Lignocellulosic Substrates Research Articles

Effects of hap2 deletion on mnp/vp transcription in Pleurotus ostreatus grown on lignocellulosic substrates.

The regulatory mechanisms governing expression of genes encoding lignin-modifying enzymes (LME) in white-rot fungi remain largely unexplored. Although molecular cloning has identified CCAAT-boxes frequently located 5'-upstream of these genes, their role in transcriptional regulation is not well understood. This study examines the function of hap2, a gene encoding a hypothetical protein homologous to a component of the CCAAT-binding Hap complex, in the white-rot fungus Pleurotus ostreatus. Deletion of hap2 resulted in significantly reduced Mn2+-dependent peroxidase activity and lignin-degrading capacity compared to the parental strain 20b grown on beech wood sawdust (BWS) medium. Real-time PCR revealed that vp2 transcript levels were significantly lower in hap2 deletants than in 20b grown when cultured on the three solid media consisting of BWS, holocellulose, or Avicel, but not on yeast-malt-glucose (YMG) agar plates. Additionally, glutathione S-transferase (GST) pulldown and electrophoretic mobility shift assays demonstrated that recombinant P. ostreatus Hap2, Hap3, and Hap5 expressed in Escherichia coli form a complex capable of binding to the CCAAT sequence 5'-upstream of vp2 in vitro. These results suggest that Hap2, as part of the CCAAT-binding complex, is essential for transcriptional upregulation of vp2 in P. ostreatus growing on lignocellulosic substrates. KEY POINTS: • P. ostreatus hap2 deletants were generated. • Lignin-degrading capacity was significantly reduced in the hap2 deletants. • vp2 was significantly downregulated upon hap2 deletion.

Microbial enzymatic indices for predicting composting quality of recalcitrant lignocellulosic substrates

Three different enzymes alkaline phosphatase, Urease and Dehydrogenase were measured during this study to monitor the organic matter dynamics during semi-industrial composting of mixture A with 1/3 sludge+2/3 palm waste and mixture B with ½ sludge+1/2 palm waste. The phosphatase activity was higher for Mix-A (398.7 µg PNP g−1 h−1) than Mix-B (265.3 µg PNP g−1 h−1), while Mix-B (103.3 µg TPF g−1d−1) exhibited greater dehydrogenase content than Mix-A (72.3 µg TPF g−1 d−1). That could contribute to the dynamic change of microbial activity together with high amounts of carbonaceous substrates incorporated with the lignocellulosic. The gradual increase in the dehydrogenase from the compost Mix-A implies that high lignocellulosic substrate requires gradual buildup of dehydrogenase activity to turn the waste into mature compost. A higher pick of urease with a maximum activity of 151.5 and 122.4 µg NH4-N g−1 h−1 were reported, respectively for Mix-A and B. Temperature and pH could also influence the expression of enzyme activity during composting. The machine learning well predicted the compost quality based on NH3/NO3, C/N ratio, decomposition rate and, humification index (HI). The root mean square error (RMSE) values were 1.98, 1.95, 4.61%, and 4.1 for NH+3/NO−3, C/N ratio, decomposition rate, and HI, respectively. The coefficient of determination between observed and predicted values were 0.87, 0.93, 0.89, and 0.94, for the r NH3/NO3, C/N ratio, decomposition rate, and HI. Urease activity significantly predicted the C/N ratio and HI only. The profile of enzymatic activity is tightly linked to the physico-chemical properties, proportion of lignocellulosic-composted substrates. Enzymatic activity assessment provides a simple and rapid measurement of the biological activity adding understunding of organic matter transformation during sludge-lignocellulosic composting.

Exploring local lignocellulosic substrates for the production of edible mushrooms in Northwestern Argentina

La Rioja province annually produces approximately 75,000 tons of agricultural residues and derived materials from agro-industrial activities, which could potentially be incorporated into oyster mushroom cultivation. This study aimed to evaluate the viability of this lignocellulosic biomass as a substrate for the cultivation of edible mushrooms belonging to the genus Pleurotus. Initially, the mycelial growth of two species (P. ostreatus and P. djamor) was assessed by formulating combinations of local substrates. Experimental crops were grown employing the most promising substrates, which were subsequently selected for chemical characterization. It was found that both strains exhibited maximum mycelial growth in the substrate formulated with jojoba leaf litter. A comparison of the two strains revealed no direct correlation between mycelium growth and productive performance. The highest biological efficiency (BE) values were obtained when P. ostreatus was cultivated in treatments combining jojoba leaf litter and grape pomace with olive pomace. Furthermore, these treatments showed suitable chemical properties and were formulated from problematic waste generated in large quantities in the region without proper processing and disposal methods. In this context, there is potential to ensure a continuous supply of this lignocellulosic biomass for cultivating these mushroom species over an extended period of time, thus providing a sustainable alternative for these regional by-products.

In-depth recognition of mixed surfactants maintaining the enzymatic activity of cellulases through stabilization of their spatial structures

Mixed surfactants improve the enzymatic hydrolysis of lignocellulosic substrates by enhancing cellulase stability against heat, pH, shear, and air–liquid interface stress. Under conditions of multiple factorial stresses (50 °C, pH 4.8, 180 rpm, and 15.5 cm2 air–liquid interface), cellulase with ternary surfactants (Tween 60/Triton X-114/CTAB, the molar ratio 14:5.5:1) retained 84 % of its activity after 48 h of incubation, representing 1.15 and 1.29 folds that of the cellulase activity with the single Tween 60 and with no surfactants, respectively. This is attributed to the fact that ternary surfactants possess better rheology modulation and air–liquid interface competitiveness. In addition, the computational approach demonstrated that the ternary surfactants were capable of forming stronger hydrophobic and hydrogen-bond interactions with cellulase enzymes, thus maintaining its secondary structure and preventing the detrimental α-helix to β-sheet transformation known to compromise cellulase activity. This synergy offers valuable insights into surfactant-cellulase interactions and supports efficient enzymatic hydrolysis in biorefineries.

Enhancing Laccase and Manganese Peroxidase Activity in White-Rot Fungi: The Role of Copper, Manganese, and Lignocellulosic Substrates

Enhancing Laccase and Manganese Peroxidase Activity in White-Rot Fungi: The Role of Copper, Manganese, and Lignocellulosic Substrates

From agricultural biomass to D form lactic acid in ton scale via strain engineering of Lactiplantibacillus pentosus

Worsening environmental conditions make lactic acid a sustainable alternative to petroleum-based plastics. This study created a genetically-engineered strain Lactiplantibacillus pentosus PeL containing a disrupted L-lactate dehydrogenase gene to produce high yield and optically pure D-lactic acid. Cellobiose was identified as the optimal sugar in the single carbon source test, yielding the highest lactic acid. In 5-L fermentation tests, pretreated wood chips hydrolysate was the best lignocellulosic substrate for PeL, resulting in a D-lactic acid yield of 900.7 ± 141.4 mg/g of consumed sugars with an optical purity of 99.8 ± 0.0 %. Gradually scaled-up fermentations using this substrate were achieved in 100-, and 9,000-L fermenters; PeL produced remarkably high D-lactic acid yields of 836.3 ± 11.9 and 915.9 ± 4.4 mg/g of consumed sugars, with optical purities of 95.0 ± 0.0 % and 93.8 ± 0.2 %, respectively. This study is the pioneer in demonstrating economical and sustainable ton-scale production of D-lactic acid.

Effect of Cold Exposure on the Biofoam Produced from Different Types of Oyster Mushroom

Mycelium-based biofoam is a sustainable material derived from the growth of fungal mycelium on lignocellulosic agricultural waste substrate, as it has potential use in a variety of applications. The main objective of this research is to advance the sustainable alternatives for various application by investigating the mycelium growth of the biofoam produced from Pleurotus florida and Pleurotus sajor-caju on rice husk substrate, in improving the properties of the biofoam through innovative cold exposure. This study showed P. florida can produce mycelium biofoam at a faster rate, 7.022mm/day compared to P. sajor-caju 6.08mm/day). By cold exposure at 0°C and 10°C for 3 hours, every 2 days and 5 days, respectively until the mycelium are fully grown in the substrate, sample exposed to the latter condition for P. florida exhibits a faster growth rate at 7.3037 mm/day. However, cold exposure on biofoam produced from P sajor-caju had not improved the mycelium growth rate. Cold exposure samples at 0°C every 5 days and 10°C every 2 days have demonstrated capability in water (103.51%) and oil absorption (143.23%), proving their effectiveness in absorbing pollutants for the purpose of environmental remediation. The FTIR analysis confirmed the presence of hydrophilic and oleophilic characteristics in the biofoam, indicating its capability to absorb water and oil. By subjecting biofoam to cold exposure, its properties can be altered, broadening its potential applications.

Insight into the mechanism of alkali-thermal pretreatment of food-waste solid residue through fluorescence spectroscopy coupled with parallel factor analysis

Food-waste solid residue is the remaining solid after food waste treatment, with high yield, high solid content, high protein and fiber content. Effective pretreatment is necessary to improve the efficiency of hydrolysis and acidification for anaerobic digestion of food-waste solid residue. In this study, fluorescence spectroscopy coupled with parallel factor analysis were used to insight into the mechanism of food-waste solid residue during three pretreatments (alkali, thermal and alkali-thermal). Pretreatments increased the solubility of lignocellulosic substrate and destroyed structure of starch, while lignocellulosic analogs were effectively cracked, changing the composition and improving the degradability. Soluble chemical oxygen demand, soluble protein and soluble polysaccharide concentrations were increased by 144.60%, 350.57% and 138.72% after pretreatment under the condition of 120 °C + 2% CaO, respectively. Three-dimensional fluorescence spectra showed the region of maximum fluorescence intensity under alkali-thermal pretreatments, indicating chemical bonds (such as OC–C) were easier broken and the solubility of organic substances were increased. Three main fluorescence components were obtained by parallel factor analysis, which were humic acid-like, lignocellulose-like and protein-like, respectively, while the lignocellulose-like had the maximum Fmax value. The fluorescence intensity of samples under alkali-thermal pretreatment varied in the range from 59.48 × 105 to 13.18 × 106, which was an increase of 174.27%–507.74% over the control (21.68 × 105), indicating that alkali-thermal pretreatment observably accelerated the breaking of chemical bonds, and thus promoted the dissolution of organic matter. This study deeply revealed the mechanism of alkali-thermal pretreatment of food-waste solid residue, which is of great significance for efficient resource utilization of food waste and food-waste solid residue.

Bioaugmentation with targeted recombinant functional consortia to improve lignocellulosic biowaste co-anaerobic digestion performance

Hydrolysis rate limitation and acid-methanogenesis phase imbalance are critical challenges in the anaerobic digestion (AD) of lignocellulosic substrates, hindering substrate utilization and methane conversion. Bioaugmentation is widely employed to promote biochemical and metabolic reactions at all stages of AD to enhance methanogenic efficiency. However, synergistic reinforcement for simultaneous enhancement of the hydrolysis and methanogenesis phases requires further elucidation. This study investigated the effect of lignocellulolytic mutualistic methanogenic culture (LMMC) obtained by targeted domestication on the AD system. The results showed that the bioaugmented reactor enhanced the methane production by 21.03%-239.12% and the peak daily methanogenesis by 6.54%-127.85%. Introducing LMMC was directly related to improving methanogenic performance and significantly affected the AD key indicator, which facilitated the timely consumption of volatile fatty acid (VFA) for utilization. Microbial analysis results showed that the enriched hydrolytic acidifying bacteria, anaerobic fungi, and acetoclastic methanogens formed a microbial synergistic reinforcement alliance, which jointly stimulated methane production, alleviated the potential risk of instability, and promoted the stable operation of the AD system. Collectively, the role of synergistic reinforcement in the microbial consortium was highlighted to improve the stability and efficiency of AD process of lignocellulosic biomass.

Comparative Evaluation of Mechanical and Physical Properties of Mycelium Composite Boards Made from Lentinus sajor-caju with Various Ratios of Corn Husk and Sawdust.

Mycelium-based composites (MBCs) exhibit varied properties as alternative biodegradable materials that can be used in various industries such as construction, furniture, household goods, and packaging. However, these properties are primarily influenced by the type of substrate used. This study aims to investigate the properties of MBCs produced from Lentinus sajor-caju strain CMU-NK0427 using different ratios of sawdust to corn husk in the development of mycelium composite boards (MCBs) with thicknesses of 8, 16, and 24 mm. The results indicate that variations in the ratios of corn husk to sawdust and thickness affected the mechanical and physical properties of the obtained MCBs. Reducing the corn husk content in the substrate increased the modulus of elasticity, density, and thermal conductivity, while increasing the corn husk content increased the bending strength, shrinkage, water absorption, and volumetric swelling. Additionally, an increase in thickness with the same substrate ratio only indicated an increase in density and shrinkage. MCBs have sound absorption properties ranging from 61 to 94% at a frequency of 1000 Hz. According to the correlation results, a reduction in corn husk content in the substrate has a significant positive effect on the reduction in bending strength, shrinkage, and water absorption in MCBs. However, a decrease in corn husk content shows a strong negative correlation with the increase in the modulus of elasticity, density, and thermal conductivity. The thickness of MCBs with the same substrate ratio only shows a significant negative correlation with the modulus of elasticity and bending strength. Compared to commercial boards, the mechanical (bending strength) and physical (density, thermal conductivity, and sound absorption) properties of MCBs made from a 100% corn husk ratio are most similar to those of softboards and acoustic boards. The results of this study can provide valuable information for the production of MCBs and will serve as a guide to enhance strategies for further improving their properties for commercial manufacturing, as well as fulfilling the long-term goal of eco-friendly recycling of lignocellulosic substrates.

Research Trends in the Recovery of By-Products from Organic Waste Treated by Anaerobic Digestion: A 30-Year Bibliometric Analysis

The prevailing extractive economic model is unsustainable due to the finite nature of resources, thereby necessitating the development of alternative models and policies. The anaerobic digestion (AD) process is key to achieving this objective, as it facilitates the conversion of organic waste into biogas and nutrient-rich digestate. This approach is aligned with the principles of a circular economy and contributes to a reduction in carbon emissions. This study aims to conduct a comprehensive bibliometric analysis of the literature published over the past three decades (1993–2023). The analysis will be based on data drawn from the Scopus database and then analysed using the VOSviewer software, which allows for the interconnection of the revised bibliography through a series of selected keywords. The results demonstrated the existence of four clusters: (i) the beneficial valorisation of waste; (ii) volatile fatty acids and biohydrogen as added value by-products resulting from AD; (iii) lignocellulosic substrates and their by-products; and iv) the main products of AD, biogas and digestate. The bibliometric analysis demonstrates a growing interest in AD within the biorefinery concept in recent years, showcasing its potential for effective waste management and integration into the production chain through the principles of the circular economy.

Genome analysis of the esca-associated Basidiomycetes Fomitiporia mediterranea, Fomitiporia polymorpha, Inonotus vitis, and Tropicoporus texanus reveals virulence factor repertoires characteristic of white-rot fungi.

Some Basidiomycete fungi are important plant pathogens, and certain species have been associated with the grapevine trunk disease esca. We present the genomes of 4 species associated with esca: Fomitiporia mediterranea, Fomitiporia polymorpha, Tropicoporus texanus, and Inonotus vitis. We generated high-quality phased genome assemblies using long-read sequencing. The genomic and functional comparisons identified potential virulence factors, suggesting their roles in disease development. Similar to other white-rot fungi known for their ability to degrade lignocellulosic substrates, these 4 genomes encoded a variety of lignin peroxidases and carbohydrate-active enzymes (CAZymes) such as CBM1, AA9, and AA2. The analysis of gene family expansion and contraction revealed dynamic evolutionary patterns, particularly in genes related to secondary metabolite production, plant cell wall decomposition, and xenobiotic degradation. The availability of these genomes will serve as a reference for further studies of diversity and evolution of virulence factors and their roles in esca symptoms and host resistance.

Upgraded cellulose and xylan digestions for synergistic enhancements of biomass enzymatic saccharification and bioethanol conversion using engineered Trichoderma reesei strains overproducing mushroom LeGH7 enzyme

Crop straws provide enormous lignocellulose resources transformable for sustainable biofuels and valuable bioproducts. However, lignocellulose recalcitrance basically restricts essential biomass enzymatic saccharification at large scale. In this study, the mushroom-derived cellobiohydrolase (LeGH7) was introduced into Trichoderma reesei (Rut-C30) to generate two desirable strains, namely GH7–5 and GH7–6. Compared to the Rut-C30 strain, both engineered strains exhibited significantly enhanced enzymatic activities, with β-glucosidases, endocellulases, cellobiohydrolases, and xylanase activities increasing by 113 %, 140 %, 241 %, and 196 %, respectively. By performing steam explosion and mild alkali pretreatments with mature straws of five bioenergy crops, diverse lignocellulose substrates were effectively digested by the crude enzymes secreted from the engineered strains, leading to the high-yield hexoses released for bioethanol production. Notably, the LeGH7 enzyme purified from engineered strain enabled to act as multiple cellulases and xylanase at higher activities, interpreting how synergistic enhancement of enzymatic saccharification was achieved for distinct lignocellulose substrates in major bioenergy crops. Therefore, this study has identified a novel enzyme that is active for simultaneous hydrolyses of cellulose and xylan, providing an applicable strategy for high biomass enzymatic saccharification and bioethanol conversion in bioenergy crops.

Efficient sugar utilization and high tolerance to inhibitors enable Rhodotorula toruloides C23 to robustly produce lipid and carotenoid from lignocellulosic feedstock

The utilization of lignocellulosic substrates for microbial oil production by oleaginous yeasts has been evidenced as an economically viable process for industrial-scale biodiesel preparation. Efficient sugar utilization and tolerance to inhibitors are critical for lipid production from lignocellulosic substrates. This study investigated the lignocellulosic sugar utilization and inhibitor tolerance characteristics of Rhodotorula toruloides C23. The results demonstrated that C23 exhibited robust glucose and xylose assimilation irrespective of their ratios, yielding over 21 g/L of lipids and 11 mg/L of carotenoids. Furthermore, C23 exhibited high resistance and efficiently degradation towards toxic inhibitors commonly found in lignocellulosic hydrolysates. The potential molecular mechanism underlying xylose metabolism in C23 was explored, with several key enzymes and signal regulation pathways identified as potentially contributing to its superior lipid synthesis performance. The study highlights R. toruloides C23 as a promising candidate for robust biofuel and carotenoid production through direct utilization of non-detoxified lignocellulosic hydrolysates.

Cellobionate production from sodium hydroxide pretreated wheat straw by engineered Neurospora crassa HL10

This study investigated cellobionate production from a lignocellulosic substrate using Neurospora crassa HL10. Utilizing NaOH-pretreated wheat straw as the substrate obviated the need for an exogenous redox mediator addition, as lignin contained in the pretreated wheat served as a natural mediator. The low laccase production by N. crassa HL10 on pretreated wheat straw caused slow cellobionate production, and exogenous laccase addition accelerated the process. Cycloheximide induced substantial laccase production in N. crassa HL10, enabling the strain to yield approximately 57 mM cellobionate from pretreated wheat straw (equivalent to 20 g/L cellulose), shortening the conversion time from 8 to 6 days. About 92% of the cellulose contained in the pretreated wheat straw is converted to cellobionate. In contrast to existing methods requiring pure cellobiose or cellulase enzymes, this process efficiently converts a low-cost feedstock into cellobionate at a high yield without enzyme or redox mediator supplementation.

Mycelium-metal hybrids: Exploring fabrication and application for reconfigurable design structures

Abstract Mycelium-bound composites (MBCs) are materials grown by fungi onto lignocellulosic substrates. MBCs are a low-cost, lightweight, valorised biomass with promising properties concerning acoustics, heat insulation and fire resistance, among others. These properties make MBCs interesting as a sustainable alternative to currently existing fossil-fuel-derived products. However, MBCs lack properties such as mechanical strength or other functional properties like electrical conductivity which could widen their range of applications. In this work, the mycelium from Pleurotus ostreatus is grown in presence of metals. First, a coating strategy is developed to grow the fungus on aluminium, copper, and stainless-steel surfaces. The coating is made of agar and cornstarch to provide nutrients for the fungus to grow. It is found that the mycelium can grow on all surfaces, even on anti-bacterial copper surface. Secondly, magnetic MBCs with 3D shapes are fabricated for making potential reconfigurable structures. For these composites, the fungus is exposed to lignocellulosic substrate and rare earth magnets. Using 3D printing to create 3D moulds to grow the composite, and by strategically placing the magnet, several structures are made. This approach is interesting for the future design and fabrication of reconfigurable panels for room partition, building thermal or insulation, or to replace plastics in toy products, among others.

Enhanced enzymatic hydrolysis of lignocellulosic substrate with less-expensive formulation of homemade cellulase cocktails

This study focused on the efficient production of fermentable sugars from high-solids enzymatic hydrolysis of the lignocellulosic substrate, using homemade cellulase preparation (from China company) combined with surfactants, plant-derived proteins, and accessory enzymes. Results show that various non-ionic and ionic surfactants worked together very effectively, resulting in the 48-h glucose yield of enzymatic hydrolysis increasing by 21.4 %, compared with a single surfactant. The surfactant mixture was particularly competitive at a lower enzyme loading and higher solid content of the enzymatic hydrolysis. Many plant-derived proteins were found to be useful in enzymatic hydrolysis. With the optimized mixture of surfactants, plant-derived protein, and accessory enzymes, the homemade cellulase preparation produced 170 g/L of fermentable sugars (∼ 90 % of the yield) from the enzymatic hydrolysis (2 mg/g dried substrate, 20 % (w/v), 72 h), which was promisingly economic and close to the starchy sugar. The homemade cellulase preparation by formulation with various surfactants and plant-derived proteins supplied a feasible solution for the development of a cost-effective enzymatic hydrolysis process.

Prediction of anaerobic degradation kinetics based on substrate composition of lignocellulosic biomass

This study presents a comprehensive biochemical predictive approach for assessing biogas production kinetics across ten lignocellulosic substrates in batch operation. The methodology employs a range of kinetic and regression models, all grounded in the substrates' chemical composition. Among the kinetic models, the cone model demonstrated superior performance, achieving an average error of 1.67 % in describing biogas production from all substrates. The quadratic Monod type model followed closely, with an error of 1.96 %. Among the regression models, on the other hand, the logistic function model exhibited enhanced predictive capabilities, yielding an average error of 6.02 %, while the Chen and Hashimoto one showed a higher error of 60.54 %. The findings underscore the potential of precise biogas production forecasting and tracking the daily rates of gas generation, rather than solely relying on cumulative gas yields at the end of the process.

Efficient methane production from agro-industrial residues using anaerobic fungal-rich consortia.

Anaerobic digestion (AD) emerges as a pivotal technique in climate change mitigation, transforming organic materials into biogas, a renewable energy form. This process significantly impacts energy production and waste management, influencing greenhouse gas emissions. Traditional research has largely focused on anaerobic bacteria and methanogens for methane production. However, the potential of anaerobic lignocellulolytic fungi for degrading lignocellulosic biomass remains less explored. In this study, buffalo rumen inocula were enriched and acclimatized to improve lignocellulolytic hydrolysis activity. Two consortia were established: the anaerobic fungi consortium (AFC), selectively enriched for fungi, and the anaerobic lignocellulolytic microbial consortium (ALMC). The consortia were utilized to create five distinct microbial cocktails-AF0, AF20, AF50, AF80, and AF100. These cocktails were formulated based on varying of AFC and ALMC by weights (w/w). Methane production from each cocktail of lignocellulosic biomasses (cassava pulp and oil palm residues) was evaluated. The highest methane yields of CP, EFB, and MFB were obtained at 337, 215, and 54 mL/g VS, respectively. Cocktails containing a mix of anaerobic fungi, hydrolytic bacteria (Sphingobacterium sp.), syntrophic bacteria (Sphaerochaeta sp.), and hydrogenotrophic methanogens produced 2.1-2.6 times higher methane in cassava pulp and 1.1-1.2 times in oil palm empty fruit bunch compared to AF0. All cocktails effectively produced methane from oil palm empty fruit bunch due to its lipid content. However, methane production ceased after 3 days when oil palm mesocarp fiber was used, due to long-chain fatty acid accumulation. Anaerobic fungi consortia showed effective lignocellulosic and starchy biomass degradation without inhibition due to organic acid accumulation. These findings underscore the potential of tailored microbial cocktails for enhancing methane production from diverse lignocellulosic substrates.

Structure–function characterization of two enzymes from novel subfamilies of manganese peroxidases secreted by the lignocellulose-degrading Agaricales fungi Agrocybe pediades and Cyathus striatus

BackgroundManganese peroxidases (MnPs) are, together with lignin peroxidases and versatile peroxidases, key elements of the enzymatic machineries secreted by white-rot fungi to degrade lignin, thus providing access to cellulose and hemicellulose in plant cell walls. A recent genomic analysis of 52 Agaricomycetes species revealed the existence of novel MnP subfamilies differing in the amino-acid residues that constitute the manganese oxidation site. Following this in silico analysis, a comprehensive structure–function study is needed to understand how these enzymes work and contribute to transform the lignin macromolecule.ResultsTwo MnPs belonging to the subfamilies recently classified as MnP-DGD and MnP-ESD—referred to as Ape-MnP1 and Cst-MnP1, respectively—were identified as the primary peroxidases secreted by the Agaricales species Agrocybe pediades and Cyathus striatus when growing on lignocellulosic substrates. Following heterologous expression and in vitro activation, their biochemical characterization confirmed that these enzymes are active MnPs. However, crystal structure and mutagenesis studies revealed manganese coordination spheres different from those expected after their initial classification. Specifically, a glutamine residue (Gln333) in the C-terminal tail of Ape-MnP1 was found to be involved in manganese binding, along with Asp35 and Asp177, while Cst-MnP1 counts only two amino acids (Glu36 and Asp176), instead of three, to function as a MnP. These findings led to the renaming of these subfamilies as MnP-DDQ and MnP-ED and to re-evaluate their evolutionary origin. Both enzymes were also able to directly oxidize lignin-derived phenolic compounds, as seen for other short MnPs. Importantly, size-exclusion chromatography analyses showed that both enzymes cause changes in polymeric lignin in the presence of manganese, suggesting their relevance in lignocellulose transformation.ConclusionsUnderstanding the mechanisms used by basidiomycetes to degrade lignin is of particular relevance to comprehend carbon cycle in nature and to design biotechnological tools for the industrial use of plant biomass. Here, we provide the first structure–function characterization of two novel MnP subfamilies present in Agaricales mushrooms, elucidating the main residues involved in catalysis and demonstrating their ability to modify the lignin macromolecule.