MnP on a larger scale using a low-cost substrate.     

Transformation of Industrial Dyes by Manganese Peroxidases from Bjerkandera adusta and Pleurotus eryngii in a Manganese-Independent Reaction

We investigated the transformation of six industrial azo and phthalocyanine dyes by ligninolytic peroxidases from Bjerkandera adusta and other white rot fungi. The dyes were not oxidized or were oxidized very little by Phanerochaete chrysosporium manganese peroxidase (MnP) or by a chemically generated Mn³⁺-lactate complex. Lignin peroxidase (LiP) from B. adusta also showed low activity with most of the dyes, but the specific activities increased 8- to 100-fold when veratryl alcohol was included in the reaction mixture, reaching levels of 3.9 to 9.6 U/mg. The B. adusta and Pleurotus eryngii MnP isoenzymes are unusual because of their ability to oxidize aromatic compounds like 2,6-dimethoxyphenol and veratryl alcohol in the absence of Mn²⁺. These MnP isoenzymes also decolorized the azo dyes and the phthalocyanine complexes in an Mn²⁺-independent manner. The reactions with the dyes were characterized by apparent K_m values ranging from 4 to 16 μM and specific activities ranging from 3.2 to 10.9 U/mg. Dye oxidation by these peroxidases was not increased by adding veratryl alcohol as it was in LiP reactions. Moreover, the reaction was inhibited by the presence of Mn²⁺, which in the case of Reactive Black 5, an azo dye which is not oxidized by the Mn³⁺-lactate complex, was found to act as a noncompetitive inhibitor of dye oxidation by B. adusta MnP1.     

Directed evolution of a temperature-, peroxide- and alkaline pH-tolerant versatile peroxidase

The VPs (versatile peroxidases) secreted by white-rot fungi are involved in the natural decay of lignin. In the present study, a fusion gene containing the VP from Pleurotus eryngii was subjected to six rounds of directed evolution, achieving a level of secretion in Saccharomyces cerevisiae (21?mg/l) as yet unseen for any ligninolytic peroxidase. The evolved variant for expression harboured four mutations and increased its total VP activity 129-fold. The signal leader processing by the STE13 protease at the Golgi compartment changed as a consequence of overexpression, retaining the additional N-terminal sequence Glu-Ala-Glu-Ala that enhanced secretion. The engineered N-terminally truncated variant displayed similar biochemical properties to those of the non-truncated counterpart in terms of kinetics, stability and spectroscopic features. Additional cycles of evolution raised the T50 8°C and significantly increased the enzyme's stability at alkaline pHs. In addition, the Km for H2O2 was enhanced up to 15-fold while the catalytic efficiency was maintained, and there was an improvement in peroxide stability (with half-lives for H2O2 of 43?min at a H2O2/enzyme molar ratio of 4000:1). Overall, the directed evolution approach described provides a set of strategies for selecting VPs with improvements in secretion, activity and stability.     

A new strain of <Emphasis Type="Italic">Bjerkandera</Emphasis> sp. production,purification and characterization of versatile peroxidase

The lignin modifying enzymes (LMEs) secreted by a new white rot fungus isolated from Chile were studied in this work. This fungus has been identified as a new anamorph of Bjerkandera sp. based on the sequences of the ribosomal DNA and morphological analysis at light microscopy showing hyaline hyphae without clamp connection, cylindrical conidia and lack of sexual forms, similar to those reported in other Bjerkandera anamorphs. The characterization of the culture medium for the highest LMEs production was performed in flask cultures, with a formulation of the culture medium containing high levels of glucose and peptone. The highest Mn-oxidizing peroxidase activity (1,400 U/L) was achieved on day 6 in Erlenmeyer flasks. Four peroxidases (named R1B1, R1B2, R1B3 and R1B4), have been purified by using ion-exchange and exclusion molar chromatographies. All of them showed typical activity on Mn²⁺ and exhibited Mn-independent activity against 2,6-dimethoxyphenol. R1B4 showed also activity on veratryl alcohol (pH 3) indicating that this enzyme belongs to the versatile peroxidase family. The high VP production capacities of this strain, as well as the enzymatic characteristics of the LMEs suggest that it may be successfully used in the degradation of recalcitrant compounds.     

DyP-like peroxidases of the jelly fungus Auricularia auricula-judae oxidize nonphenolic lignin model compounds and high-redox potential dyes

The jelly fungus Auricularia auricula-judae produced an enzyme with manganese-independent peroxidase activity during growth on beech wood (∼300 U l⁻¹). The same enzymatic activity was detected and produced at larger scale in agitated cultures comprising of liquid, plant-based media (e.g. tomato juice suspensions) at levels up to 8,000 U l⁻¹. Two pure peroxidase forms (A. auricula-judae peroxidase (AjP I and AjP II) could be obtained from respective culture liquids by three chromatographic steps. Spectroscopic and electrophoretic analyses of the purified proteins revealed their heme and peroxidase nature. The N-terminal amino acid sequence of AjP matched well with sequences of fungal enzymes known as “dye-decolorizing peroxidases”. Homology was found to the N-termini of peroxidases from Marasmius scorodonius (up to 86%), Thanatephorus cucumeris (60%), and Termitomyces albuminosus (60%). Both enzyme forms catalyzed not only the conversion of typical peroxidase substrates such as 2,6-dimethoxyphenol and 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) but also the decolorization of the high-redox potential dyes Reactive Blue 5 and Reactive Black 5, whereas manganese(II) ions (Mn²⁺) were not oxidized. Most remarkable, however, is the finding that both AjPs oxidized nonphenolic lignin model compounds (veratryl alcohol; adlerol, a nonphenolic β-O-4 lignin model dimer) at low pH (maximum activity at pH 1.4), which indicates a certain ligninolytic activity of dye-decolorizing peroxidases.     

Calorimetric experiments of Mn2+-binding to α-lactalbumin

We measured by batch microcalorimetry the standard enthalpy change ΔH° of the binding of Mn²⁺ to apo-bovine α-lactalbumin; ΔH° = −90 ± 4kJ·mol⁻¹. The binding constants, K_Mn²⁺, calculated from the calorimetric and circular dichroism titration curves, are (4.6±1) · 10⁵M⁻¹, respectively. Batch calorimetry confirms the competitive binding of Ca²⁺, Mn²⁺ and Na⁺ to the same site. The relatively small enthalpy change for Mn²⁺ binding compared to Ca²⁺ binding favours a model of a rigid and almost ideal Ca²⁺-complexating site, different from the well-known EF-hand structures. Cation binding to the high-affinity site most probably triggers the movement of an α-helix which is directly connected to the complexating loop.     

Depolymerization of low-rank coal by extracellular fungal enzyme systems. II. The ligninolytic enzymes of the coal-humic-acid-depolymerizing fungus Nematoloma frowardii b19

The production of ligninolytic enzymes was studied in surface cultures of the South American white-rot fungus Nematoloma frowardii b19 and four other strains of this ecophysiological group (Clitocybula dusenii b11, Auricularia sp. m37a, wood isolates u39 and u45), which are able to depolymerize low-rank-coal-derived humic acids with the formation of fulvic-acid-like compounds. The fungi produced the three crucial enzymes of lignin degradation – lignin peroxidase, manganese peroxidase and laccase. In the case of N. frowardii b19, laccase and the two peroxidases could be stimulated by veratryl alcohol. Manganese (II) ions (Mn²⁺) caused a rapid increase of Mn peroxidase activity accompanied by the complete repression of lignin peroxidase. Under nitrogen-limited conditions the growth as well as the production of ligninolytic enzymes was partly repressed. During the depolymerization process of coal humic acids using solid agar media, gradients of ligninolytic enzyme activities toward 2,2′-azinobis(3-ethylbenzthiazoline-6-sulphonate) and syringaldazine were detectable inside the agar medium. Received: 5 August 1996 / Received revision: 13 November 1996 / Accepted: 15 November 1996     

Lignin Peroxidase Activity Is Not Important in Biological Bleaching and Delignification of Unbleached Kraft Pulp by Trametes versicolor

The discovery in 1983 of fungal lignin peroxidases able to catalyze the oxidation of nonphenolic aromatic lignin model compounds and release some CO₂ from lignin has been seen as a major advance in understanding how fungi degrade lignin. Recently, the fungus Trametes versicolor was shown to be capable of substantial decolorization and delignification of unbleached industrial kraft pulps over 2 to 5 days. The role, if any, of lignin peroxidase in this biobleaching was therefore examined. Several different assays indicated that T. versicolor can produce and secrete peroxidase proteins, but only under certain culture conditions. However, work employing a new lignin peroxidase inhibitor (metavanadate ions) and a new lignin peroxidase assay using the dye azure B indicated that secreted lignin peroxidases do not play a role in the T. versicolor pulp-bleaching system. Oxidative activity capable of degrading 2-keto-4-methiolbutyric acid (KMB) appeared unique to ligninolytic fungi and always accompanied pulp biobleaching.