Fungal Lytic Polysaccharide Monooxygenases (LPMOs): Functional Adaptation and Biotechnological Perspectives

Abstract

Fungal lytic polysaccharide monooxygenases (LPMOs) have revolutionized the field of biomass degradation by introducing an oxidative mechanism that complements traditional hydrolytic enzymes. These copper-dependent enzymes catalyze the cleavage of glycosidic bonds in recalcitrant polysaccharides such as cellulose, hemicellulose, and chitin, through the activation of molecular oxygen (O₂) or hydrogen peroxide (H₂O₂). Their catalytic versatility is intricately modulated by structural features, including the histidine brace active site, surface-binding loops, and, in some cases, appended carbohydrate-binding modules (CBMs). The oxidation pattern, whether at the C1, C4, or both positions, is dictated by subtle variations in loop architecture, amino acid microenvironments, and substrate interactions. LPMOs are embedded in a highly synergistic fungal enzymatic system, working alongside cellulases, hemicellulases, lignin-modifying enzymes, and oxidoreductases to enable efficient lignocellulose decomposition. Industrial applications of fungal LPMOs are rapidly expanding, with key roles in second-generation biofuels, biorefineries, textile processing, food and feed industries, and the development of sustainable biomaterials. Recent advances in genome mining, protein engineering, and heterologous expression are accelerating the discovery of novel LPMOs with improved functionalities. Understanding the balance between O₂- and H₂O₂-driven mechanisms remains critical for optimizing their catalytic efficiency while mitigating oxidative inactivation. As the demand for sustainable biotechnological solutions grows, this narrative review highlights how fungal LPMOs function as indispensable biocatalysts for the future of the Circular Bioeconomy and green industrial processes.

1. Introduction

The efficient degradation of recalcitrant polysaccharides such as cellulose, hemicellulose, and chitin represents one of the most significant biochemical challenges in natural ecosystems and industrial biotechnology [1,2,3]. For decades, the canonical understanding of polysaccharide depolymerization was predominantly centered around the concerted action of hydrolytic enzymes, including glycoside hydrolases (GHs) and carbohydrate esterases (CEs), collectively classified under the Carbohydrate-Active enZYmes (CAZy) framework (https://www.cazy.org/) [4,5,6]. However, the discovery of lytic polysaccharide monooxygenases (LPMOs) has fundamentally reshaped this paradigm, introducing a powerful oxidative dimension to polysaccharide breakdown that complements hydrolytic mechanisms with remarkable efficiency [7,8].

Originally misannotated as GH family members due to their superficial sequence similarities, LPMOs were only correctly identified in the late 2000s as a distinct class of copper-dependent monooxygenases capable of oxidatively cleaving glycosidic bonds in crystalline polysaccharides [9]. Their ability to introduce chain breaks through oxidation at the C1 and/or C4 positions of glycosidic linkages enables synergistic collaboration with hydrolases, significantly enhancing the deconstruction of highly ordered polymeric substrates that are otherwise resistant to enzymatic attack [9,10].

The catalytic mechanism of LPMOs is particularly intriguing [9,11,12]. It relies on a highly conserved histidine-brace copper active site, which coordinates molecular oxygen or hydrogen peroxide to drive oxidative cleavage [9,13]. This discovery resolved long-standing questions about biomass recalcitrance and opened novel avenues in biocatalysis, renewable energy, and materials science [14].

While LPMOs are widespread across bacteria, archaea, viruses, and fungi [15,16,17,18], the fungal LPMOs exhibit unique structural diversity, functional specialization, and evolutionary trajectories that are tightly linked to the ecological roles and lifestyles of fungi [19,20]. Fungi occupy many ecological niches, from saprotrophic decomposers of plant and insect biomass to symbiotic partners and aggressive pathogens [21,22]. Their unparalleled enzymatic repertoire reflects these diverse ecological strategies, and LPMOs are no exception [23,24].

LPMOs are categorized into eight Auxiliary Activity (AA) families within the CAZy database, AA9–AA11 and AA13–AA17. Fungal LPMOs are predominantly AA9 (formerly GH61), AA10, AA11, AA13, AA14, AA15, and AA16. Among these, AA9 is exclusive to fungi and represents the most extensively studied group, primarily involved in cellulose oxidation. Conversely, AA13 targets starch, AA11 acts on chitin, while AA14 exhibits activity on xylan, highlighting the substrate versatility that fungi have evolved [25].

Despite the rapidly growing body of knowledge surrounding bacterial LPMOs, the fungal counterparts remain relatively underexplored outside of a few model species, such as Trichoderma reesei, Neurospora crassa, Thermothelomyces thermophilus (formerly Myceliophthora thermophila), and Thermothielavioides terrestris (formerly Thielavia terrestris) [23,26,27,28]. Nevertheless, genomic analyses reveal that a vast, largely untapped diversity of LPMO genes exists across fungi, especially in lineages adapted to extreme environments, specialized ecological niches, or unusual substrates [29,30,31]. This points to an enormous, uncharacterized reservoir of enzymatic diversity with potential biotechnological value.

The expansion and diversification of LPMO gene families within the fungal kingdom appear tightly coupled to evolutionary pressures related to substrate complexity, competition, and habitat specialization [8,32,33]. For example, white-rot fungi, capable of fully degrading lignin, often exhibit an expanded arsenal of AA9 LPMOs, synergizing with ligninolytic enzymes such as laccases and peroxidases [34]. In contrast, brown-rot fungi, which selectively degrade cellulose and hemicellulose while leaving lignin largely intact, display distinct LPMO complements with potential adaptations to their unique decay strategies [35].

Furthermore, fungi that form mutualistic associations, such as mycorrhizal species, possess LPMO repertoires that are significantly reduced or specialized, reflecting a shift away from aggressive polysaccharide degradation toward more subtle modifications of plant cell walls [21]. Similarly, pathogenic fungi leverage LPMOs not only for nutrient acquisition but also for breaching host barriers, suggesting a dual role in both metabolism and virulence [36].

These evolutionary patterns underscore the importance of examining fungal LPMOs not merely as isolated biocatalysts but as dynamic components of larger metabolic and ecological networks [19,37]. Understanding these connections could provide critical insights into the fundamental biology of fungi and inform the rational design of enzyme cocktails for industrial processes [38].

Fungal LPMOs are of paramount interest to the biotechnology sector, particularly in biorefineries, biofuel production, textile processing, and functional materials development [36,39,40,41]. Their capacity to disrupt crystalline cellulose and hemicellulose’s crystalline regions drastically improves the efficiency of saccharification processes, reducing enzyme loadings and process costs [42]. Moreover, generating oxidized oligosaccharides with unique chemical functionalities opens new avenues in synthesizing bio-based materials, fine chemicals, and nutraceuticals [43,44,45].

Nevertheless, most industrial applications rely on a narrow set of LPMOs derived from thermophilic filamentous fungi, leaving the broader functional potential of the fungal kingdom vastly underutilized [46]. Emerging genomic and metagenomic resources, coupled with advances in structural biology and enzyme engineering, now offer unprecedented opportunities to explore and exploit this diversity [47].

This review aims to provide a comprehensive overview of the diversity, evolution, mechanisms, and biotechnological potential of fungal LPMOs. We will explore how these enzymes are distributed across fungal phylogeny, how they correlate with different ecological strategies, and how their biochemical properties can be leveraged in industrial applications. By focusing exclusively on the fungal kingdom, this work seeks to fill a critical gap in the current literature, offering new perspectives on the role of LPMOs beyond the confines of standard model systems.

2. Classification of Fungal LPMO Families

The classification of LPMOs is based on the CAZy database, which organizes enzymes involved in the assembly, modification, and degradation of complex carbohydrates. LPMOs are categorized within the Auxiliary Activities (AA) class, reflecting their oxidative mechanism that assists traditional hydrolytic enzymes in biomass deconstruction [25].

Fungal LPMOs exhibit remarkable diversity in substrate specificity, structural features, oxidation patterns, and ecological roles [8,29,32,48]. This diversity results from evolutionary adaptations that have enabled fungi to access and degrade a wide range of recalcitrant polysaccharides present in plant and animal biomass [19]. While the AA9 family is the most studied due to its leading role in cellulose degradation, other families such as AA10, AA11, AA13, AA14, AA15, and AA16 represent functionally specialized enzymes with distinct substrate preferences, often tied to specific ecological niches or metabolic requirements [25].

Table 1. Comparative classification of fungal lytic polysaccharide monooxygenases (LPMO) families.

AA Family	Taxonomic Distribution	Substrate Specificity	Oxidation Pattern	Structural Features	Biochemical Features	Ecological and Functional Roles	Fungi Examples	References
AA9	Fungi	Primarily cellulose; also acts on hemicellulose (e.g., xylan, xyloglucan, galactoglucomannan)	C1, C4, or both (C1/C4)	β-sandwich fold with conserved histidine brace; variable loops (L2, L3, L8, LC) modulate substrate interaction; many members have carbohydrate-binding modules (CBMs)	High catalytic diversity; some thermostable; accept multiple electron donors (CDHs, lignin, H2O2); work synergistically with cellulases	Leading role in cellulose deconstruction; key in saprotrophic fungi for lignocellulose breakdown; industrial relevance in biofuels and biorefineries	Aspergillus fumigatus, Aspergillus nidulans, Malbranchea cinnamomea, Neurospora crassa, Pycnoporus sanguineus, Thermothelomyces fergusii Thermothelomyces thermophilus, Thermothielavioides terrestris, Thielavia australiensis, Trichoderma reesei	[23,25,26,27,28,49,50,51,52,53,54]
AA10	Mainly bacteria, some viruses and archaeans, minor reports in fungi	Cellulose, chitin	C1, C4, or both (C1/C4)	Similar β-sandwich fold to AA9; monocopper site; some contain CBMs	Oxidizes chitin and cellulose (C1/C4); requires O2 or H2O2; electron donors such as ascorbate or CDH	Originally bacterial; contributes to degradation of recalcitrant polysaccharides; cross-kingdom relevance, including minor fungal representatives	Ustilago maydis	[8,25,55]
AA11	Fungi, bacteria	Chitin (especially crystalline chitin from crustaceans, fungal cell walls)	Predominantly C1	β-sandwich fold; distinct loop regions compared to AA9; conserved histidine brace with unique surrounding residues	Lower catalytic rates; electron donor systems less characterized; potential interaction with small molecule donors or fungal redox partners	Chitin degradation for nutrient acquisition; possible roles in fungal cell wall remodelling and defense against other fungi or arthropods	Aspergillus fumigatus, Trichoderma guizhouense	[25,56,57]
AA13	Fungi	Retrograded (gelatinized) starch; inactive on crystalline cellulose	Mainly C1	β-sandwich fold; shallow binding groove adapted for α-glucans rather than flat crystalline surfaces	High efficiency on retrograded starch; activity enhanced by small molecule donors; natural redox partners still unclear	Enables fungi to utilize recalcitrant starch in natural or anthropogenic environments; adaptation to transient starch-rich substrates	Aspergillus nidulans, Aspergillus oryzae	[25,58,59,60]
AA14	Fungi	Xylan, but only when associated with cellulose microfibrils	C1	β-sandwich fold with surface adapted to interact with xylan helices embedded in cellulose	Requires xylan to be in the context of the cellulose-xylan network; facilitates loosening of plant cell wall matrices	Plays a role in disrupting hemicellulose-cellulose interactions; facilitates access for other glycoside hydrolases; niche role in complex biomass degradation	Phlebia radiata, Sordaria brevicollis Talaromyces rugulosus. Trametes coccinea (syn. Pycnoporus coccineus)	[25,61,62,63,64,65]
AA15	Fungi, viruses, archaeans, animals, plants	Cellulose, chitin	C1 and C4 (dual oxidation possible)	β-sandwich fold; higher structural plasticity; maintains histidine brace; adaptable binding surfaces	Poorly characterized in fungi; functional versatility seen in arthropods suggests potential for broad substrate recognition	Ecological role in fungi remains unclear; may be involved in specific symbiotic or pathogenic interactions, or degradation of insect-derived biomass	No robust biochemical characterizations have been published yet confirming the expression, enzymatic activity, and specific substrate of AA15 in fungi. In most databases, such as CAZy and JGI MycoCosm, AA15 are often annotated as “putative” or “hypothetical” enzymes in fungi	[25,66,67]
AA16	Fungi, viruses, oomycetes	Cellulose, chitin	C1	β-sandwich fold; monocopper active site	Acts primarily on cellulose; C1 oxidation predominant; works synergistically with cellulases	Contributes to cellulose breakdown; improves saccharification yields in industrial enzyme blends	Peniophora sp., Pleurotus eryngii, Thermothelomyces thermophilus	[26,68,69,70,71]

synthesizes current knowledge on these seven fungal LPMO families, providing a comparative framework highlighting their biochemical properties, structural distinctions, and ecological relevance.

The presence of AA15 family members in fungi is notably rare and, when identified, appears to reflect possible events of horizontal gene transfer or highly specialized niche adaptations [72]. Unlike its widespread distribution in arthropods, where AA15 LPMOs play roles in chitin and cellulose degradation, the occurrence in fungi may be linked to ecological pressures requiring the utilization of substrates derived from insect biomass or other specific environmental conditions [72,73]. The functional role of AA15 in fungi remains largely unexplored and warrants further investigation.

A particularly intriguing aspect of the AA14 family is its strict functional requirement for the co-localization of xylan with cellulose microfibrils to exhibit enzymatic activity. This dependency is unique among LPMOs and highlights a specialization toward modifying the structural heterogeneity of plant cell walls [62,64]. Rather than acting on soluble xylan, AA14 LPMOs facilitate the loosening of the hemicellulose-cellulose matrix, enabling more efficient access for other hydrolytic enzymes involved in lignocellulose degradation [61,62].

The AA13 family exemplifies an evolutionary adaptation that diverges from the typical β-glucan-targeting paradigm of most fungal LPMOs [74]. Instead, AA13 enzymes are specialized for the oxidative cleavage of retrograded (gelatinized) starch, an α-glucan, thereby allowing fungi to exploit starch-rich substrates that become recalcitrant after physical or thermal treatment [75,76]. This functional shift represents a significant expansion of the LPMO enzymatic repertoire beyond plant cell wall polysaccharides.

In contrast, the AA11 family represents an evolutionary solution tailored to the degradation of chitin, a highly crystalline β-1,4-linked polysaccharide found in fungal cell walls and arthropod exoskeletons [56,77]. While functionally analogous to the bacterial AA10 family of chitin-active LPMOs, fungal AA11s exhibit distinct structural adaptations and biochemical properties that reflect the unique evolutionary pressures and ecological contexts faced by fungi [56,78]. This specialization underscores the diversification of LPMOs in fungal lineages to accommodate different structural polysaccharides beyond cellulose.

3. Phylogenetic Distribution of Fungal LPMOs

The phylogenetic distribution of LPMOs within the fungal kingdom reveals intricate evolutionary patterns that are tightly linked to ecological strategies, substrate availability, and the diversification of fungal lineages [19,32,48,79,80]. Among the eight currently classified LPMO families (AA9–AA17), seven are present in fungi, AA9, AA10, AA11, AA13, AA14, AA15, and AA16 [25]. Their distribution is far from homogeneous across the two largest fungal phyla: Ascomycota and Basidiomycota [81].

The AA9 family is by far the most widespread, abundant, and phylogenetically diverse group of LPMOs in fungi [82]. It is extensively represented in both Ascomycota and Basidiomycota, reflecting its leading role in lignocellulose degradation [19]. Phylogenetic studies indicate that AA9 enzymes likely originated alongside the emergence of filamentous fungi capable of exploiting plant biomass, particularly during the colonization of terrestrial ecosystems [83,84]. In Ascomycota, AA9 genes are highly represented in saprotrophic species such as N. crassa, Aspergillus spp., and T. thermophilus, as well as in some plant pathogens [27,50,85,86]. These enzymes often show specialization towards crystalline cellulose, but display expanded activity towards hemicelluloses such as xyloglucan in specific lineages [87,88]. In Basidiomycota, AA9s are equally abundant, particularly in white-rot fungi like Pycnoporus sanguineus and Pleurotus ostreatus, which are capable of complete lignin mineralization [53,89]. Brown-rot fungi, though lacking extensive ligninolytic systems, also retain AA9 LPMOs, emphasizing the universal role of oxidative cellulose cleavage across divergent fungal lifestyles [35,90].

The AA10 family, originally characterized in bacterial systems (formerly known as CBM33), targets both chitin and crystalline cellulose through C1 and/or C4 oxidative cleavage [15,91,92]. Phylogenomic analyses suggest that fungal AA10 genes likely originated via horizontal gene transfer from bacterial sources [56]. Functionally, fungal AA10s may provide complementary oxidative activity alongside AA9 enzymes, particularly in mixed polysaccharide substrates or environments rich in chitinous and cellulosic materials [55,93,94].

The AA11 family is predominantly found in filamentous ascomycetes such as Aspergillus fumigatus, N. crassa, and Trichoderma spp. [31,56,57]. Despite being broadly distributed taxonomically, AA11 exhibits low diversification relative to AA9, possibly reflecting functional constraints tied to its narrow substrate specificity, crystalline chitin [31,95].

Similar to the AA11 family, the AA13 family is found exclusively in fungi [69]. It is particularly prevalent in species of the genera Aspergillus and Neurospora, which are known for their saprotrophic or opportunistic lifestyles [27,74].

The evolutionary emergence of AA14 is hypothesized to be relatively recent compared to AA9, representing a lineage highly specialized for acting on xylan that is physically associated with cellulose microfibrils [62,96]. This strict substrate constraint likely limits the ecological spread of AA14, confining it to fungi that occupy specific ecological niches requiring synergistic hemicellulose-cellulose deconstruction [23,62].

The AA15 family majority is found in arthropods, which play essential roles in chitin and cellulose degradation [66,97,98]. When detected, fungal AA15 genes show strong phylogenetic affinity to arthropod AA15 sequences, suggesting that horizontal gene transfer events are the most plausible explanation for their occurrence [69,83,99].

The ecological significance of AA15 in fungi remains largely speculative. Hypotheses include roles in niche-specific degradation of insect-derived biomass, symbiotic interactions, or opportunistic metabolic adaptations. However, the rarity and limited functional characterization of fungal AA15 enzymes prevent robust information about their evolutionary trajectory within the fungal kingdom [69,83,99].

The AA16 family represents one of the most recent additions to the CAZy LPMO classification, being initially identified in Aspergillus aculeatus [69]. Phylogenetic analyses indicate that AA16 likely evolved from a duplication and divergence event within fungal AA9 ancestors, acquiring distinct structural motifs that confer substrate specificity and potentially different redox partner interactions [100,101]. Their limited distribution suggests a specialization linked to specific ecological niches, potentially those involving highly recalcitrant plant biomass or environments with fluctuating oxidative conditions [77].

4. Relationship Between LPMOs and Fungal Lifestyle

LPMOs are widespread among lignocellulolytic fungi [18,23,46,102]. The capacity of fungi to degrade lignocellulosic biomass represents a cornerstone of carbon cycling in terrestrial ecosystems [103,104,105]. This ability has driven the evolution of highly specialized enzymatic systems capable of degrading the complex matrix of cellulose, hemicellulose, and lignin found in plant cell walls [106,107,108]. The functional deployment of LPMOs is tightly linked to the physicochemical challenges posed by lignocellulose, especially the crystalline regions of cellulose and tightly bound hemicelluloses that are highly recalcitrant to hydrolytic enzymes alone [42,109]. Then, LPMOs are recognized as central components of the lignocellulolytic machinery in fungi, providing oxidative cleavage mechanisms that complement classical hydrolytic pathways [52,110].

In fungal lignocellulolytic systems, LPMOs function as molecular oxidizers capable of introducing chain breaks in crystalline polysaccharides through the oxidative cleavage of glycosidic bonds [77]. This mechanism differs fundamentally from that of glycoside hydrolases, as it relies on the activation of molecular oxygen (or hydrogen peroxide) to hydroxylate carbon atoms at C1 and/or C4 positions, leading to destabilization and fragmentation of the polysaccharide chains [111]. This oxidative mode of action enhances the accessibility of cellulose microfibrils and hemicellulose fractions to hydrolases [43]. As a result, LPMOs serve as synergistic partners that significantly boost the efficiency of fungal lignocellulose degradation, particularly in complex plant materials such as wood, leaf litter, and agricultural residues [112,113,114].

Among the LPMO families, AA9 is the most prevalent in fungi involved in plant biomass degradation [56]. While AA14, though less common, is specialized for targeting xylan when it is physically associated with cellulose, a feature frequently encountered in hardwoods and grasses [27,78]. This specialization enhances the deconstruction of complex plant cell walls by disrupting the hemicellulose-cellulose interface [62].

The efficiency of LPMOs in lignocellulolytic fungi is further enhanced by their integration with other oxidative enzymes and redox systems [8]. Many fungi co-produce extracellular oxidoreductases such as cellobiose dehydrogenase (CDH), glucose-methanol-choline (GMC) oxidoreductases, or copper radical oxidases, which supply the electrons necessary for LPMO catalytic cycles [25,115]. Additionally, the generation of hydrogen peroxide, either enzymatically or through Fenton-type reactions, can serve as an alternative co-substrate, highlighting a flexible oxidative toolkit that complements hydrolytic enzymology [116].

The presence and functional roles of LPMOs in phytopathogenic and endophytic fungi have emerged as an intriguing area of study that bridges plant-fungal interactions, microbial ecology, and enzymatic evolution [83,117]. While LPMOs were initially characterized for their role in saprotrophic lignocellulose degradation [118], mounting evidence demonstrates that these oxidative enzymes are also employed by fungi engaged in intimate relationships with living plants, whether antagonistic (pathogenic) or mutualistic (endophytic) [83,118].

Phytopathogenic fungi leverage LPMOs as part of their arsenal to breach and colonize plant tissues [117,119]. The plant cell wall constitutes the primary defensive barrier, composed of complex networks of cellulose microfibrils embedded within matrices of hemicellulose, pectin, and lignin [120]. While classical hydrolytic enzymes (e.g., cellulases, pectinases, xylanases) have long been recognized as virulence factors [121], LPMOs provide a complementary oxidative mechanism that enhances cell wall degradation, particularly in crystalline regions that are otherwise resistant to hydrolysis [122].

AA9 LPMOs are the most widespread among plant pathogenic fungi, including species such as Magnaporthe oryzae, Botrytis cinerea, Fusarium graminearum, and Verticillium dahlia [83,123,124,125]. These enzymes facilitate the oxidative cleavage of cellulose, generating chain breaks that weaken cell wall integrity and release oligosaccharides that can serve as nutrients for the pathogen [126]. Importantly, the action of LPMOs may help create physical entry points or exacerbate mechanical damage during host colonization [127,128].

Moreover, some phytopathogens possess AA11 LPMOs, which are chitin-active [95,129]. Although chitin is absent from plant cell walls, AA11 enzymes may participate in self-cell wall remodelling or play roles in inter-microbial competition, particularly in the complex rhizosphere or phyllosphere environments [81,130].

A critical observation is that the expression of LPMO genes in phytopathogenic fungi is often highly regulated and induced during host infection stages [119,126]. Transcriptomic and proteomic studies have shown that LPMOs are upregulated during appressorium formation, tissue penetration, and necrotrophic growth, suggesting that these enzymes are integral to the infection process [83,86,119,131].

Endophytic fungi establish symbiotic or commensal relationships with plants, inhabiting internal tissues without causing visible disease symptoms [132]. Unlike phytopathogens, endophytes generally avoid eliciting strong plant defense responses, which raises questions about the presence and roles of LPMOs in these organisms [133].

Genome analyses reveal that many endophytic fungi encode AA9 LPMOs, albeit often in reduced numbers compared to saprotrophic or pathogenic relatives. This suggests a selective retention of LPMOs for functions that do not compromise host viability [81,129]. For endophytes, LPMOs may be involved in subtle modifications of the plant cell wall that facilitate colonization of intercellular spaces without triggering defense mechanisms [134,135]. Rather than aggressive degradation, LPMOs in this context may loosen cell wall structures to allow hyphal penetration or movement [115].

Mycorrhizal fungi, including both ectomycorrhizal (ECM) and arbuscular mycorrhizal (AM) species, are generally considered poor decomposers of lignocellulosic material compared to saprotrophs [136]. Nevertheless, genomic studies have revealed that certain ECM fungi, such as Laccaria bicolor, Hebeloma cylindrosporum, Paxillus involutus, and Suillus luteus, retain genes encoding AA9-type LPMOs [115,118,137,138]. These LPMOs are typically present in reduced numbers relative to saprotrophic relatives, reflecting an evolutionary transition towards a symbiotic lifestyle with diminished reliance on extensive plant biomass degradation [136].

Unlike their saprotrophic counterparts that deploy LPMOs for large-scale cellulose depolymerization, mycorrhizal fungi may use LPMOs for subtler modifications of plant cell walls [139]. During the establishment of symbiosis, fungi must penetrate the root epidermis and navigate the apoplastic space without triggering strong plant defense responses [133,140]. LPMOs may facilitate this process by loosening cell wall components, particularly cellulose microfibrils and hemicellulose, to allow hyphal ingress and the formation of fungal structures such as the Hartig net in ECM associations [141].

5. Catalytic Pathways and Specific Modulations of Fungal LPMOs

LPMOs are oxidative enzymes that revolutionized the understanding of polysaccharide depolymerization [7,25,142]. Unlike hydrolytic GHs that cleave glycosidic bonds via hydrolysis, LPMOs utilize an oxidative mechanism to break the crystalline regions of polysaccharides such as cellulose, chitin, starch, and hemicelluloses [25]. This oxidative cleavage introduces chain breaks that dramatically increase biomass accessibility to hydrolases [10]. The mechanistic versatility of fungal LPMOs is intimately connected to their substrate preferences, cofactor requirements, and synergistic interactions with complementary enzymes in complex enzyme cocktails.

5.1. Catalytic Mechanism Overview

At the heart of LPMO catalysis is a conserved active site architecture featuring a mononuclear copper center coordinated by the histidine brace, where the N-terminal histidine coordinates the copper via both its terminal amine and the imidazole side chain, and a second histidine provides the other imidazole ligand [13]. This highly conserved motif enables LPMOs to activate molecular oxygen (O₂) or hydrogen peroxide (H₂O₂) to perform oxidative cleavage of glycosidic bonds [143].

The catalytic cycle can follow two primary oxidative routes:

• Oxygen-Driven (O₂) Mechanism: Traditionally, it was believed that LPMOs functioned as monooxygenases, employing molecular oxygen (O₂) as the oxygen source [144]. In this pathway, the copper in the resting Cu(II) state must first be reduced to Cu(I) via an external electron donor (e.g., cellobiose dehydrogenase—CDH or phenolic compounds). The reduced Cu(I) center reacts with molecular oxygen to form a reactive oxygen species, which may be a Cu(II)-superoxide or Cu(II)-peroxide intermediate. This intermediate performs hydrogen atom abstraction (HAA) from the C–H bond of the polysaccharide substrate. The subsequent rebound of the hydroxyl group to the carbon leads to cleavage of the glycosidic bond, typically at the C1 or C4 position, depending on the enzyme’s regioselectivity. Although well-characterized in vitro, this O₂-driven pathway is kinetically slow, as it requires two sequential electron transfers per catalytic event, one for reducing Cu(II) to Cu(I) and another to complete the O₂ reduction [31,116,145].
• Peroxide-Driven (H₂O₂) Mechanism: In this route, the Cu(I) center reacts directly with hydrogen peroxide (H₂O₂), forming a highly reactive Cu(II)-oxyl species or a related high-valent intermediate. This pathway is significantly faster than the O₂ route because only one electron is required to reduce Cu(II) to Cu(I) for catalytic turnover, and the reaction with H₂O₂ produces a potent oxidant capable of immediate hydrogen abstraction and glycosidic bond cleavage. It is important to highlight that in the H₂O₂-driven mechanism, LPMO stays in the reduced state (Cu(I)) at the end of the catalytical cycle, then multiple cycles are possible without additional reductant being necessary. However, this route carries inherent risks. Excessive or uncontrolled H₂O₂ levels can lead to enzyme self-inactivation through oxidative damage to the histidine brace or other nearby residues. Consequently, fungi have evolved intricate systems to regulate H₂O₂ production, including tight coupling with oxidoreductases (like CDH, glucose oxidase, and aryl-alcohol oxidases) and enzymes that scavenge excess H₂O₂ (such as catalases and peroxidases) [116,142,146,147,148,149].

This dual mechanism provides LPMOs with remarkable flexibility under varying environmental conditions or within different enzymatic systems.

Table 2. Differences between oxygen-driven (O₂) and hydrogen peroxide-driven (H₂O₂) catalytic mechanisms in lytic polysaccharide monooxygenases (LPMOs) [31,116,142,145,146].

Aspect	O2-Driven Pathway	H2O2-Driven Pathway
Speed (Turnover Rate)	Slow	Fast (10–100× faster)
Electron Requirement	Two electrons per cycle	One electron per cycle
Control Complexity	Lower (less prone to ROS damage)	Higher (requires strict H2O2 control)
Risk of Inactivation	Minimal	High if H2O2 is in excess
Physiological Prevalence	Secondary, backup under low H2O2 conditions	Dominant under most natural biomass degradation
Evolutionary Implication	Possibly ancestral or for oxygen-rich conditions	Adaptation to lignocellulose-rich microenvironments

summarizes the key differences between oxygen-driven (O₂) and hydrogen peroxide-driven (H₂O₂) catalytic mechanisms in LPMOs, highlighting their biochemical, ecological, and industrial implications.

5.2. Cofactor Requirements and Electron Donors

LPMOs are strictly copper-dependent enzymes, with their catalytic mechanism fundamentally relying on the redox cycle of a mononuclear copper center [115]. The copper ion within the active site must transition from its oxidized Cu(II) resting state to the reduced Cu(I) form prior to each catalytic turnover, enabling the activation of molecular oxygen or hydrogen peroxide for the oxidative cleavage of polysaccharide substrates [143]. This redox cycle necessitates a continuous supply of external electrons, and the nature of these electron donors varies considerably depending on the ecological niche and metabolic capabilities of the fungus [122,150].

One of the most prominent and well-studied electron donors in fungal pathways is cellobiose dehydrogenase (CDH), a flavocytochrome enzyme co-secreted alongside LPMOs, particularly in wood-decaying fungi [151]. CDH possesses both a flavin domain, which oxidizes cellobiose or other sugars, and a cytochrome domain, which directly transfers electrons to the copper center of LPMOs [152]. This creates an exceptionally efficient and synergistic enzymatic partnership, where CDH facilitates LPMO catalysis and couples lignocellulose oxidation with sugar metabolism [25].

In addition to CDH, pyrroloquinoline-quinone-dependent pyranose dehydrogenases (PQQ-PDHs) have also emerged as potential fungal redox partners for LPMOs. These enzymes catalyze the oxidation of a wide range of aldopyranoses and transfer electrons via PQQ cofactors, contributing to LPMO activation in certain fungal systems. While less characterized than CDH, evidence of fungal expression of PQQ-PDHs and their redox activity suggests they may play an auxiliary role in sustaining oxidative polysaccharide degradation under specific ecological or developmental conditions [153].

Beyond CDH and PPQ-PDH, a variety of small phenolic compounds (e.g., caffeic acid, vanillic acid, catechol, guaiacol, among others) generated during lignin degradation serve as natural, diffusible redox mediators capable of donating electrons to LPMOs [154]. These phenolics arise as by-products of the oxidative action of lignin-modifying enzymes such as laccases and peroxidases [155]. Their role extends beyond mere electron donation; they contribute to an intricate redox network within the fungal extracellular space, linking lignin depolymerization with polysaccharide oxidation [156]. This crosstalk between lignin breakdown and carbohydrate degradation is a hallmark of fungal biomass deconstruction strategies [157].

In addition to enzymatic and phenolic electron donors, fungi can exploit simpler small-molecule reductants such as ascorbic acid, gallic acid, and reduced glutathione. While these molecules are often employed in vitro to study LPMO activity, they are also present in fungal microenvironments under certain physiological conditions [43]. These reductants provide flexible electron supply options, particularly when dedicated enzymes like CDH are absent or less active [158,159].

Remarkably, recent studies have uncovered that some fungi utilize light-driven photoredox systems to sustain LPMO activity [84,113,160]. In these systems, photoactive pigments, such as melanin-like compounds or quinone-containing molecules, absorb visible light and generate excited states capable of transferring electrons to LPMOs [161]. This photochemical strategy allows fungi to harness environmental light as an auxiliary energy source to drive oxidative degradation, an adaptation particularly relevant in surface-exposed or shallow-soil fungal habitats [84,113,160,161]. In addition to serving as electron donors, these light-activated systems have also been shown to produce hydrogen peroxide (H₂O₂), which can act as a direct co-substrate for LPMO catalysis. This dual function, electron donation and H₂O₂ generation, provides a powerful mechanism for enhancing LPMO activity under light-exposed conditions, potentially enabling multiple oxidative cycles without the need for continuous external reductant supply. Such findings expand our understanding of how photochemical processes are integrated into fungal redox metabolism and polysaccharide degradation [85].

5.3. Substrate Specificity

Fungal LPMOs are classified into multiple Auxiliary Activity (AA) families [25], each with distinct substrate specificities:

• AA9: the most studied in fungi, primarily targets cellulose, with some members also acting on hemicelluloses like xyloglucan, glucomannan, and mixed-linkage β-glucans [25]. Oxidation patterns vary between C1, C4, and mixed C1/C4, depending on the enzyme [162]. This broad substrate range is largely attributed to the flexible architecture of AA9 surface loops (especially L2, L3, LC, and LS), which adapt the conserved LPMO β-sandwich fold to accommodate both crystalline and amorphous substrates [163,164].
• AA10 (rare in fungi): Fungal AA10s exhibit activity on both crystalline cellulose and chitin. They are capable of C1, C4, or mixed oxidation, similar to AA9 [25]. This dual specificity stems from a structurally open substrate-binding surface and conserved residues in the flat catalytic plane of the typical LPMO fold, allowing alignment with both β-1,4-glucans and β-1,4-N-acetylglucosamine chains [165,166].
• AA11: specializes in chitin, particularly in the modification of crystalline chitin [95], with oxidative cleavage at the C1 position being most common [31]. This specificity is associated with a narrower substrate-binding groove and distinct electrostatic surface potentials, tailored to interact with the acetylated chitin chains rather than the more hydrophobic cellulose [31,95].
• AA13: targets starch (α-1,4-glucans), specifically disrupting the crystalline regions of amylose and amylopectin [167]. Its substrate preference reflects adaptations in the catalytic interface that favor the helical conformation of starch polysaccharides, diverging structurally from other LPMOs by modifications to loops around the active site cleft [58,168].
• AA14: exhibits activity on xylan but uniquely requires the presence of cellulose for activity, suggesting that it acts on xylan tightly bound to cellulose fibrils within the plant cell wall. The specificity of AA14 is hypothesized to rely on the positioning of the substrate-binding loops and electrostatic complementarity that favor xylan–cellulose complexes, consistent with the LPMO β-sandwich fold but with added surface constraints [62].
• AA15 (rare in fungi): demonstrates broad activity on both chitin and cellulose, with evidence of oxidation on both C1 and C4 carbons [25]. This functional versatility may reflect its evolutionary origin from arthropods and a more flexible binding surface, superimposed onto the typical LPMO fold, that tolerates multiple polysaccharide geometries [67,98].
• AA16: acts predominantly on cellulose, with oxidation typically at the C1 position. It may be complementary to AA9 enzymes, especially in fungal species specialized in degrading highly recalcitrant plant biomass [25]. The substrate preference of AA16 is linked to specific loop configurations and redox partner preferences that distinguish it from AA9, despite sharing the core β-sandwich fold and catalytic mechanism [70,71].

The substrate specificity of fungal LPMOs is not solely determined by the nature of their catalytic copper center. However, it is profoundly influenced by a series of structural features that fine-tune their interaction with complex polysaccharide substrates [48]. One of the key determinants involves the configuration of surface-binding loops that surround the copper active site [169]. These flexible loops, commonly designated L2, L3, LS (short loop), L8, and LC (C-terminal loop) in AA9 enzymes, play a pivotal role in defining the shape and chemistry of the substrate-binding surface [32,170]. The loop nomenclature is based on their relative sequence positions and structural location in the enzyme: for instance, LC refers to a C-terminal loop that often contributes to substrate positioning, while LS denotes a short loop located near the active site cleft. Variations in the length, amino acid profile, and conformation of these loops dictate how the enzyme aligns with crystalline polysaccharide surfaces, thereby affecting both substrate recognition and oxidation regioselectivity (i.e., C1, C4, or mixed oxidation) [95]. For instance, enzymes with extended loops often display enhanced binding to rough or heterogeneous surfaces, such as amorphous regions or hemicellulose-decorated cellulose fibrils [23,171,172].

In addition to the loop architecture, several LPMOs incorporate carbohydrate-binding modules (CBMs), which are non-catalytic domains specialized in targeting specific polysaccharides. CBMs function as molecular anchors that tether the catalytic domain to the substrate surface, significantly enhancing the local concentration of the enzyme on the target polymer [173]. The presence of CBMs not only increases catalytic efficiency but can also shift substrate preference [174,175]. For example, some AA9 LPMOs with CBM1 domains exhibit improved action on crystalline cellulose, whereas those lacking CBMs may preferentially act on soluble oligosaccharides or less crystalline regions [51,87,176].

Furthermore, the electrostatic surface properties of LPMOs exert a subtle yet critical influence on substrate binding [32]. The distribution of charged residues around the active site can either attract or repel specific polysaccharide surfaces, depending on the chemical environment of the substrate [172]. Cellulose, being relatively hydrophobic and neutral, tends to favor LPMOs with hydrophobic or neutral surfaces in the binding region [177]. In contrast, chitin, which carries acetyl groups and occasional charges, may favor different charge distributions [178].

These structural features do not act in isolation but synergistically modulate the affinity of enzymes, orientation, and reactivity toward diverse carbohydrate substrates [178]. Moreover, subtle point mutations or natural sequence variations in these regions can profoundly alter the catalytic performance, oxidation pattern, and even tolerance to oxidative stress, underscoring the evolutionary plasticity of fungal LPMOs in adapting to specific ecological niches and substrates [179,180,181]. As a result, understanding these structural determinants is essential for deciphering fungal biomass degradation strategies and guiding the engineering of LPMOs with tailored properties for industrial applications [178,182].

5.4. Oxidation Pattern Modulation (C1, C4, Mixed)

The regioselectivity of oxidation in fungal LPMOs is a defining biochemical feature that greatly influences both the mechanism of polysaccharide cleavage and the types of degradation products generated [32,111]. This oxidation can occur at different carbon positions of the glycosidic unit, primarily at the C1 position, the C4 position, or, in some enzymes, at both (mixed C1/C4 oxidation) [183]. Each oxidation mode has distinct biochemical consequences and ecological functions [184].

C1 oxidation generates a terminal lactone that spontaneously hydrolyzes under aqueous conditions to yield an aldonic acid [185]. This process effectively opens the sugar ring, destabilizes the glycosidic bond, and facilitates hydrolysis by exo- or endo-acting enzymes [180,186,187]. C1-oxidizing LPMOs are exceptionally efficient in synergizing with β-glucosidases or cellobiohydrolases that target the resulting oxidized chain ends [42].

C4 oxidation, conversely, introduces a keto group at the C4 position of the glucose unit [178]. This oxidation creates a ketoaldose at the non-reducing end, which also destabilizes the glycosidic linkage but differs chemically from the products of C1 oxidation [188]. The keto functional group can influence downstream enzymatic processing differently, sometimes resulting in slower hydrolysis by standard hydrolases due to altered ring chemistry [178].

Mixed C1/C4 oxidation endows certain LPMOs with enhanced flexibility to act on structurally heterogeneous or highly recalcitrant substrates [32]. These enzymes can introduce oxidative breaks at multiple sites within the same polysaccharide chain, improving accessibility for hydrolytic enzymes and promoting more efficient biomass turnover [162,176].

The determinants of the oxidation pattern in fungal LPMOs are deeply rooted in the structural architecture of the enzyme, particularly in the geometry of the active site relative to the substrate plane [189]. One of the primary factors influencing regioselectivity is the configuration of surface-exposed loops [88]. These loops form the structural framework surrounding the catalytic copper center and critically define how the enzyme orients itself over the polysaccharide chain [164,190].

Substrate surface properties further modulate oxidation patterns. The physical and chemical characteristics of the polysaccharide, whether it is crystalline, amorphous, highly ordered, or chemically modified, directly impact how the LPMO interacts with it [191]. C1 oxidation tends to dominate when the enzyme engages with tightly packed, crystalline cellulose surfaces, where the enzyme binds in a planar orientation that aligns the active site for C1 targeting [186]. In contrast, more flexible, amorphous regions may allow alternative binding modes that favor C4 oxidation or enable mixed C1/C4 cleavage [169]. Furthermore, the presence of accessory plant cell wall components such as hemicelluloses, lignin fragments, or acetylated sugars can influence the binding surface and, consequently, the oxidative outcome [122,192].

Electron transfer dynamics also play a subtle but significant role in oxidation pattern modulation. The nature, availability, and redox potential of electron donors, including cellobiose dehydrogenase (CDH), small aromatic compounds, and even photoexcited systems, can impact how efficiently electrons are delivered to the LPMO’s copper center [78,122,142].

5.5. Synergy with Other Enzymes

LPMOs do not function as standalone depolymerization agents but rather operate in tight synergy with a complex ensemble of hydrolytic and oxidative enzymes present in the fungal secretome (Figure 1). This orchestrated cooperation is one of the key factors underlying the remarkable efficiency of fungi in deconstructing plant biomass, particularly recalcitrant lignocellulosic substrates [187,193].

A vital component of this synergy involves classical cellulases, particularly members of glycoside hydrolase families such as GH6 and GH7. These enzymes catalyze the hydrolytic cleavage of β-1,4-glycosidic bonds in cellulose. However, their efficiency is severely hampered when cellulose is in its native, highly crystalline form. Here, LPMOs play a transformative role by introducing oxidative cleavage at specific positions within the crystalline cellulose microfibrils; LPMOs create novel chain ends and disrupt the tight packing of cellulose chains. This structural loosening dramatically increases the accessibility of the cellulose to exo- and endo-cellulases, thereby enhancing their catalytic turnover and accelerating overall cellulose hydrolysis [19,193,194].

This synergistic effect extends beyond cellulose to hemicellulose components of the plant cell wall. Hemicellulases, including xylanases (GH10, GH11) and arabinofuranosidases or mannanases (GH43 and others), benefit indirectly from LPMO activity. The oxidative disruption of the cellulose scaffold by LPMOs exposes previously inaccessible hemicellulose domains embedded within or strongly associated with cellulose fibrils. As a result, the action of hemicellulases becomes more effective, facilitating a more comprehensive deconstruction of plant polysaccharides [72,194,195].

The synergy is not confined to carbohydrate-active enzymes but extends to lignin-degrading oxidative systems. Fungi produce a suite of lignin-modifying enzymes, such as laccases and class II peroxidases (including manganese peroxidase and lignin peroxidase), which initiate oxidative cleavage of the complex lignin polymer. A key by-product of lignin degradation is the generation of low-molecular-weight phenolic compounds, which can function as effective electron donors for LPMOs. This biochemical crosstalk creates a functional linkage between lignin depolymerization and polysaccharide oxidation, integrating oxidative degradation pathways across different components of the plant cell wall matrix [196,197,198,199].

Additionally, oxidoreductases such as CDH and members of the GMC oxidoreductase family are vital for sustaining the LPMO catalytic cycle. These enzymes facilitate the continuous reduction of the copper active site in LPMOs, delivering electrons necessary for the activation of molecular oxygen or hydrogen peroxide. By maintaining the redox balance, these oxidoreductases ensure that LPMO activity is sustained over prolonged periods during biomass conversion [25,200].

Altogether, the synergistic interplay among LPMOs, glycoside hydrolases, lignin-modifying oxidoreductases, and auxiliary redox enzymes constitutes a highly integrated fungal biomass-deconstruction system. This cooperative enzymatic network is significantly more effective than hydrolytic processes operating alone, particularly when targeting the most recalcitrant forms of plant biomass. Such synergy not only enhances the biological efficiency of fungal decomposition in natural ecosystems but also represents a fundamental principle leveraged in industrial biorefineries for the sustainable conversion of lignocellulosic feedstocks into biofuels and biochemicals [187,193,201].

5.6. Biological Modulation and Regulation

The biological modulation and regulation of LPMOs in fungi is a highly sophisticated and tightly controlled process, reflecting the delicate balance between the energetic benefits of biomass degradation and the potential cellular risks associated with oxidative chemistry [86]. Fungal deployment of LPMOs is primarily governed by substrate availability [32]. The presence of specific polysaccharides, particularly crystalline cellulose or hemicellulose, acts as a potent inducer of LPMO gene expression. This substrate-driven regulation ensures that fungi invest metabolic resources into producing LPMOs only when the appropriate target polysaccharides are present in the environment, thereby maximizing enzymatic efficiency while minimizing unnecessary energy expenditure [202,203].

An additional layer of regulation stems from the intrinsic risks associated with the oxidative nature of LPMO catalysis. Since LPMOs can utilize both molecular oxygen and hydrogen peroxide (H₂O₂) as co-substrates, their activity inherently generates reactive oxygen species (ROS). Unchecked ROS production can be highly detrimental to fungal cells, leading to oxidative stress and potential cellular damage. As a result, fungi have evolved mechanisms to carefully modulate LPMO activity, balancing the need for efficient biomass degradation with the imperative to maintain redox homeostasis and protect cellular integrity. This modulation likely involves transcriptional regulation and post-transcriptional controls, including feedback from redox sensors and the availability of electron donors [86,204,205].

Transcriptomic and proteomic studies across several fungal species, including N. crassa, T. reesei, T. thermophilus, and P. chrysosporium, consistently reveal that LPMO genes are co-expressed alongside genes encoding cellulases, hemicellulases, CDH, and lignin-modifying enzymes such as laccases and peroxidases. This coordinated expression pattern underscores the integrated role of LPMOs within the broader fungal secretome and highlights their functional interdependence with both hydrolytic and oxidative enzymes. Such regulatory coherence ensures that LPMO deployment is synchronized with the overall strategy for plant biomass decomposition, reflecting a finely tuned adaptation to ecological pressures and resource availability [18,206,207,208].

6. Fungal LPMOs with Industrial Applications

Fungal LPMOs have emerged as pivotal biocatalysts in several industrial sectors due to their unparalleled ability to oxidatively cleave recalcitrant polysaccharides, such as crystalline cellulose and hemicelluloses. Their capacity to disrupt the crystalline architecture of lignocellulosic biomass positions them as indispensable components in modern enzyme formulations designed for biomass valorization. Unlike classical glycoside hydrolases, which rely solely on hydrolytic mechanisms, LPMOs introduce oxidative cleavage at specific carbons, generating chain breaks that dramatically improve the accessibility of polysaccharides to other enzymes [10,77,182].

The most significant industrial application of fungal LPMOs is in the biorefinery sector, particularly in the enzymatic hydrolysis of plant biomass to produce second-generation biofuels (cellulosic ethanol) and biochemicals [10,184]. LPMOs, especially those belonging to the AA9 family, are widely incorporated into commercial cellulase cocktails produced by leading biotechnology companies [46]. Their inclusion has been shown to boost saccharification yields, particularly when processing highly crystalline biomass like pretreated corn stover, sugarcane bagasse, or wheat straw [46,201,209].

Beyond biofuels, LPMOs play an emerging role in the pulp and paper industry, where they facilitate fiber modification, enhancing delignification and improving paper quality with reduced chemical inputs. Their oxidative action disrupts cellulose crystallinity, making fibers more amenable to subsequent chemical or enzymatic treatments, thereby contributing to more sustainable and energy-efficient processing [112,210].

The textile industry also benefits from LPMOs, particularly in cotton processing and denim finishing. Here, LPMOs contribute to fiber modification, offering a greener alternative to harsh chemical treatments. Their ability to weaken cellulose fibers in a controlled manner enables more sustainable bio-polishing, improving fabric softness and appearance while reducing water and chemical usage [211,212].

A rapidly growing frontier for LPMO applications is in the food and feed industries. For example, LPMOs assist in the degradation of plant-based feedstocks, improving digestibility and nutrient accessibility in animal feed formulations [213,214]. Additionally, in the production of plant-based proteins and functional food ingredients, LPMOs contribute to the extraction and modification of polysaccharides from complex biomass [215,216].

From an industrial enzyme engineering perspective, fungal LPMOs have become targets for protein engineering to enhance stability, catalytic efficiency, and oxidative robustness under harsh industrial conditions, including elevated temperatures, variable pH, and oxidative stress. Advances in understanding the H₂O₂-driven catalytic mechanism have led to optimized process designs where controlled peroxide dosing maximizes LPMO activity while minimizing enzyme inactivation [217,218].

Furthermore, integrated biorefineries are beginning to exploit LPMOs in conjunction with oxidative lignin-modifying enzymes, such as laccases and peroxidases. This synergy improves carbohydrate conversion and opens pathways for lignin valorization into biopolymers, resins, and aromatic compounds, reinforcing the Circular Bioeconomy model [122,161].

In the context of emerging green technologies, LPMOs are being explored for novel applications such as bioplastic degradation, enzymatic textile dye bleaching, and even in the field of carbon capture via engineered fungal systems that degrade plant biomass into stable soil organic carbon [178,219,220].

In summary, fungal LPMOs have transitioned from being a fundamental enzymatic curiosity to becoming a cornerstone of industrial biotechnology. Their incorporation into commercial processes is a direct consequence of their unique catalytic capabilities, offering sustainable solutions for biomass conversion, material processing, and the development of bio-based products. Continued research into their mechanisms, regulation, and interaction with electron donors will further expand their applicability across diverse industrial landscapes.

7. Future Perspectives

The growing industrial relevance of LPMOs continues to drive a surge of interest in unlocking their full potential beyond current applications. As the demand for efficient and sustainable biotechnological processes escalates, future perspectives for fungal LPMOs revolve around three interconnected pillars: (i) the exploration of underexplored fungal genomes, (ii) the engineering of strains and heterologous expression systems, and (iii) the expansion of their applications in biorefineries and biomaterials [7,19,202].

Despite considerable progress in characterizing LPMOs from well-studied fungi, the fungal kingdom harbours an enormous untapped reservoir of enzymatic diversity [24]. Numerous fungal taxa, particularly those adapted to extreme environments (e.g., thermophilic, halophilic, acidophilic fungi) or specialized ecological niches (e.g., marine fungi, endophytes, and soil saprobes), remain largely unexplored for their oxidative enzyme repertoires [221]. Advances in next-generation sequencing and metagenomics are revealing a vast array of novel LPMO genes from these underexplored lineages [47]. Comparative genomics combined with machine-learning-guided enzyme mining is expected to uncover LPMOs with unique properties such as higher thermal stability, broader substrate specificity (e.g., activity on non-cellulosic polysaccharides like chitin or pectin), and enhanced oxidative resistance [222]. These novel biocatalysts could significantly broaden the applicability of LPMOs in industrial contexts that demand more robust enzymes. Furthermore, the exploration of fungal microbiomes associated with decaying lignocellulosic biomass, symbiotic relationships (e.g., lichens, mycorrhizae), or polluted environments may yield LPMOs adapted to challenging chemical conditions, offering new functionalities for biotechnology [223].

A significant bottleneck in the industrial deployment of LPMOs lies in their production yield, oxidative stability, and control over their activity. Future research is increasingly focused on synthetic biology and strain engineering strategies to overcome these limitations [224]. Optimizing heterologous expression systems, such as Komagataella pastoris (formerly Pichia pastoris), Aspergillus niger, and Saccharomyces cerevisiae, is essential for scalable production of tailored LPMO variants. Codon optimization, promoter engineering, and secretory pathway enhancement are being combined to boost yields and ensure proper copper loading and folding [219,225,226]. Additionally, protein engineering approaches targeting improvements in LPMO stability under oxidative stress, tuning their regioselectivity (C1 vs. C4 oxidation), and modifying their interaction with hydrogen peroxide to minimize self-inactivation [227]. Directed evolution, structure-guided mutagenesis, and computational enzyme design are expected to yield next-generation LPMOs with superior catalytic profiles tailored to specific industrial processes. Coupling LPMOs with engineered redox partners, such as optimized CDHs or artificial electron transfer systems, is another frontier poised to enhance operational stability and efficiency in biocatalytic systems [228,229,230].

As the bioeconomy shifts toward zero-waste, circular models, LPMOs are poised to play an increasingly vital role in integrated biorefineries. Their oxidative cleavage ability synergizes with both hydrolytic and lignin-modifying enzymes, enabling comprehensive biomass deconstruction and valorization. Future biorefinery models will leverage LPMOs not only for the saccharification of lignocellulose into fermentable sugars but also for the selective extraction of functional oligosaccharides, nanocellulose, and lignin-derived aromatic compounds. These high-value intermediates are key feedstocks to produce bio-based chemicals, bioplastics, resins, and advanced biomaterials. In parallel, LPMOs are gaining attention for applications in biomaterial processing, such as enhancing fiber properties in textiles, facilitating the production of biodegradable composites, and even contributing to enzymatic recycling strategies for composite materials that blend natural fibers with synthetic polymers. Emerging applications are also exploring the role of LPMOs in the biodegradation of environmental pollutants, including complex plastics, where LPMO-driven oxidative mechanisms may aid in breaking down polymeric structures when coupled with other oxidative enzymes [10,46,122,161,178,184,201,209,210,211,212,213,214,215,216,217,218,219,220].

8. Conclusions

Fungal lytic polysaccharide monooxygenases (LPMOs) have emerged as pivotal enzymes in the global carbon cycle and in industrial biotechnology, owing to their unique oxidative mechanism that complements classical hydrolytic enzymes in lignocellulose degradation. Their ability to cleave recalcitrant polysaccharides such as crystalline cellulose, hemicellulose, and chitin through oxidative reactions has fundamentally reshaped our understanding of fungal biomass conversion. The structural sophistication of these enzymes, particularly the histidine brace copper active site, combined with substrate-binding loops and CBMs, orchestrates a highly selective and efficient catalytic process. Furthermore, the capacity of LPMOs to utilize both molecular oxygen (O₂) and hydrogen peroxide (H₂O₂) as co-substrates adds a remarkable degree of flexibility to their function. However, it necessitates precise cellular regulation to mitigate oxidative stress. Their activity is deeply intertwined with that of other fungal enzymes, including cellulases, hemicellulases, oxidoreductases, and lignin-modifying enzymes, forming an integrated enzymatic arsenal that facilitates effective deconstruction of lignocellulosic biomass in both natural ecosystems and industrial processes.

Looking forward, the biotechnological potential of fungal LPMOs continues to expand. Advances in genome mining, transcriptomics, and metagenomics are uncovering a wealth of untapped fungal diversity, particularly from ecological niches that remain poorly explored. This opens avenues for the discovery of LPMOs with novel substrate specificities, enhanced thermal or chemical stability, and improved oxidative performance. Concurrently, protein engineering and synthetic biology approaches are enabling the development of tailored LPMOs and optimized microbial hosts for heterologous expression, facilitating their integration into enzyme cocktails for biorefineries, biofuel production, and emerging sectors such as sustainable biomaterials and green chemistry. As mechanistic understanding deepens, particularly regarding the balance between O₂- and H₂O₂-driven catalysis, electron transfer dynamics, and enzyme stabilization, fungal LPMOs are poised to play an increasingly significant role in advancing the Circular Bioeconomy and sustainable biotechnological solutions for the future.