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# Fungal cell walls: the rising importance of carbohydrate-active enzymes

Roseline Assiah Yao, Jean-Guy Berrin, Lauren Mckee, Bastien Bissaro

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## To cite this version:

Roseline Assiah Yao, Jean-Guy Berrin, Lauren Mckee, Bastien Bissaro. Fungal cell walls: the rising importance of carbohydrate-active enzymes. Trends in Microbiology, 2025, 10.1016/j.tim.2025.05.001. hal-05239120

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# Fungal Cell Walls: the rising importance of carbohydrate-active enzymes

Roseline Assiah Yao¹, Jean-Guy Berrin¹, Lauren S. McKee²* and Bastien Bissaro¹*

¹INRAE, Aix Marseille Univ, BBF, Biodiversité et Biotechnologie Fongiques, Marseille, France

²Division of Glycoscience, Department of Chemistry, KTH Royal Institute of Technology, AlbaNova University Centre, 106 91, Stockholm, Sweden

*correspondence to:

mckee@kth.se (L.S. McKee) or bastien.bissaro@inrae.fr (B. Bissaro)

## Abstract

As the interface between the fungal cell and its surroundings, the fungal cell wall (FCW) plays an essential physiological role in a myriad of biological processes. It provides support, protection, and enables material exchange with the environment, playing a key role in microbiome and host-microbe interactions. The fact that FCWs are mainly composed of complex carbohydrates makes carbohydrate-active enzymes (CAZymes) the main players in FCW remodelling and degradation. Despite the biological importance of these processes, our understanding of the underlying enzymology remains limited. In this review, we discuss the role of FCW-active CAZymes in various contexts, including fungal physiology, pathogenesis, human gut microbiomes, and the global carbon cycle, while highlighting knowledge gaps and potential applications in agriculture, biotechnology, and health.

**Key words:** Agriculture, Bioeconomy, CAZymes, Health, Host-microbe interactions, Polysaccharides.
## HIGHLIGHTS

* Models of FCW architecture are evolving; previously seen as a highly-structured wall it is today shown to be flexible and dynamic.

* FCWs are diverse in terms of composition and structure, and this biodiversity is still largely underexplored.

* FCW-active CAZymes play important roles in fungal cell biology, at various stages of the life cycle.

* CAZymes play a major role in modulation of the immunogenicity of the FCW in various fungus-host interplays

* Fungal necromass contributes to the build-up of soil organic carbon, the recycling of which is mainly ensured by the action of FCW-active CAZymes produced by fungi and bacteria.
The Fungal Cell Wall: A versatile and vital interface

The fungal cell wall (FCW) is one of the most essential and well-conserved structures throughout the fungal kingdom. Indeed, whether they be involved in plant biomass decay, pathogenesis, or symbiotic associations, fungi need to remodel/degrade their own cell wall to adapt to their environment [1]. Also, as a major component of microbial necromass (i.e. dead biomass), the FCW contributes to the build-up of soil organic carbon and therefore to the global carbon cycle [2,3]. However, the diversity of FCW structures has yet to be fully explored, as most studies have focused on a few species, mainly human pathogens [4] such as *Aspergillus fumigatus* [5] or *Cryptococcus neoformans* [6], the causative agents of aspergillosis and meningoencephalitis, respectively. Current knowledge teaches that the FCW is an intricate and dynamic multi-layer matrix of polymers, composed of an inner wall of chitin and β-1,3/β-1,6-glucans, with an outer wall of species-dependent glycoproteins and polysaccharides [5,7–9] (Fig. 1). While the outer wall is more involved in interactions with the environment, the inner wall contributes cell strength and shape, and the proportions and fine structural details (e.g. substitutions) of the polymers of both walls can vary with environmental stress or life cycle stage [10], adding an extra source of complexity and diversity.

As with plant cell walls (PCWs), FCW carbohydrates are synthesized, modified, and
hydrolysed by so-called carbohydrate-active enzymes (CAZymes; **Box 1**; [11]), which play a
pivotal role in the metabolism of FCW. While research on CAZymes produced by fungi has
mainly focused on finding those efficient at degrading and/or modifying PCW components for
industry, the study of CAZymes acting on FCW-derived substrates has principally focused on
biosynthetic enzymes (i.e. glycosyl transferases; GT; [12]), which are seen as promising
therapeutic targets to fight fungal pathogens [13]. Although most aspects of FCW enzymology
remain uncharted, there is an increasing corpus of studies highlighting the role of FCW-active
CAZymes in biological and environmental contexts. Our aim here is to give an account of the
diversity of these roles in fungal physiology, in pathogenesis of human, plant, and insect hosts,
in the global carbon cycle, and in the human gut microbiome. Finally, we highlight outstanding
questions in the field and exciting perspectives for future work.
FCW-active CAZymes in cell biology

CAZymes involved in FCW biosynthesis, belonging mainly to the GT class (see overview in Table 1), have been studied for long [14–17], and covering properly this class would be beyond the scope of the present review. Beyond biosynthesis, fungi also secrete a vast array of CAZymes catalyzing modifications and rearrangements of the polysaccharides constituting the FCW, ensuring wall plasticity. Among these enzymes, chitin-hydrolysing CAZymes, glycoside hydrolases (GHs) such as GH18 chitinases play a crucial role during cell division and morphogenesis, as demonstrated in the maize pathogen *Ustilago maydis* [18] and the human pathogen *Histoplasma capsulatum* [19]. The role of chitinases (GH18) and glucanases (GH5) in conidia germination has also been highlighted, as their genes are up-regulated during isotropic and polarized growth [20–22]. The products of chitin degradation can also have developmental effects: N-acetylglucosamine (GlcNAc) released by the action of chitobioses (GH20) on chito-oligosaccharides (CHOS), induced species-dependent morphological transitions in some *Candida* species [23]. Glucosamine (GlcN), released by the joint action of chitin deacetylases (CE4) and chitosanases (GH8), inhibited growth, reduced sporulation, and delayed germination and biofilm formation when exogenously applied to *A. fumigatus* [24]. We can speculate that the regulatory role of these FCW degradation products may also be relevant when produced by the enzymatic machinery of the fungus itself.

FCW-active CAZymes are not only harnessed for cell morphogenesis but also in autophagy and cell wall autolysis [25]. Indeed, when nutrient sources become scarce, the FCW of the aging hyphae represents a carbon source directly available to the growing fungus. For instance, under carbon starvation, *A. nidulans* produces a significant amount of *endo*-β-1,3-glucanase (“EngA”; GH81) and *endo*-chitinase (“ChiB”; GH18), releasing nutrients from dead hyphal segments [26–29]. During their life cycle, fungi can also be exposed to other types of stresses, such as osmotic pressure between the intra- and extra-cellular environments, exemplified by fungi able to live in harsh environments such as salterns [30]. As a case in point, when exposed to a hypersaline environment, *Aspergillus sydowii* strengthens its cell wall by increasing its chitin content via the down-regulation of β-1,3-glucan elongases (aka “Gel” family transglycosylases, GH72) [31]. A similar remodelling strategy has been observed when the fungus is exposed to inhibitors of β-1,3-glucan synthesis, such as echinocandin [32].

Chitin and β-glucans that provide stiffness to the FCW [1] form a cross-linking network, referred to as the chitin-glucan complex, important for the resilience of the FCW architecture. The cross-linking within and between these polymers is ensured by GHs with transglycosylation activity (notably GH16, GH17 and GH72) [33–35]. Deletion of the corresponding genes increased cell sensitivity to cell wall interfering compounds [36,37]. In
*Neurospora crassa*, β-1,3/1,4-glucan-active GH72 transglycosylases can, in synergy with GH76 α-1,6-mannanases, crosslink β-1,3/1,4-glucans with cell wall glycoproteins (CWP) [38]. CWPs can be covalently linked to the FCW, notably by GPI-anchors [39], the maturation of which is mediated by GH76 endo-α-1,6-mannanases. These ubiquitous transmembrane enzymes are suggested to be involved in membrane-to-cell wall transfers of GPI-anchor proteins, an important step in cell wall biogenesis [40]. These GH76 enzymes are able to discriminate between CWP and secreted glycoproteins [41]. They are important for osmotic stress resistance and hyphal morphology as demonstrated for *Trichoderma atroviride* where gene deletion led to cell wall disorganisation and an increase in non-covalent protein attachment to the cell surface [42]. In other fungi, GH76 enzymes were shown to be involved in cell separation and hyphal morphology [43], vegetative and colony growth, conidiation and appressorium maturation [41,44].

In their natural environment, fungi establish intercellular communication with the same or different species [45], ensuring maintenance and propagation of populations [46]. In *N. crassa*, a chitin-active lytic polysaccharide monooxygenase (LPMO; AA11) is involved in a so-called cell fusion checkpoint, blocking cell fusion upon contact between cells with nonidentical genetic backgrounds [47]. This regulatory function can protect the colony against the spread of infectious elements or defective organelles [48]. Intriguingly, this function did not depend on the enzyme's catalytic activity *per se*, suggestive of a moonlighting, non-catalytic mode of action.

Altogether, these examples show that endogenous FCW-active CAZymes are diverse and crucial contributors to fungal growth and development, playing key roles at every significant stage of the fungal life cycle, including in interaction with hosts, as further developed in the following sections.

## FCW-active CAZymes in pathogenicity

**Plant pathogens.** Depending on their lifestyle, phytopathogenic fungi can produce either a large set of PCW-active CAZymes (**necrotrophs and hemibiotrophs**), or a smaller set (**biotrophs**) [49] (see **Glossary**). Regardless of the lifestyle, infectious fungi must adopt strategies to make themselves undetectable by the plant's immune system. Hydrolysis of FCW-derived chitin and β-1,3-glucans by plant hydrolases releases oligosaccharides recognised by the plant as **pathogen-associated molecular patterns (PAMPs)**, triggering a reactive oxygen species (ROS) burst in plant leaves [50,51]. As a defensive strategy, (hemi)biotrophic fungi produce protein effectors that bind to and/or degrade these PAMPs. Examples include β-1,3-glucanases from the biotroph *U. maydis* (“Erc1”, predicted as an unusual GH51 family member) [52] and the hemibiotroph *Colletotrichum graminicola*
("CgEC124", GH128) [53], as well as chitin binding modules (CBM18; aka **lectins**) with high affinity for chitin oligomers that outcompete plant chitin receptors such as in the hemibiotroph *Verticillium nonalfalfae* [54] and the biotrophic flower-infecting *Ustilaginoidea virens* [55]. Alternatively or complementarily, some fungi use FCW camouflage strategies such as Mg1LysM (CBM50, aka **lectins**), a single LysM domain-containing effector from the wheat pathogen *Zymoseptoria tritici*, which upon oligomerization shields hyphal FCW chitin against plant chitinases [56]. In addition to sequestering oligosaccharides, the fungus can produce enzymes that render the PAMP non-immunogenic. As an illustration, the biotroph *Podosphaera xanthii* co-expresses endo-chitinases and *PxLPMO1* (a non-classified LPMO) during the first stages of melon infection, where the LPMO oxidises CHOS, making them undetectable by plant receptors [57]. Another strategy to evade chitin-triggered plant immunity is chitin modification to chitosan by CE4 chitin deacetylases, as shown in various plant pathogens including the hemibiotroph *Verticillium dahliae* [58], the wheat pathogen *Puccinia striiformis f. sp. tritici* [59], the rice blast fungus *Magnaporthe oryzae* [60], or the corn pathogen *U. maydis* [61,62]. Recently, *U. maydis* has been shown to secrete various AA class carbohydrate oxidases active on FCW components [63,64], but their biological functions are yet to be unveiled.

**Human pathogens.** Millions of people around the world are affected by life-threatening fungal diseases, which cause more than 3 million deaths every year [65]. The most significant are aspergillosis, candidiasis, cryptococcosis, and pneumocystic pneumonia. Numerous studies have investigated the processes by which the corresponding pathogenic fungi infect and overwhelm their hosts' immune system. While a common strategy of human pathogens is to modify their chitin into chitosan via CE4 chitin deacetylases [66–68], each pathogen displays its own specificities. *Cryptococcus neoformans*, one of the most infamous causative agents of cryptococcosis [69], undergoes morphological changes during its infectious cycle, with an increase in cell size leading to the formation of so-called titan cells [70]. Comparative analysis of the FCW of titan cells and normal cells revealed that the former contain a higher proportion of mannose, xylose, and chitin, and a reduction in glucans. These observations suggest a major enzymatic remodelling process in *C. neoformans* cell wall, which likely involves glucanases, chitinases, transglycosylases, and other CAZymes [71]. It has also recently been shown that an AA9 LPMO (*CnCel1*) whose gene is induced in host-relevant condition, is associated with the cell surface of *C. neoformans* and is required for cell wall remodelling, stress resistance, and virulence [72]. However, its actual substrate and activity remain to be identified.

*Candida albicans*, the causative agent of candidiasis, can pose a vital threat, becoming systemic when it crosses the blood barrier in immunocompromised patients or causes
disruption of the mucosal microbiome. In response to some environmental stimuli, such as acidic pH, hypoxia, or lactate [73], and during hyphal development [74], β-1,3-glucans will be exposed and recognized as major PAMP by the human lectin receptor Dectin-1 inducing host immune response. To evade this response, *C. albicans* secretes β-1,3-glucanases (GH81, GH5_9) in order to trim the exposed β-1,3-glucans and escape recognition by the immune system [74–76].

A. fumigatus, the main causative agent of aspergillosis, can germinate and lead to invasive pulmonary infections when inhaled by immunocompromised individuals [77,78]. As β-1,3-glucans are an essential component of the core structure of the A. fumigatus cell wall, their remodelling is a vital process, and several families of β-1,3-glucanase have been identified as crucial agents of infection [79]. Multiple deletion of genes coding for GH81 (ENG1) and GH16 enzymes (ENG2-5, ENG6, and ENG7) leads to defects in conidial separation and altered morphology during conidial swelling due to the inability to degrade primary septa rich in β-1,3-glucans [80]. In contrast, the GH55 family of β-1,3-glucanases, present in six copies in A. fumigatus, has been shown to be involved in conidial maturation and germination processes [81]. The outermost part of the A. fumigatus wall is formed of so-called **fungal-type galactomannans (FTGM)** and O-mannose-type galactomannans (OMGM) [82]. Two members of the GT15 family of mannosyltransferases are involved in the biosynthesis of the α-1,2-mannan segments of FTGM backbone. Deleting these genes caused hyperbranched mycelium, impaired growth, reduced conidiation and viability, and decreased virulence in mice [83]. Family members of α-1,6-mannosyltransferase (GT62), involved in the biosynthesis of the α-1,6-mannan segments of FTGM backbone, are not crucial for A. fumigatus pathogenicity but help maintain the structural strength of the cell wall, conidia formation, and mycelial elongation [84]. Additionally, deletion of β-1,5-galactofuranosyltransferases (GT31) involved in the polymerization of FTGM side chains - composed of β-1,5-/β-1,6-galactofuranosyl chains - led to reduced conidium formation and hyphal growth, abnormal hyphal branching, and increased surface hydrophobicity [14].

**Insect pathogens.** Today, entomopathogenic fungi represent an environmentally-friendly alternative to chemical insecticides, which are toxic for both wildlife and humans. The mechanisms by which these fungi infect and kill their hosts involve FCW-acting enzymes [85]. In *Beauveria bassiana*, the causative agent of white muscardine disease in arthropods, fungal chitin deacetylases (CDAs, CE4) are involved in spore formation and germination processes, and delayed cuticle penetration [86]. Like with the plant pathogens discussed above, chitin deacetylation reduces the pathogen's susceptibility to the chitinases produced by its host. *B. bassiana* can also evade host immunity by suppressing the production of FCW-derived PAMPs. BbEng1 is a cell-surface β-1,3-glucanase (GH16) whose gene is specifically
expressed during infection and under conditions of oxidative or osmotic stress. This enzyme degrades glucan PAMPs, promoting colonisation of the host by the fungus [87]. In the same vein, an *N*-acetyl-β-D-glucosaminidase (GH20) from *B. bassiana* hydrolyses CHOS released upon action of fungal chitinases on the insect cuticle, rendering them non-immunogenic and thereby contributing to a successful infection [88].

FCW-active CAZymes in carbon cycle

Decomposition of organic matter by fungal CAZymes is key to carbon cycling in fertile soils [89]. In addition, the accumulation of fungal necromass contributes significantly to the build-up of organic carbon in the soil, but the enzymatic recycling of FCW biopolymers has been under-studied [90,91]. Researchers have proposed the concept of a “necrobiome” with bacteria and fungi enriched during phases of fungal decomposition [92,93]. Transcriptomic analyses have shown that *T. reesei* upregulates the expression of CAZyme genes specific to different exposed layers of a decomposing Ascomycota cell wall, to enable the step-wise degradation of melanin, proteins, and polysaccharides in the FCW [94].

The phenomenon of mycoparasitism, where organisms kill fungi, is relevant to the turning over of fungal biomass and necromass in the soil, as well as the biocontrol of pathogens [95]. *Trichoderma* species have been well studied in this context and are used in some biocontrol initiatives [96,97]. Although mycoparasitism often involves the production of toxic secondary metabolites, the secretion of FCW-degrading CAZymes is also key, as the cell wall is the outer shield protecting the fungus from the environment [98,99]. Chitinases secretion and specificities are key, such as the hydrolytic *N*-acetyl-β-D-glucosaminidases (GH20), which underpins some of the selectivity seen in biocontrol by *Trichoderma* [100]. Indeed, supplementing soil with chitin or chitin-derivatives enriches the microbiome for chitin-degraders to improve soil health and decrease plant disease [101–103]. Recent data suggest that chitinolytic mycoparasitic fungi may prevent self-inflicted damage via an enrichment of chitosan in the cell wall [104]. Other FCW-active enzymes such β-1,3- and β-1,6-glucanases (GH55) are also secreted by *T. virens*, conveying potent anti-fungal activity, as well as control of the oomycete phytopathogen *Pythium ultimum*, whose cell wall lacks chitin but is enriched in glucans [105,106].

Beyond fungal enzymes, research has been clarifying the importance of bacterial enzymes in the recycling of chitin-rich biomass in diverse ecosystems [107]. There is a complex interplay between fungal and bacterial CAZymes in the turnover of necromass [3]. Bacterial genera proficient in complex carbohydrate degradation, especially the Bacteroidota [108] are known to be enriched in specific microhabitats in soil, so-called hotspots of secreted CAZyme activity
where dead fungal mycelium is being decomposed [109]. Fluctuations in the bacterial
community were associated with changing CAZyme activity levels and the eventual
degradation of recalcitrant biopolymers. A similar study used metagenomic sequencing,
proteomic profiling, and enzyme assays to identify *Ewingella americana* and two
*Pseudomonas* isolates as the key drivers of FCW decomposition [110]. These bacteria used
various chitin-active CAZymes (incl. GH5, GH8, GH18, GH19, GH20, AA10), while some
abundant species either released β-glucanases (incl. GH5, GH17, GH73) or relied on
oligosaccharides released by others [110]. For a closer look at this phenomenon, recent work
assembled a co-culture of *Cellvibrio japonicus*, *Chitinophaga pinensis*, and *Serratia marcescens*, all of which abundant in soil and capable of necromass degradation [111]. This
study compared CAZyme-encoding gene expression and overall growth rates of these bacteria
on high-melanin and low-melanin forms of necromass, finding that production levels of FCW-
active CAZymes like chitinases, mannanases, and glucanases, were similar despite the slower
growth in high melanin conditions [111]. By considering the growth profiles of the three
bacteria, as well as the nature and rapidity of their transcriptional response to necromass, the
authors proposed that *C. pinensis* likely dominates during the early stages of fungal
decomposition, due to the rapid production of a broad swathe of FCW-active CAZymes [112–
115]. By contrast, *C. japonicus* is proposed as a high-efficiency FCW degrader during mid-
stage fungus decomposition, as a substantial lag phase preceded the strong growth and high
production of relevant CAZymes by this species [116–119].

FCW-active CAZymes in the human gut

Microbes dwelling in complex, competitive environments are often difficult to isolate, which is
why our understanding of the functions of the human gut microbiome has grown in step with
the development of meta-omic sequencing technologies. Although the bacteria of the human
gut have been examined in more detail, we now know that there is a so-called mycobiome, a
fungal community resident in the human intestines. Higher gut fungal abundance and diversity
has been connected with certain markers of good health [120], whereas dysbiosis of the
mycobiome is being explored in colorectal cancer [121]. Certain species seem to correlate with
disease states [122] such as Crohn's disease [123,124]. In addition, diverse strains of
opportunistic *C. albicans* become enriched during periods of inflammation in Inflammatory
Bowel Disease (IBD) patients [125]. Changes in diet, including alterations in dietary
polysaccharide intake, are also relevant to mycobiome modulation around IBD, hinting at the
importance of fungal CAZyme specificities in determining fungal proliferation in the gut
(reviewed in [126]).
While the mycobiome has been mostly studied in sequencing-based [127,128] and immune-
focussed [129,130] investigations, the bacterial gut microbiome has been subjected to closer
biochemical examination. Although most research into the degradation of carbohydrates by
gut bacteria has focussed on plant-derived substrates from the human diet, a few key studies
have shown the importance of FCW-derived carbohydrates. Indeed, bacteria in the human gut
are exposed to FCW substrates from several sources. Dietary sources include edible fungi like
the mushroom *Agaricus bisporus* [131], baker's and brewer's strains of the yeast
*Saccharomyces cerevisiae* [132], and industrially-produced functional food ingredients like
mycoproteins [133]. Meanwhile, both fungal pathogens and symbionts of the mycobiome itself
can also represent sources of FCW-derived substrates.

It seems likely that all these sources of FCW represent accessible nutrition for gut bacteria, but few examples of the CAZymes involved have so far been described. One important study showed that a model gut symbiont, *Bacteroides thetaiotaomicron*, can feed on yeast-derived α-mannans using CAZymes encoded by a Polysaccharide Utilisation Locus (PUL) [134]. This study was the first to explicitly describe the use of PUL-based CAZymes as “selfish”, evidenced by total retention of mannose and manno-oligosaccharides released enzymatically from yeast FCW substrates. In the same bacterial species, a small PUL encoding just two enzymes was shown to support β-1,6-glucan catabolism by *B. thetaiotaomicron* [135]. Syntenic PULs in other gut-dwelling *Bacteroides* were observed and indicated a widespread ability to consume β-1,6-glucans. In some cases, CAZymes capable of hydrolysing fungal β-glucans are also able to hydrolyse PCW-derived β-glucans, complicating our understanding of the niche specialisation of enzymatic pathways [136].

More recently, cross-feeding studies with *Bacteroides* and *Bifidobacterium* species of known
β-glucan-hydrolysing capacities revealed extensive sharing of released oligosaccharides
[137], showing how FCW-derived substrates can support a complex mutually interdependent
bacterial community. Moreover, consumption of processed mycoprotein foods that contain
fungal β-glucans correlated with an enrichment of known β-glucan-degrading *Bacteroides*
species in gut-mimicking fermentation experiments [138].
Concluding remarks and future perspectives

Through this short review, we have attempted to show that deciphering the role of FCW-
targeting enzymes, and understanding how their functions are regulated, is of utmost
importance for a deeper biochemical understanding of fungal biology. While we focused on
CAZymes, as they are likely the main players, other types of enzymes such as proteases,
should not be overlooked and will deserve attention in the future.

The prediction of protein function from sequence is still a major bottleneck in modern biology, and CAZymes are no exception. The study of enzymes secreted at the interface between the fungus and its environment presents special difficulties: the substrate versatility of some CAZymes complicates the identification of their preferential substrate (e.g. PCW vs FCW in the case of glucans), and the often-unclear role of co-factors (e.g. metals) and protein partners in catalysis add an extra layer of complexity. Relying on function predictions is not sufficient and can even be misleading. Huge efforts therefore still need to be made to unravel the true functional role of CAZymes in their ecological context. This calls for highly interdisciplinary approaches, where “old-school” enzymology still has a central role to play. We urge for systematic biochemical testing of presumed enzymatic activities in biological studies and also request that we keep our minds open with regards to possible ‘moonlighting’ functions in proteins, where the activity of an enzyme observed under *in vitro* conditions might not reflect its true biological function. In the same vein, the availability of adequate “biological” substrates is a true bottleneck for enzymology as commercial polysaccharides (but also those extracted in-house) are often not pure and their natural properties might be altered by the extraction process. Evaluating enzymatic activities on as natural as possible substrates, or, ideally, *in situ*, constitutes thus a challenging necessity.

It is truly fascinating how fungi have evolved a myriad of strategies to adapt, invade, recycle,
and collaborate within their ecosystem. This vast biological diversity serves as an invaluable
source of bioinspiration. To illustrate just a few possibilities, understanding the enzymatic
processes at the interface between plants/humans and their fungal pathogens could allow the
design of new biological control and biostimulation strategies. We also anticipate that the
growing understanding of the human gut microbiota, and the role of CAZymes in releasing
prebiotic FCW components or in combatting pathogenic fungi, will contribute to improving
human health. Although we have not discussed the topic here, better understanding of FCW
architecture and the CAZymes that interact with it may also allow the design of new
environmentally-friendly biomaterials or new feed sources, and may offer better mastery of the
behaviour of yeast/fungi in bioprocesses. Given the biodiversity of “fungus-environment”
interfaces, and the largely uncharted structural diversity of the FCW, we foresee that both
ongoing and future research on FCW enzymology herald discoveries across a variety of fields, from agriculture to industrial biotechnology and medicine.
Box. Outstanding Questions

* How broad is the sequence-activity space of FCW-active CAZymes?

* What is the exact role of CAZymes in FCW remodelling during various physiological processes, including stage transitions, vesicles secretion or adaptation to host?

* What is the contribution of FCW-active CAZymes in the soil carbon cycle in different parts of the world and under different types of land use?

* How do FCW-active CAZymes impact the gut-brain axis?

* How and to what extent can FCW-active CAZymes contribute to the development of a new generation of FCW-based biomaterials?
**List of Figures:**

**Figure 1.** Schematic overview of FCW architecture

**Figure 2.** Case studies on the use of FCW-active CAZymes by well-known human and plant fungal pathogens.

**Figure 3.** FCW-active CAZymes in fungal necromass turnover and global carbon cycle.

**Figure 4, Key Figure.** Overview of biological processes in which FCW-active CAZymes play a crucial role.

**Table 1.** Inventory of FCW-active CAZymes.

**Box 1.** The CAZymes nomenclature.
<figure><img src="image_3.png" /><figcaption>**Figure 1. Schematic overview of FCW architecture.** Models of yeast cell wall (C. albicans, on the left) and filamentous fungus cell wall (hyphal form of A. fumigatus, on the right), prepared on the basis of models and data reported in [5,7,139]. Of note, these models are not to scale. The FCW of these two organisms are the best known to date, as a result of the combination of biochemical and biophysical analyses. They display common polysaccharides such as chitin and β-glucans, and specific polysaccharides (written in red) such as α-glucans, mannans or galactosaminogalactans.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Case studies on the use of FCW-active CAZymes by well-known human (left) and plant (right) fungal pathogens. A. *fumigatus* causes lung infections in immunocompromised individuals. Spore germination, which includes changes in FCW composition, involves a set of CAZymes that are active on the polysaccharides of the fungal wall. The same is true for the plant pathogen *Ustilago maydis*, the causative agent of corn smut disease. The white bold stars highlight enzymes with a proven involvement in FCW remodeling during infection [18,62,79,81,140], while the other CAZymes displayed on the figure need deeper investigations. See **Table 1** for details on activities for each family.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Figure 3. FCW-active CAZymes in fungal necromass turnover and global carbon cycle.**</figcaption></figure>

Alive microbial biomass secretes a range of CAZymes targeting the different polysaccharides of dead fungal mycelium, releasing thereby carbohydrates used as nutrient source by living microorganisms, and contributing to soil organic carbon turnover. The fungal necromass can represent up to 60% of the microbial necromass. The latter can also be stabilized by association with minerals (not shown). Of note, on the right-hand side of the figure, cell wall proteins are not shown for the sake of clarity. Bacteria have different enzymatic arsenal, which, will act at different stages of the degradation process. Here, we show the main CAZymes detected as upregulated during fungal necromass degradation by soil bacteria [111]. See **Table 1** for a more extensive list of CAZymes potentially involved in FCW degradation. A GH20 is shown as an example of CAZyme acting on oligosaccharides while other families will be recruited to generate the various monosaccharides.
<figure><img src="image_6.png" /><figcaption>**Figure 4, Key Figure.** Overview of biological processes in which FCW-active CAZymes play a crucial role. The circle in the center of the figure shows the interface between the FCW (in purple) and its environment (in green). CAZymes secreted by the fungus (purple arrows) can act on exogenous targets or on the FCW itself. Alternatively, environmental CAZymes (e.g. from bacteria or plants) can also act on the FCW. Around the central circle, the green boxes illustrate some of the major biological contexts in which FCW-active CAZymes are instrumental and where research is highly needed.</figcaption></figure>
<table><caption>Table 1. Inventory of FCW-active CAZymes.</caption><thead><tr><th>FCW substrates</th><th>CAZymes Family</th><th>Activities*</th></tr></thead><tbody><tr><td rowspan="14">Chitin</td><td>GH16_18</td><td>Chitin β-1,3/1,6-glucanosyltransferase</td></tr><tr><td>GH16_19</td><td>Chitin β-1,3/1,6-glucanosyltransferase</td></tr><tr><td>GH18</td><td>Chitin endo-β-1,4-N-acetylglucosaminidase</td></tr><tr><td>GH20</td><td>β-N-acetylhexosaminidase</td></tr><tr><td>GT2</td><td>N-acetylglucosaminyltransferase / chitin synthase</td></tr><tr><td>GT4</td><td>Phosphatidylinositol N-acetylglucosaminyltransferase</td></tr><tr><td>CE4</td><td>Chitin deacetylase</td></tr><tr><td>CE9</td><td>N-acetylglucosamine-6-phosphate deacetylase</td></tr><tr><td>CBM14</td><td>Chitin binding module (Avirulence protein 4, Avr4)</td></tr><tr><td>CBM18</td><td>Chitin binding module (lectin)</td></tr><tr><td>CBM50</td><td>Chitin binding module (LysM effector)</td></tr><tr><td>AA7</td><td>Chitooligosaccharide oxidase</td></tr><tr><td>AA10</td><td>Lytic chitin monooxygenase</td></tr><tr><td>AA11</td><td>Lytic chitin monooxygenase</td></tr><tr><td rowspan="3">Chitosan</td><td>GH8</td><td>Chitosanase</td></tr><tr><td>GH46</td><td>Chitosan endo-β-1,4-N-glucosaminase</td></tr><tr><td>GH75</td><td>Chitosan endo-β-1,4-N-glucosaminase</td></tr><tr><td rowspan="16">Glucans</td><td>GH5_9</td><td>Exo-β-1,3-glucanase</td></tr><tr><td>GH5_15</td><td>Endo-β-1,6-glucanase</td></tr><tr><td>GH16_1</td><td>β-1,3-1,4-glucanase / lichenase / β-1,3-glucanase</td></tr><tr><td>GH16_3</td><td>β-1,3-glucanase</td></tr><tr><td>GH17</td><td>β-1,3-glucanosyltransferase / β-1,3-glucanase</td></tr><tr><td>GH30_3</td><td>β-1,6-glucanase</td></tr><tr><td>GH55</td><td>Glucan exo/endo-β-1,3-glucosidase</td></tr><tr><td>GH71</td><td>Endo-α-1,3-glucanase</td></tr><tr><td>GH72</td><td>β-1,3-glucanosyltransferase</td></tr><tr><td>GH81</td><td>Endo-β-1,3-glucanase</td></tr><tr><td>GH128</td><td>Glucan exo-β-1,3-glucosidase</td></tr><tr><td>GH131</td><td>Bifunctional exo-β-1,3-glucanase and endo-β-1,4-glucanase</td></tr><tr><td>GH132</td><td>Exo-β-1,3-glucanase</td></tr><tr><td>AA3_2</td><td>β-1,3/ β-1,6-glucooligosaccharides dehydrogenases</td></tr><tr><td>GT48</td><td>β-1,3-glucan synthase</td></tr><tr><td>GT5</td><td>α-1,3-glucan synthase</td></tr><tr><td>CBM43</td><td>β-1,3-glucan binding module</td></tr><tr><td rowspan="7">Mannan – Galactomannan-Galactosaminogalactan</td><td>GH76</td><td>Mannan endo-α-1,6-mannosidase</td></tr><tr><td>GH114</td><td>Endo-α-1,4-galactosaminidase</td></tr><tr><td>GH135</td><td>α-1,4-galactosaminogalactan hydrolase</td></tr><tr><td>GT15</td><td>α-1,2-mannosyltransferase</td></tr><tr><td>GT31</td><td>β-galactofuranoside-β-1,5-galactosyltransferase</td></tr><tr><td>GT62</td><td>α-1,6-mannosyltransferase</td></tr></tbody></table>

<footnote>*Activities are based according to description made in the CAZy database (www.cazy.org; [141]), which rely on biochemical evidences. For format constraints, each original article cannot be cited here and we orient the reader towards the CAZy database where all references are duly cited.</footnote>
Box. 1. The CAZymes nomenclature

Carbohydrate-active enzymes (CAZymes) (www.cazy.org) [11,141,142] are organized on the basis of sequence similarity into five enzyme classes and one class of associated carbohydrate binding modules (CBMs):

- **Glycoside Hydrolases (GH):** involved in the hydrolysis and/or rearrangement of glycosidic bonds). Of note, while most GHs are hydrolytic enzymes, some have the capacity to catalyse a so-called transglycosylation (TG) reaction (i.e. formation of glycosidic bonds, via a mechanism that differs from glycosyl transferases (see [143]), and the ratio between hydrolase and transglycosylase activities varies depending on the family and the reaction conditions.

- **Glycosyl Transferases (GT):** involved in the formation of glycosidic bonds.

- **Polysaccharide Lyases (PL):** involved in the non-hydrolytic cleavage of glycosidic bonds.

- **Carbohydrate Esterases (CEs):** involved in the hydrolysis of carbohydrate ester bonds.

- **Auxiliary Activities (AA):** oxidoreductases that act, in general, in synergy with other CAZymes.

Today (as of March 2025), there are 189 GH families, 138 GT families, 44 PL families, 20 CE families and 17 AA families, which belong to 20 different clans on the basis of their 3D structure (clans A to T). Of note, some CAZy families have been divided into subfamilies (e.g. GH5, GH16) to optimize the prediction of functions.

Importantly, facing the observation that enzymes naming is too heterogenous in the community studying FCW-active enzymes, we would like to deeply encourage the community to employ the CAZy nomenclature as it allows a better identification of enzymes activities and their comparison, as well as a better trackability of enzymes throughout the literature.
## Glossary

**Lectin:** A class of protein that bind to carbohydrate and can be responsible of agglutination or precipitation of glycoconjugates and polysaccharide. They are standalone CBMs not appended to any catalytic domain.

**Mannan structure of FTGM:** A polymer made up of a main α-1,2/ α-1,6-mannan chain and a number of side chains of β-1,5/ β-1,6-linked Galf residues.

**Necrotrophs and (hemi)biotrophs.** Phytopathogenic fungi adopt varying strategies to infect their hosts: biotrophs are dependent on and develop in living tissues; necrotrophs kill their hosts and develop on dead tissue; and hemibiotrophs start their infection cycle with a biotrophic phase followed by a necrotrophic phase [144].

**pathogen-associated molecular patterns (PAMPs):** Small molecules with a conserved motif, specific of the pathogen and not found in the host, recognized by the host pattern recognition receptors (PRRs) as a signal of the presence of an aggressor or of an occurring infection.

**Polysaccharide Utilization Locus (PUL):** Strictly regulated, colocalized gene clusters that encode enzymes and protein ensembles required for the degradation, capture and transport of complex carbohydrates. PULs are prevalent in the Bacteroidetes bacterial phylum.

## Abbreviations

**CBM:** Carbohydrate binding module

**CHOS:** Chitooligosaccharides

**CWP:** Cell wall proteins

**FCW:** Fungal cell wall

**FTGM:** fungal-type galactomannans

**GM:** Galactomannans

**GPI:** Glycosylphosphatidylinositol

**OMGM:** O-mannose-type galactomannans

**PAMP:** Pathogen-associated molecular patterns

**PCW:** Plant cell wall

**PUL:** Polysaccharide Utilisation Locus

## Author contributions

R.A.Y. conceptualised, wrote, edited, and revised the manuscript; and conceptualised and generated the figures. J.-G.B. conceptualised, edited and revised the manuscript; L.S.M. conceptualised, wrote, edited, and revised the manuscript; B.B. conceptualised, wrote, edited, and revised the manuscript; and conceptualised and generated the figures.

## Acknowledgements

We thank current and past members of our research groups that have contributed to fungal cell wall enzymology research. We apologize in advance to all investigators whose research cannot be appropriately discussed and cited owing to space limitations. R.Y. thanks Aix-Marseille University for funding her PhD thesis. B.B. thanks the French National Research Agency (ANR) for support on FCW enzymology research (OXIWALL project, Grant Number ANR-23-CE44-0023).
**Declaration of interests**

The authors declare no competing interests.
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