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Adsorption Capacity of CBM104s Appended to Different Types of Catalytic Domains
2025 年 72 巻 4 号 論文ID: 7204101
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2025 年 72 巻 4 号 論文ID: 7204101
Filamentous fungi use various enzymes to degrade cellulose, some of which contain cellulose-binding domains (CBDs), most of which belong to carbohydrate-binding module family 1 (CBM1). We recently identified the novel fungal CBD, CBM104, from Gloeophyllum trabeum. Reportedly, CBM104 specifically binds to native crystalline cellulose, not to amorphous or artificially modified crystalline cellulose, exhibiting a unique adsorption characteristic. To gain further insights into CBM104, the adsorption properties of six different CBM104s, each appended to a different catalytic domain, were investigated. The adsorption tests illustrated that all CBM104s predicted to possess a three-dimensional structure in which two α-helices were crosslinked by disulfide bonds specifically adsorbed onto cellulose I. Conversely, CBM104 lacking these disulfide bonds failed to adsorb onto any form of cellulose used in this study, suggesting the importance of the fixed pair of α-helices for specific binding to cellulose I. To identify CBM104 homologs in which the disulfide bonds are conserved, a homology search was performed against fungal genomes, resulting in 144 hits. These CBM104 homologs were primarily appended to auxiliary activities (AA) family 9 or to domains that work cooperatively with AA9 enzyme. CBM104s were found only in certain orders of Agaricomycetes, and the majority of these fungi are suggested to have the ability to degrade plant cell walls. These results suggest that some Agaricomycetes utilize plant cell wall degradation systems involving CBM104-attached proteins. This study provides detailed insights into the structural factors involved in the adsorption capacity of CBM104, as well as its phylogenetic distribution.
AA, auxiliary activities; CAZy, Carbohydrate-Active enZyme; CBH, cellobiohydrolase; CBM, carbohydrate-binding module; CBD, cellulose-binding domain; GH, glycoside hydrolase; JGI, Joint Genome Institute; LPMO, lytic polysaccharide monooxygenase; PASC, phosphoric acid-swollen cellulose; RFP, red fluorescent protein.
Cellulose, a primary component of plant cell walls, is among the most abundant biopolymers on Earth [1, 2]. Filamentous fungi, which are the predominant organisms responsible for cellulose decomposition in nature, secrete several cellulose-degrading enzymes. These include glycoside hydrolases, such as cellobiohydrolases (CBHs), endoglucanases, and β-glucosidases, as well as oxidoreductases, such as lytic polysaccharide monooxygenases (LPMOs), which cleave cellulose chains oxidatively [3, 4]. These enzymes often contain cellulose-binding domains (CBDs) at their N- or C-termini. Most CBDs derived from fungi have been classified into family 1 carbohydrate-binding module (CBM1) in Carbohydrate-Active enZyme (CAZy) database [5, 6]. CBM1 members feature three conserved aromatic amino acid residues forming a planar surface, enabling hydrophobic interactions with the pyranose rings of cellulose chains [7, 8, 9]. A previous study revealed that the artificial removal of CBM1 from CBH, which continuously degrades cellulose chains to form crystalline regions, led to reduced hydrolytic activity toward crystalline cellulose and had no effect on activity against amorphous cellulose [10]. Furthermore, deletion of CBM1 from fungal auxiliary activity (AA) family 9 LPMOs, such as PaLPMO9H from Podospora anserina, reduced the yield of soluble products from crystalline cellulose, amorphous cellulose, and cellulose nanofibers [11]. These observations strongly suggest that CBM1 promotes the activity of glycoside hydrolases and oxidoreductases by facilitating the localization of their catalytic domains to the cellulose surface.
Wood-rotting fungi are filamentous fungi that degrade wood cell walls and serve as the primary decomposers of wood in forest ecosystems. Among them, brown rot fungi are the primary decomposers in northern coniferous forests. The fungi selectively degrades cellulose and hemicellulose and chemically modifies lignin, which remains as a polymer [12]. Despite their remarkable cellulose-degrading capabilities, most brown rot fungi, excluding those in the order Boletales, lack CBM1-appended cellulases [13, 14, 15, 16]. Thus, it is recognized that the significance of CBDs in the enzymatic degradation system is lower in brown rot fungi than in other cellulolytic filamentous fungi. However, we recently discovered that the LPMO from Gloeophyllum trabeum (GtLPMO9A-2), a species of brown rot fungus, possesses a C-terminal CBD, which has been newly classified as CBM104 [17]. Unlike CBM1 that adsorbs onto natural crystalline cellulose (cellulose I), artificial crystalline celluloses (cellulose II and III), and artificial amorphous cellulose (phosphoric acid-swollen cellulose), CBM104 specifically adsorbs onto cellulose I, a unique characteristic not observed in any previously known CBMs [17].
CBM104 has been found in certain wood-decaying Agaricomycetes species, and is appended by AA9 as well as AA8, AA16, and GH131. These CBM104s not only contain highly conserved amino acid residues and regions but also exhibit relatively high diversity. Therefore, it is unclear whether CBM104 appended to different types of catalytic domains exhibits the same function as that appended to GtLPMO9A-2. In this study, six types of CBM104 appended to different catalytic domains derived from various fungi were heterologously expressed as fusion proteins with red fluorescent protein (RFP) in the yeast Komagataella phaffii (Pichia pastoris), and their adsorption abilities were investigated. Based on the results, a comprehensive analysis of amino acid sequences that conserve the characteristics of CBM104 was conducted, providing new insights into the distribution of CBM104 in fungi.
Substrates
Cellulose was prepared using a previously described method [17]. To prepare cellulose Iα and Iβ from Cladophora and cellulose Iβ from Halocynthia, Cladophora sp. and Halocynthia roretzi were repeatedly treated with 5 % KOH and 0.3 % NaClO2 for purification. The Cladophora cellulose was further hydrothermally treated in 0.1 M NaOH solution at 260 °C to obtain a cellulose Iβ. Subsequent hydrolysis was performed using 4 M hydrochloric acid, followed by centrifugation, washing with water, and collection of the supernatant [18]. Phosphoric acid-swollen cellulose (PASC) was prepared by mixing Avicel (Funakoshi Co., Ltd., Tokyo, Japan) with 85 % (w/w) phosphoric acid, followed by stirring to yield a completely clear solution. After overnight incubation at 4 °C, the cellulose was regenerated in water, and a suspension was created using a high-speed blender. The cellulose suspension was washed with water prior to use [19].
Strains
Escherichia coli strain TOP10 (Thermo Fisher Scientific Inc., Waltham, MA, USA) and K. phaffii strain KM71H (Invitrogen Corp., Waltham, MA, USA) were used as hosts for subcloning experiments and heterologous production of recombinant proteins, respectively.
Sequence analysis
The amino acid sequence of CBM104 appended to GtLPMO9A-2 (GtAA9-CBM104) has been already determined in previous studies [17, 20]. The three-dimensional structures were predicted using AlphaFold 3 [21]. To explore the distribution of CBM104 domains in nature, homology searches were conducted against the masked assembly and filtered model proteins in the fungal genome database of the Joint Genome Institute (JGI) Genome Portal (https://genome.jgi.doe.gov/programs/fungi/index.jsf) using the tblastn and blastp algorithms, respectively. In these searches, the amino acid sequence of GtAA9-CBM104 was used as the query, with the BLOSUM62 substitution matrix and an E-value threshold of 1 × 10−3. In the tblastn search, nucleotide sequences of approximately 2,000 bp, including 1,000 bp upstream and downstream of the hit nucleotide positions, were used to predict exon sequences with the gene structure prediction program Fgenesh plus (RRID:SCR_018928) [22]. After excluding predicted amino acid sequences that did not retain the CBM104 structure or featured incomplete full-length sequences, 97 sequences identified by the blastp search were integrated, resulting in the analysis of 144 sequences as CBM104s. The characteristics of fungi possessing these sequences were obtained from the JGI database and previous research [23, 24]. The domains appended to CBM104s were predicted for each sequence using National Center for Biotechnology Information (NCBI) blastp [25] and MAFFT Multiple Sequence Alignment Software Version 7 [26]. Totally, 144 sequences were aligned again using MAFFT, and a phylogenetic tree was generated using the neighbor-joining method in MEGA X with 1,000 bootstrap replicates [27, 28].
Construction of expression vectors
The DNA fragments encoding RFP-fused CBM104s were artificially synthesized according to the sequence from the JGI database for heterologous expression in the P. pastoris expression system (Table S1; see J. Appl. Glycosci. Web site). The synthesized DNAs were inserted into the XhoI and NotI sites of pPICZαA (Invitrogen Corp.). Each CBM104 was fused to RFP via its original linker region. The nucleotide sequences of the inserted fragments were confirmed by sequence analysis. RFP-fused GtAA9-CBM104 was prepared as previously described [17].
Heterologous expression and purification of recombinant proteins
Recombinant proteins were expressed in K. phaffii and purified as previously described [17]. Following purification, the proteins were analyzed by SDS-PAGE to confirm that their purity was sufficient for the adsorption assay.
Adsorption onto various celluloses
The adsorption tests for various celluloses were conducted as previously reported [17]. Cellulose Iα and Iβ from Cladophora, cellulose Iβ from Halocynthia, and PASC were used as substrates. Reaction mixtures (100 μL) contained 0.1 % substrate, 100 mM sodium acetate buffer (pH 5.0), and 1 μM recombinant protein. After incubation at 4 °C for 2 h, the reaction solution was filtered through a 96-well filter plate (MilliporeSigma, Burlington, MA, USA) to remove the substrate and adsorbed recombinant proteins. The fluorescence of the filtrate was measured using a microplate reader (Fluoroskan Ascent, Thermo Fisher Scientific Inc.) at excitation and emission wavelengths of 559 and 599 nm, respectively. The concentration of protein in the filtrate was calculated from the fluorescence intensity of RFP. Each measurement was performed in triplicate. Error bars represented the standard error (n = 3) and asterisks indicated samples that differ significantly from RFP, as determined by a t-test (p < 0.05). To calculate the adsorption parameters for cellulose I, the levels of adsorption at various protein concentrations (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 μM) were measured using the same experimental conditions. The adsorption data were fitted to Hill equation using Delta Graph software (version 7.07), and adsorption parameters, namely the apparent association constant (
CBM104 exhibits a unique adsorption property in that it binds to native crystalline cellulose but does not adsorb onto amorphous cellulose (PASC) or artificial crystalline cellulose with different crystalline forms, such as cellulose II and cellulose III. It has also been shown that CBM104 can adsorb onto cellulose I regardless of its biological origin, and that it binds to both cellulose Iα (triclinic structure) and cellulose Iβ (monoclinic structure), which differ in their crystalline structures [17]. However, the only reported CBM104 to date is the CBM104 domain of GtLPMO9A-2, an LPMO9 derived from G. trabeum. Thus, the present study investigated whether other CBM104s also exhibit this unique adsorption property.
Six CBM104s, each of which was appended to a different catalytic domain, were selected from sequences homologous to the CBM104 appended to GtLPMO9A-2 (GtAA9-CBM104) identified in previous research (Table 1), and their adsorption properties were investigated. A CBM104 appended to AA9, as in GtAA9-CBM104, was selected from an amino acid sequence that was reported to belong to a different clade than GtAA9-CBM104 [17]. Figure 1 shows the results of the multiple amino acid sequence alignment for the six selected CBM104s and GtAA9-CBM104. Among these seven compared sequences, four cysteine residues were conserved in six sequences. However, in CBM104 appended to AA3 (AeAA3-CBM104), only two cysteine residues were conserved, whereas the other two were replaced with tyrosine. For all CBM104s, excluding AeAA3-CBM104, the three-dimensional structures predicted using AlphaFold 3 based on the amino acid sequences suggested that two α-helices are bridged by the two pairs of disulfide bonds, and these disulfide bonds appear to stabilize the relative orientation of the two α-helices at a fixed angle (Fig. 2). Conversely, AeAA3-CBM104 was predicted to lack these two disulfide bonds, resulting in the two α-helices not being fixed in position (Fig. 2). All CBM104 homologs were expressed as recombinant proteins fused with RFP in K. phaffii (Fig. S1; see J. Appl. Glycosci. Web site). Adsorption tests on celluloses demonstrated that all proteins, excluding AeAA3-CBM104, adsorbed onto three types of crystalline cellulose (Cladophora Iα, Cladophora Iβ, and Halocynthia Iβ) and not onto the amorphous cellulose PASC (Fig. 3). This suggest that the ability to adsorb specifically onto native crystalline cellulose is a widely conserved feature of CBM104. Conversely, AeAA3-CBM104 did not adsorb onto cellulose I or PASC (Figs. 3 and 4). Previous studies have shown that CBM1 adsorbs to the hydrophobic surface of cellulose via aromatic amino acid residues, whereas GtAA9-CBM104 may adsorb to cellulose chains forming the corners of hydrophilic regions [17]. Although the amino acid residues involved in the adsorption of CBM104 are currently under investigation, the present study reveals that the adsorption capacity of CBM104 is lost when the two α-helices are not properly oriented by disulfide bonds. This suggests that the angles and distances between certain amino acid residues in the α-helices play crucial roles in this specific adsorption of CBM104.
Table 1. List of six selected CBM104s.
| Organism | JGI protein ID | Catalytic domain | Abbreviation |
| Armillaria ectypa FPL83.16 v1.0 | 1387449 | AA3 | AeAA3-CBM104 |
| Armillaria gallica 21-2 v1.0 | 1012473 | AA8 | AgAA8-CBM104 |
| Schizophyllum commune 225.1 v1.0 | 216510 | AA9 | ScAA9-CBM104 |
| Schizophyllum commune H4-8 v3.0 | 2256397 | AA16 | ScAA16-CBM104 |
| Armillaria tabescens CCBAS 213 v1.0 | 630390 | AA8-AA3 | AtAA8-AA3-CBM104 |
| Schizophyllum commune ZB1 v1.0 | 125785 | GH131 | ScGH131-CBM104 |
The organism names are presented as registered in JGI.
Black background: identical amino acid residues conserved across all sequences. Gray background: amino acid residues with similar properties conserved across all sequences. Cysteine residues that are crucial for maintaining the three-dimensional structure, and they are conserved in all sequences are indicated by black arrowheads. Meanwhile, those conserved in all sequences excluding AeAA3-CBM104 are indicated by white arrowheads.
Amino acid residues predicted to form α-helices are highlighted with a gray background. Cysteine residues that form disulfide bonds linking α-helices are indicated in bold blue and shown as blue sticks. Arginine residues, which are abundant in the flexible regions at the C-termini of α-helices, are also indicated in bold red and shown as red sticks.
The amounts of protein adsorbed on various celluloses were measured after adding 1 μM protein. Each CBM104 and CBM1 was fused with RFP. RFP and TrCBM1 were used as controls. Error bars represent the standard error (n = 3). Asterisks (*) indicate samples differ significantly from RFP, as determined by a t-test (p < 0.05).
The concentration of recombinant CBM104s fused with RFP in the supernatant was measured after 2 h of incubation with each cellulose. Each measurement was performed in triplicate, and the plot represents the average of the three measurements. The data were fitted to the Hill equation.
Then, to compare the adsorption capacity in detail, the adsorption parameters in the presence of three types of cellulose I at different protein concentrations were investigated. The plots of the obtained protein adsorption concentrations and free concentrations were fitted to Hill equation, which was previously demonstrated to best fit the adsorption isotherms when CBM1 adsorbs onto cellulose [29]. The maximum adsorption capacity (Amax), the Hill coefficient (n), the apparent association constant (
Table 2. Adsorption parameters obtained via fitting to the Hill equation.
| Cellulose | CBM104s | Amax [μmol/g cellulose] | n | [μM-1] | Kd [μM] | Adsorption efficiency [L/g-cellulose] |
| Cladophora Iα | GtAA9-CBM104 | (2.37 ± 0.36) × 100 | (7.20 ± 1.90) × 10−1 | (3.48 ± 2.42) × 100 | 1.77 × 10−1 | 1.34 × 101 |
| ScAA9-CBM104 | (2.03 ± 0.11) × 100 | (8.80 ± 1.30) × 10−1 | (6.64 ± 2.69) × 100 | 1.16 × 10−1 | 1.74 × 101 | |
| AtAA8-AA3-CBM104 | (2.66 ± 0.44) × 100 | (5.40 ± 0.80) × 10−1 | (1.18 ± 0.45) × 100 | 7.36 × 10−1 | 3.61 × 100 | |
| ScGH131-CBM104 | (4.31 ± 4.52) × 100 | (5.80 ± 1.80) × 10−1 | (3.00 ± 4.20) × 10−1 | 7.97 × 100 | 5.41 × 10−1 | |
| AgAA8-CBM104 | (9.31 ± 16.4) × 100 | (6.10 ± 1.30) × 10−1 | (1.10 ± 2.10) × 10−1 | 3.73 × 101 | 2.50 × 10−1 | |
| ScAA16-CBM104 | (7.00 ± 1.40) × 10−1 | (7.50 ± 5.30) × 10−1 | (4.31 ± 5.55) × 100 | 1.43 × 10−1 | 4.90 × 100 | |
| AeAA3-CBM104 | (4.46 ± 321) × 100 | (6.00 ± 19.7) × 10−1 | (3.00 ± 242) × 10−2 | 3.45 ×102 | 1.29 × 10−2 | |
| Cladophora Iβ | GtAA9-CBM104 | (2.95 ± 0.15) × 100 | (1.00 ± 0.12) × 100 | (8.04 ± 2.97) × 100 | 1.24 × 10−1 | 2.37 × 101 |
| ScAA9-CBM104 | (3.27 ± 0.32) × 100 | (7.20 ± 0.80) × 10−1 | (2.10 ± 0.63) × 100 | 3.57 × 10−1 | 9.16 × 100 | |
| AtAA8-AA3-CBM104 | (2.95 ± 0.47) × 100 | (6.10 ± 0.90) × 10−1 | (1.44 ± 0.58) × 100 | 5.50 × 10−1 | 5.36 × 100 | |
| ScGH131-CBM104 | (6.033 ± 89.6) × 101 | (5.50 ± 1.50) × 10−1 | (2.00 ± 27.0) × 10−2 | 1.23 × 103 | 4.91 × 10−2 | |
| AgAA8-CBM104 | (6.82 ± 13.5) × 100 | (5.70 ± 2.10) × 10−1 | (1.90 ± 4.50) × 10−1 | 1.84 × 101 | 3.70 × 10−1 | |
| ScAA16-CBM104 | (1.22 ± 0.41) × 100 | (5.60 ± 2.40) × 10−1 | (1.35 ± 1.15) × 100 | 5.85 × 10−1 | 2.08 × 100 | |
| AeAA3-CBM104 | (4.07 ± 153) × 100 | (6.70 ± 9.90) × 10−1 | (3.00 ± 97.0) × 10−2 | 1.87 × 102 | 2.17 × 10−2 | |
| Halocynthia Iβ | GtAA9-CBM104 | (3.09 ± 0.11) × 100 | (1.28 ± 0.12) × 100 | (1.95 ± 0.69) × 101 | 9.82×10−2 | 3.15 × 101 |
| ScAA9-CBM104 | (3.18 ± 0.27) × 100 | (8.80 ± 1.30) × 10−1 | (4.92 ± 2.15) × 100 | 1.64 × 10−1 | 1.94 × 101 | |
| AtAA8-AA3-CBM104 | (2.56 ± 0.24) × 100 | (8.00 ± 1.40) × 10−1 | (4.02 ± 1.83) × 100 | 1.76 × 10−1 | 1.46 × 101 | |
| ScGH131-CBM104 | (6.06 ± 91.3) × 101 | (6.20 ± 1.80) × 10−1 | (2.00 ± 28.0) × 10−2 | 5.50 × 102 | 1.10 × 10−1 | |
| AgAA8-CBM104 | (3.56 ± 1.24) × 100 | (7.00 ± 1.30) × 10−1 | (6.20 ± 3.60) × 10−1 | 1.98 × 100 | 1.80 × 100 | |
| ScAA16-CBM104 | (2.20 ± 0.95) × 100 | (7.30 ± 3.20) × 10−1 | (1.22 ± 1.27) × 100 | 7.62 × 10−1 | 2.89 × 100 | |
| AeAA3-CBM104 | (0.140 ± 91.1) × 102 | (1.96 ±16.4) × 101 | (2.02 ± 0.92) × 10−13 | - | - |
The adsorption parameters obtained by fitting the adsorption plot to the Hill equation are rounded to two decimal places.
Subsequently, the distribution of CBM104 that conserve four cysteines in nature was investigated. Previous studies reported that the gene encoding GtAA9-CBM104 is regulated by selective splicing, which controls the attachment of CBM104 to the catalytic domain LPMO9 [17, 33]. To comprehensively explore CBM104s, including those potentially associated with unannotated transcripts generated through alternative splicing, tblastn searches were conducted using GtAA9-CBM104 as a query against publicly available fungal genome data in JGI. In total, 144 sequences with a hit identity of ≥ 54.55 % were obtained. Of these, 14 sequences were excluded because they lacked conservation of cysteine residues, did not preserve the CBM104 structure, or had incomplete full-length sequences. Additionally, 14 sequences that only hit in blastp searches were included, giving 144 sequences for the analysis (Table S2; see J. Appl. Glycosci. Web site). The analysis revealed that 39 species of Agaricomycetes belonging to the orders Agaricales, Gloeophyllales, and Russulales possessed CBM104. These species included one saprotroph and one involved in algae decomposition, whereas the remaining 37 were wood-rotting fungi, which were identified as white or brown rot fungi (Table S3; see J. Appl. Glycosci. Web site). This confirms that CBM104s are primarily conserved in fungi capable of degrading plant cell walls, similar to the findings of a previous research [17]. The most common catalytic domain to which CBM104 is attached was AA9, with all 39 Agaricomycetes possessing at least one gene that encodes an AA9-CBM104. The second most common catalytic domain was AA16, followed by AA8 and GH131, found at similar frequencies. Only one type of CBM104 possessed AA8-AA3 (Table S3; see J. Appl. Glycosci. Web site). Phylogenetic analysis of the 144 CBM104 homologs revealed that clades were differentiated according to the type of attached domain rather than the biological species possessing them (Fig. 5). This suggests that the amino acid sequences of CBM104s have evolved to be optimized based on the function of their attached domain. Most Agaricales possess multiple genes encoding CBM104s, which are attached not only to AA9 but also to AA8, AA16, or GH131 (Fig. 6 and Table S3; see J. Appl. Glycosci. Web site). It has been reported that AA16 supplies H2O2 to AA9 [34], and that AA8 transfers electrons that AA3 takes from cellobiose to LPMO9 [35, 36]. This suggests that Agaricales utilize CBM104 to selectively localize its attached domains in close proximity on crystalline cellulose within structurally complex and compositionally diverse plant cell walls, thereby enabling the cooperative degradation of crystalline cellulose. Although GH131 is reported to have hydrolytic activity against a broad range of β-glucans, GH131-CBM104 might also have functions related to AA9. Furthermore, among the fungi in Agaricales, those with combinations of AA8-CBM104, AA9-CBM104, and AA16-CBM104 genes, such as Armillaria sp. and Guyanagaster necrorhizus, were distinguished from those with combinations of AA9-CBM104, AA16-CBM104, and GH131-CBM104 genes, such as Auriculariopsis ampla, Cylindrobasidium torrendii, and Schizophyllum sp. (Fig. 6). This suggests the existence of plant cell wall degradation systems mediated by CBM104 that are adapted to different survival strategies.
The unrooted topology tree was constructed by neighbor-joining method the sequences of 144 CBM104s after excluding their linker and attached domain sequences. The sequence names are displayed as “domain type appended to CBM104_JGI abbreviation of the organism|scaffold number where the CBM104 sequence was identified.” The names are color-coded according to the type of attached domain: AA8 is indicated in light green, AA8-AA3 in blue, AA9 in red, AA16 in light blue, and GH131 in magenta.
The names of the fungi are presented as registered in JGI.
In this study, six CBM104s, each attached to a different catalytic domain, were selected, and their adsorption capacity was analyzed in detail. The results revealed that two conserved pairs of disulfide bonds are essential for the specific adsorption of CBM104 onto cellulose I. The differences in adsorption efficiency of CBM104 for cellulose I are likely influenced by the flexible C-terminal region. However, further investigation is required. Genomic analysis using the JGI database confirmed that CBM104 is conserved in plant cell wall-degrading filamentous fungi belonging to Agaricomycetes. All fungi harboring genes encoding CBM104 possessed at least one AA9-CBM104 gene, and most Agaricales species contained additional CBM104-encoding genes. These genes were primarily categorized into two combinations: AA8-CBM104 and AA16-CBM104, or AA16-CBM104 and GH131-CBM104. These findings suggest the existence of plant cell wall degradation systems involving CBM104-attached proteins that vary depending on survival strategies.
These findings provide important insights into the functional characteristics of CBM104, and they are expected to be valuable for future research on plant biomass degradation and industrial applications. Future research should focus on elucidating the specific factors contributing to adsorption properties of CBM104 for cellulose I, as well as the interactions between the domains to which CBM104 is attached.
The authors declare no conflicts of interest.
This study was supported by Grant-in-Aid for Scientific Research (B) (Grant Number 23K23668), Grant-in-Aid for Challenging Research (Exploratory) (Grant Number 23K18046), Grant-in-Aid for Early-Career Scientists (Grant Number 24K17938) and by JST Grant Number JPMJPF2104 from the Japan Science and Technology Agency. Additionally, we thank the 1000 Fungal Genomes consortia for access to unpublished genome data. The genome sequence data were produced by the US Department of Energy Joint Genome Institute in collaboration with the user community.
