Transcription factor MtCLR-2 regulates cellulase production via direct modulation of Mtegl2 and Mtbgl1 expression in Myceliophthora thermophila

Abstract

Background

The thermophilic fungus Myceliophthora thermophila can secrete large amounts of lignocellulolytic enzymes, such as cellulases and xylanases, which are regulated by multiple transcription factors. However, the understanding of the regulatory mechanism of cellulase gene expression in M. thermophila is limited. Here, we characterized the function of MtCLR-2, a M. thermophila ortholog of CLR-2, a key cellulolytic transcriptional regulator initially identified in Neurospora crassa.

Results

Deletion of Mtclr-2 significantly reduced cellulase activities, particularly affecting endoglucanase production, whereas overexpression of Mtclr-2 led to elevation in cellulase secretion when M. thermophila was grown on Avicel. Subcellular localization assay of MtCLR-2 fused to green fluorescent protein (GFP) indicated that MtCLR-2 is localized to the nucleus. Real-time quantitative reverse transcription PCR (RT-qPCR) analysis revealed that disruption of Mtclr-2 caused a decrease in transcript levels of the β-glucosidase gene bgl1 (MYCTH_66804) and the endoglucanase gene egl2 (MYCTH_86753) throughout the stages of growth in cellulose medium. Furthermore, electrophoretic mobility shift assays (EMSAs) demonstrated that MtCLR-2 directly binds to the promoter regions of bgl1 and egl2 in a zinc-dependent manner. The comparative transcriptomic analysis also showed that MtCLR-2 positively regulates the expression of ribosomal protein genes under cellulosic conditions.

Conclusions

These findings contribute to a better understanding of the regulatory network governing cellulase gene expression and provide a potential target for boosting cellulase biosynthesis in M. thermophila.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12934-026-02976-1.

Background

Lignocellulose, the most abundant renewable energy source on Earth, can be converted into fermentable sugars for the production of biofuels and value-added biochemicals in biorefineries [1]. The primary components of lignocellulosic material are cellulose, hemicellulose, and lignin, with cellulose accounting for the highest proportion (40–50%) [2]. The complete hydrolysis of cellulose requires the concerted activities of three complementary cellulase enzymes, endo-1,4-β-glucanase (EC 3.2.1.4), cellobiohydrolase (EC 3.2.1.91), and β-glucosidase (EC 3.2.1.21). These three cellulases act synergistically to decompose cellulose to release glucose as the final product that can be subsequently fermented into ethanol [3]. In addition, lytic polysaccharide monooxygenases (LPMOs; AA9) have been shown to work in synergy with cellulolytic enzymes during the decomposition of the recalcitrant cellulose [4]. These copper-dependent enzymes oxidatively cleave glycosidic bonds of the cellulose when reducing agents are present, thus improving the efficiency of cellulases and promoting the enzymatic conversion of cellulose [5].

Filamentous fungi are highly efficient decomposers of lignocellulose in the terrestrial biosphere due to their ability to secrete a broad spectrum and large amounts of extracellular enzymes associated with the deconstruction of plant-derived biomass [6, 7]. Among others, Myceliophthora thermophila is known to produce a wide array of thermostable carbohydrate-active enzymes (CAZymes) involved in lignocellulose degradation [8, 9]. Recently, this fungus has been genetically modified to produce cellulases and xylanases, as well as biochemicals and biofuels, such as fumaric acid, malic acid, and ethanol [10–13], based on the current omics technologies and fungal genome editing tools [10, 14]. The M. thermophila genome encodes at least eight β-glucosidases (BGLs), seven endoglucanases (EGLs), and seven cellobiohydrolases (CBHs) [15]. Among them, egl2 (MYCTH_86753), cbh1 (MYCTH_109566) and cbh2 (MYCTH_66729), and bgl1 (MYCTH_66804), which encode endoglucanase, cellobiohydrolases, and β-glucosidase, respectively, exhibit high transcript levels and secretion quantities when grown on various sources of lignocellulosic biomass compared to glucose [8, 16, 17]. These extracellular enzymes thus represent the major cellulases of M. thermophila. Therefore, a better understanding of the regulatory mechanism governing the expression of these cellulase genes would facilitate fungal strain engineering and elevate cellulase production in M. thermophila.

The transcriptional induction of cellulases or xylanases is strictly controlled by transcription factors [18, 19]. In M. thermophila, a couple of regulators that are involved in the lignocellulose depolymerization were identified, including MtXyr1, MtCre1, MtCLR-4, and MtTRC-1. MtXyr1 not only regulates the expression of genes encoding xylanolytic enzymes but also controls genes associated with pentose transport and catabolism, while having no noticeable effect on cellulase secretion [20, 21]. MtCre1 acts as the primary transcriptional repressor of cellulolytic genes, and its silencing or disruption upregulates the transcription of cellulase genes and enhances cellulase production under inducing conditions [10, 22]. Analogous to its homolog CLR-4 in Neurospora crassa, MtCLR-4 plays a positive role in regulating cellulase gene expression by modulating its downstream targets, MtCLR-2 and MtXyr1, and the cAMP signaling pathway [23]. MtTRC-1 functions as a transcriptional activator, regulating cellulase production by modulating the expression of the transcription factor gene Mthac-1 and the cellobiohydrolase gene Mtcbh-1 [24]. Recently, the Zn(II)2Cys6 transcription factor MtClr-5 was reported to be crucial for cellulose decomposition, as its deletion reduced endoglucanase activity and protein secretion under cellulolytic conditions [25]. In addition, our recent study demonstrated that the forkhead regulator MtFkh1 represses major cellulase and xylanase gene expression by directly binding to their promoter regions [17].

The transcription factor CLR-2 was first identified in N. crassa which is an essential regulator of cellulase, but not hemicellulase gene expression [26]. The deletion of clr-2 caused severe growth defects and completely abolished cellulase activity on Avicel, but not on xylan [26]. Since then, homologs of NcCLR-2 were fully characterized in other filamentous ascomycete fungi. The Aspergillus nidulans mutant carrying a disruption of clrB, the ortholog of clr2, failed to elicit cellulase gene expression and lost cellulolytic activity during growth on cellulose [26]. Similarly, the ClrB (homolog of CLR-2) is indispensable in the production of cellobiohydrolases and endoglucanases in Aspergillus niger [27, 28]. A recent study further showed the involvement of A. niger ClrB in the utilization of xyloglucan and galactomannan [29]. Also, ClrB acts as an activator of the cellulolytic gene expression in Penicillium oxalicum [30, 31]. In Aspergillus oryzae, the CLR-2 ortholog (ManR) was reported as a regulator of both mannanolytic and cellulolytic enzyme genes [32, 33]. However, the homologue of CLR-2 was described in Trichoderma reesei and Talaromyces cellulolyticus but had only a minor impact on cellulase activity [34–36]. These results indicate both similarities and differences in the functions of NcCLR-2 orthologs. In our previous analysis of the M. thermophila transcriptome profiling data, we noted that Mtclr-2 (MYCTH_38704), the homolog of N. crassa clr-2, was highly upregulated under cellulosic conditions [17], implying that this gene may play roles in cellulose degradation. Indeed, the regulator MtCLR-2 has been found to be important for cellulase expression [37]. However, the detailed molecular basis of its role in the cellulase expression has still not been fully uncovered.

In the present study, we further characterized the function of MtCLR-2 in cellulose utilization in M. thermophila. By a combination of molecular genetic approaches and comparative transcriptomic analyses, we found that MtCLR-2 positively regulates cellulase production by regulating the expression of the major endoglucanase gene (egl2) and β-glucosidase gene (bgl1) by directly binding to their promoter regions. In addition, we also demonstrated that MtCLR-2 is involved in ribosome biosynthesis, suggesting it regulates cellulase expression at both the transcriptional and translational levels.

Results

Identification of the MtCLR-2 protein

A BLASTP search of the M. thermophila genome, using the N. crassa CLR-2 (NCU08042, NcCLR-2) protein sequence as a query, revealed MYCTH_38704 as a putative homolog of NcCLR-2, sharing 81% sequence identity with this close ortholog. The M. thermophila Mtclr-2 (MYCTH_38704) is 2570 bp in length and contains three exons encoding a protein composed of 798 amino acids (aa) (Fig. 1). In addition, the InterPro analysis (https://www.ebi.ac.uk/interpro/) showed the presence of a typical fungal transcription factor domain (IPR007219) between residues 259 and 434 of MtCLR-2 (Fig. 1). However, compared to NcCLR-2, MtCLR-2 lacks 14 aa in its Zn(II)2Cys6 fungal-type DNA-binding domain (DBD), between residues 44 and 75, which may explain why InterPro analysis did not detect this domain in MtCLR-2.

Fig. 1.

Schematic representation of the Mtclr-2 gene structure and the predicted functional domains of the MtCLR-2 protein. MtCLR-2 lacks 14 aa in its putative Zn(II)2Cys6 fungal-type DNA-binding domain compared to NcCLR-2. NcCLR-2, N. crassa CLR-2

Positive effect of MtCLR-2 on the production of cellulase in M. thermophila

To confirm the influence of MtCLR-2 on cellulase induction, we constructed Mtclr-2 deletion mutants by using CRISPR/Cas9 system (Fig. S1). The protospacer sequence targeting Mtclr-2 was designed for gene editing (Table S1). The Cas9-U6p-Mtclr-2-sgRNA plasmid was co-transformed into M. thermophila wild-type (WT) protoplasts along with a donor DNA vector carrying the neomycin resistance gene cassette (Pgpd-neo) flanked by the 5′ and 3′ homologous arms of Mtclr-2. Deletion of the Mtclr-2 gene was accomplished via homologous recombination mediated by the CRISPR/Cas9 system. The obtained disruption strains were verified by genomic PCR with three specific primer pairs (Fig. S2). After 48–96 h growth in Avicel medium, the ΔFMtclr-2 mutants displayed a marked reduction in filter paper enzyme activity (by 56% to 60%) and β-glucosidase activity (by 26% to 40%), while endoglucanase activity was almost abolished (reduced by 90% to 92%) compared with those of the WT (Fig. 2A-C). Moreover, ΔMtclr-2 secreted 48% to 64% less protein than the WT under cellulosic conditions (Fig. 2D), as confirmed by SDS-PAGE analysis of extracellular proteins (Fig. S3).

Fig. 2.

Cellulase activities and protein production of M. thermophila WT strain, ΔMtclr-2 mutants, and OE-Mtclr-2 strain. A Filter paper cellulase (FPase) activity. B Endoglucanase (CMCase) activity. C β-glucosidase (pNPGase) activity. D Extracellular protein concentration. These M. thermophila strains were pre-grown in glucose for 36 h and then transferred to 2% (w/v) Avicel medium and cultured for 48–96 h. **p < 0.01, and ***p < 0.001 indicate significant differences between the WT and ΔMtclr-2 mutants or OE-Mtclr-2 strain (Two-way ANOVA), ns indicates not significant. Error bars represent the SD from three replicates

Meanwhile, the Mtclr-2 overexpression strain OE-Mtclr-2 was created, in which Mtclr-2 was driven by the strong constitutive pyruvate decarboxylase (MYCTH_112121) promoter Ppdc. The positive overexpression mutant was validated by genomic PCR using specific primer set (Fig. S2). As expected, OE-Mtclr-2 strain exhibited a significant elevation in filter paper enzyme activity (83% to 216%), β-glucosidase activity (132% to 143%), endoglucanase activity (138% to 190%), and secreted protein (150% to 293%), compared to those of the WT when cultivated on Avicel (Fig. 2A-D). These results demonstrate that MtCLR-2 is an activator of cellulase production under cellulose induction in M. thermophila.

Furthermore, the role of MtCLR-2 in fungal development was explored. As shown in Fig. 3A, the ΔMtclr-2 mutant showed similar colony phenotype to that of the WT and OE-Mtclr-2 strains when grown on PDA and solid Avicel media. In addition, both the Mtclr-2 disruption and overexpression strains produced comparable numbers of asexual spores to the WT after 7 days of growth on Avicel plates (Fig. 3B). These observations suggest that MtCLR-2 is dispensable for hyphal radial growth or sporulation on solid cellulose medium. However, the ΔMtclr-2 mutant grew poorly in liquid cellulose culture, characterized by aberrant mycelial morphology with few hyphal branches after 36 h (Fig. 3C), but exhibited normal growth in liquid glucose medium (Fig. S4), which was similar to N. crassa Δclr-2 and A. nidulans ΔclrB mutants [26]. This growth impairment implies a specific defect associated with cellulose utilization, which is in accordance with the substantially decreased cellulase production observed in the mutant ΔMtclr-2.

Fig. 3.

Phenotypic analyses of M. thermophila WT, ΔMtclr-2, and OE-Mtclr-2 strains and subcellular localization of MtCLR-2 protein. A Colony phenotype on PDA and 1% (w/v) Avicel solid plates after 3 (PDA) or 4 days (Avicel) of growth at 45°C. B Quantitative determination of conidiation on plates containing 1% (w/v) Avicel cultivated at 45°C after 7 days. ns indicates no noticeable differences between WT and ΔMtclr-2, or OE-Mtclr-2 strains (Student’s t tests). Error bars represent the SD from three replicates. C Microscopic observations of fungal mycelia after 36 h of cultivation in liquid shaking flasks containing 2% (w/v) Avicel as the sole carbon source. Aberrant hyphal branches in the ∆Mtclr-2 strain are indicated by red arrows. Scale bar = 10 µm. D Subcellular location of MtCLR-2 in M. thermophila. MtCLR-2-GFP strain was cultivated in liquid medium containing 2% (w/v) glucose as the sole carbon source at 45°C for 36 h. Scale bar = 20 µm

Subcellular localization of MtCLR-2 in M. thermophila

To assess the subcellular localization of MtCLR-2, the Mtclr-2 open reading frame was fused to the gfp (green fluorescent protein encoding gene) under the control of the Mtpdc promoter (Fig. S2). This expression cassette was introduced into the WT strain, generating a strain expressing MtCLR-2-GFP. After growth in both glucose and Avicel media for 36 h, the MtCLR-2-GFP signal was found to be accumulated in the nuclei across the fungal hyphae. This signal overlapped with the blue fluorescence of the nucleic acid-specific dye DAPI (Fig. 3D), confirming that MtCLR-2 localizes to the M. thermophila nucleus under both glucose-repressing and cellulose-inducing conditions (Fig. S5).

MtCLR-2 regulates the expression of major cellulase genes

To test whether MtCLR-2 exerts its impact on cellulase production at the transcriptional level, we analyzed the expression of cellulase genes in the WT strain and ΔMtclr-2 mutant using real-time quantitative reverse transcription PCR (RT-qPCR). As shown in Fig. 4, the β-glucosidase gene bgl1 (MYCTH_66804) exhibited 29% to 32% reductions in transcription levels in the Mtclr-2 deletion strain compared with the WT during the entire induction period (24–72 h) on Avicel. Similarly, transcript level of the endoglucanase gene egl2 (MYCTH_86753) was substantially decreased (by 32-fold) in ΔMtclr-2 mutant after 24 h of growth in Avicel medium, and was reduced by 47% to 80% at 48 and 72 h. These results agree with the observed reduction in cellulase activity in the ΔMtclr-2 strain. Additionally, the effects of MtCLR-2 on the transcriptional levels of key regulatory genes, including cre1 (MYCTH_2310085) and xyr1 (MYCTH_2310145), were examined. The transcription of cre1 and xyr1 was not significantly altered by the deletion of Mtclr-2 throughout the cultivation period under cellulose-inducing conditions (Fig. 4). These observations imply that MtCLR-2 influences cellulase production primarily through regulating cellulase gene transcription.

Fig. 4.

Relative transcription levels of major cellulase genes bgl1 and egl2 as well as the regulatory genes cre1 and xyr1 in M. thermophila WT and ΔMtclr-2 strains. Strains were pre-grown in glucose for 36 h, washed and transferred to 2% (w/v) Avicel medium and cultivated for 24, 48, and 72 h. The expression level of each gene in ΔMtclr-2 was normalized to the level of the corresponding gene in WT. **p < 0.01, and ***p < 0.001 represent significant differences between the ΔMtclr-2 and WT strains (Two-way ANOVA), ns indicate no significant difference. Error bars represent the SD from three replicates

MtCLR-2 binds to the promoter regions of Mtbgl1 and Mtegl2 in a zinc-dependent manner

To determine whether MtCLR-2 directly regulates the expression of bgl1 and egl2, electrophoretic mobility shift assays (EMSAs) were performed. Given that MtCLR-2 lacks 14 aa within its DBD compared to NcCLR-2, we wondered whether this loss affects its DNA-binging capability. To answer this question, Strep II-fused DNA binding domain of MtCLR-2 (MtCLR-2₃₀₋₈₉) and MtCLR-2_{(30−89)+14aa}, which restores the missing 14 aa, were expressed and purified from E. coli BL21(DE3) (Fig. S6). DNA probes corresponding to the promoter regions of bgl1 (P2, − 650 to − 300) and egl2 ( P2, − 650 to – 300; P3, − 1000 to − 600) were obtained by PCR. In the initial EMSAs, neither MtCLR-2₃₀₋₈₉ nor MtCLR-2_{(30−89)+14aa} exhibited detectable binding to the upstream regions of bgl1 or egl2 (Fig. 5A-B). Considering that MtCLR-2 is a predicted Zn(II)2Cys6-type TF, we hypothesized that zinc ions may be required for its DNA-binding activity. To test this, zinc ions were supplemented in the EMSA reaction buffer. Under these conditions, both MtCLR-2₃₀₋₈₉ and MtCLR-2_{(30−89)+14aa} bound to the promoter regions of bgl1 and egl2 and the size of protein-DNA complex increased along with increasing amounts of MtCLR-2₃₀₋₈₉ and MtCLR-2_{(30−89)+14aa} (Fig. 5A-B). These findings suggest that MtCLR-2 binds its target genes in the presence of zinc ions and the absence of the 14 aa in its DBD has no impact on its ability to recognize and bind target promoters.

Fig. 5.

Electrophoretic mobility shift assays (EMSAs) of the interactions between MtCLR-2 and the promoter regions of major genes encoding cellulases. A EMSAs of the binding between MtCLR-2₃₀₋₈₉ and the promoter regions of bgl1 and egl2 in the presence or absence of zinc ions. B EMSAs of the binding between MtCLR-2_{(30−89)+14aa} and the promoter regions of bgl1 and egl2 in the presence or absence of zinc ions. The concentration of zinc ions in the reaction buffer was 12.5 mM. C Schematic diagram of the putatively conserved MtCLR-2 binding motif in the promoter regions of bgl1 and egl2. MtCLR-2 may identify and bind the CGG_N11CCG motif in the promoter regions of its target genes, box in green indicates variable N₁₁ nucleotides

Previous ChIP-seq (chromatin immunoprecipitation and next-generation sequencing) analysis revealed a conserved CGG_N11CCG motif in the upstream regions of NcCLR-2-bound genes, which is nearly identical to the DNA-binding motif of its closest paralog, Gal4p, in Saccharomyces cerevisiae [38]. To determine whether MtCLR-2 recognizes a similar motif, we searched for the promoter regions bgl1-P2, egl2-P2, and egl2-P3. The conserved CGG_N11CCG sequences were found in all these promoters, with egl2-P2 and egl2-P3 sharing identical motif fragments within their overlapping region (Fig. 5C). Additionally, the promoter region of xyn1 (P1, − 350 to − 1), which lacks the CGG_N11CCG motif, was used as a negative control. The EMSA results showed that MtCLR-2₃₀₋₈₉ did not form a protein–DNA complex with the xyn1 promoter, regardless of the presence of zinc ions (Fig. S7). Thus, MtCLR-2 may identify and bind the CGG_N11CCG motif in the promoter regions of its target genes, analogous to its homolog NcCLR-2.

Transcriptomic analysis of the Mtclr-2 deletion mutant on Avicel

To obtain a genome-wide understanding of the molecular mechanism by which MtCLR-2 regulates cellulase production, transcriptional profile analysis was performed using the ΔMtclr-2 mutant grown on Avicel with the WT as comparison strain. When compared to WT, transcript levels of 2228 genes were clearly altered in mutant ΔMtclr-2 after 48 h of growth, with 1323 genes showing significantly decreased transcription and 905 genes displaying substantial upregulation (Fig. 6A and TableS2). A Gene Ontology (GO) analysis revealed that these differentially expressed genes (DEGs) in ΔMtclr-2 were enriched in the functional categories of cytosolic ribosome, polysaccharide metabolic process, and cellulose binding (Fig. 6B), in accordance with the phenotypes observed in ΔMtclr-2 in response to cellulose.

Fig. 6.

Comparative transcriptomic analysis of strains ΔMtclr-2 and WT cultivated in 2% (w/v) Avicel medium for 48 h after a shift from glucose. A Volcano plots demonstrating differentially expressed genes (DEGs) between ΔMtclr-2 and WT strains. Genes that were significantly upregulated and downregulated in the ΔMtclr-2 mutant compared to the WT are plotted in red and green, respectively. B Gene Ontology (GO) enrichment of 2228 DEGs in the biological process, cellular component, and molecular function categories. C Heatmap analysis of expression profiles for genes encoding cellulases, LPMOs, and putative transcription factors between ΔMtclr-2 and WT strains. D Heatmap analysis of expression profiles for genes encoding putative ribosomal proteins between ΔMtclr-2 and WT strains. Heatmaps show the Z-score–normalized expression levels of selected genes across the ΔMtclr-2 mutant and WT samples

Comparative transcriptomic analysis revealed that three endoglucanase genes including egl2, MYCTH_52068, and MYCTH_111372, were remarkably downregulated in ΔMtclr-2 in comparison to WT, except for MYCTH_116157 (Fig. 6C), which was in line with the endoglucanase secretion phenotype when exposed to Avicel. Three intracellular β-glucosidase genes (MYCTH_115968, MYCTH_62925, and MYCTH_38200) showed lower expression levels by the deletion of Mtclr-2. Likewise, the transcript levels of two extracellular β-glucosidase genes, bgl1 and MYCTH_58882, were reduced by approximately 25% (log₂ fold change = – 0.42 and – 0.41, respectively) in the ΔMtclr-2 mutant, which was consistent with RT-qPCR results showing lower transcript of bgl1 in the mutant strain, although the expression changes did not reach the strict differential analysis criteria. In addition, nine genes encoding LPMOs (AA9 family) exhibited lower transcription levels in the ΔMtclr-2 mutant than in the WT, except for MYCTH_2311323 (Fig. 6C). Among these, MtLPMO9B (MYCTH_80312), MtLPMO9C (MYCTH_100518), MtLPMO9D (MYCTH_92668), and MtLPMO9J (MYCTH_79765), were found to be active against cellulose [39–41], and MtLPMO9I (MYCTH_46583) was shown to have strong synergistic effects with endoglucanase, resulting in elevated enzymatic hydrolysis yields toward cellulose [42]. Similar observation was reported in N. crassa, where disruption of Ncclr-2 led to decreased expression of several secreted endoglucanase genes and one predicted intracellular β-glucosidase gene as well as one extracellular β-glucosidase gene [26, 43], while its constitutive expression caused the upregulation of several LPMO-encoding genes [38], confirming the conserved role of CLR-2 orthologs in regulating genes involved in cellulose decomposition.

Besides these cellulose degrading genes, 56 genes encoding putative transcription factors (TFs) were also observed showing altered expression levels, with 19 genes being downregulated and 37 genes being upregulated in the ΔMtclr-2 mutant (Fig. 6C). The majority of these TFs can be classified into Fungal Zn(II)2Cys6, Fungal_TF, and Zinc finger C2H2 families. Of these, the gene encoding CLR-4 (MYCTH_2296492), which positively regulates cellulase gene expression [23], was downregulated (log2 fold change = – 1.02) in the absence of Mtclr-2. In contrast, one gene encoding AmyR (MYCTH_2301920), a repressor of cellulase production [44], was highly induced in strain ΔMtclr-2 (log₂ fold change = 1.83). Therefore, the downregulation of Mtclr-4 and upregulation of MtamyR may partially account for the reduced cellulase production seen in the ΔMtclr-2 mutant. Consistent with the RT-qPCR analysis above, the essential regulatory genes Mtcre1 and Mtxyr1 displayed no differential expression between ΔMtclr-2 and WT strains.

Genes encoding predicted sugar transporters were also differentially expressed in the Mtclr-2 disruption strain (Fig. S8). Among 15 genes annotated as transporters for glucose, xylose, cellodextrin, cellobionic acid, or other sugars, two genes increased their transcription in ΔMtclr-2, whereas the remaining genes decreased. Notably, MYCTH_2302958 and MYCTH_84164, showed transcriptional changes, with the former downregulated (log₂ fold change = − 4.7) and the latter upregulated (log₂ fold change = 2.3). These two genes encode proteins that share 55% and 54% sequence similarity, respectively, with cellobionic acid transporter CBT-1 in N. crassa, which suppresses cellulase induction on both cellobiose and Avicel [45, 46]. Whether the altered expression of these putative cbt-1 homologs contributes to the decreased cellulase induction observed in the ΔMtclr-2 mutant remains to be elucidated.

Interestingly, a set of ribosomal protein genes exhibited reduced transcript abundance in the mutant ΔMtclr-2, including 25 potential genes encoding 40 S small ribosomal subunit proteins and 36 encoding 60 S large ribosomal subunit proteins (Fig. 6D). Additionally, seven genes encoding putative mitochondrial ribosomal proteins, including three small ribosomal protein genes and four large ribosomal protein genes, were also downregulated upon Mtclr-2 disruption (Fig. 6D). The transcriptional repression of ribosomal protein genes has not been reported in other fungal species where the roles of MtCLR-2 homologs have been explored. These findings suggest that MtCLR-2 may regulate the production of cellulases not only at the transcriptional level but potentially also at the translational level.

Discussion

Myceliophthora thermophila is a thermophilic fungus able to secrete a complete set of thermostable enzymes involved in plant biomass degradation, including cellulases and xylanases. In recent years, this fungus has been developed into a platform to produce industrially relevant enzymes [47, 48] and used as a model system for exploring basic fungal cell biology, such as the mechanism of expression and secretion of lignocellulolytic enzymes [24, 49, 50]. A few transcription regulators related to lignocellulose deconstruction have been identified in this fungus, including MtFkh1, MtXyr1, MtCre1, MtClr-4, and MtTrc-1 [17, 21–24]. However, more transcription factors involved in inducing the expression of lignocellulolytic enzyme-encoding genes in M. thermophila remain to be elucidated. In this paper, we present the molecular basis underlying cellulase expression regulated by MtCLR-2, which is the ortholog of NcCLR-2 in N. crassa. The assay of cellulase production of Mtclr-2 deletion and overexpression strains indicated that MtCLR-2 serves as an activator of cellulase production. The disruption of this nuclear localized transcription factor MtCLR-2 drastically decreased cellulolytic enzyme production, whereas the overexpression of MtCLR-2 resulted in cellulase enzyme activities that were elevated approximately two-fold compared with the WT when M. thermophila was grown on cellulose. This role was supported by our RT-qPCR and transcriptomic analyses, which uncovered downregulated expression of the major endoglucanase and β-glucosidase genes and several genes encoding crucial LPMOs (AA9 family) in the ΔMtclr-2 strain. In addition, the severely affected growth of ΔMtclr-2 in liquid cellulose medium but normal growth in glucose culture suggested that MtCLR-2 is specifically involved in cellulose catabolism. However, no obvious difference was observed in the cellobiohydrolase activity between the ΔMtclr-2 and WT strains for growth on Avicel (Fig. S9), despite transcriptomic data revealed the downregulation of five cellobiohydrolase genes and upregulation of one cellobiohydrolase gene in the ΔMtclr-2 mutant (Fig. S8), indicating a self-equilibrating mechanism that modulates production of cellulase. Overall, herein, we provide a potential target for rational fungal strain engineering for hyperproduction of cellulase.

Previous studies showed that the absence of Ncclr-2 resulted in no detectable endoglucanase activity and almost abolished secreted protein production when N. crassa was grown on cellulose [26]. Our research also demonstrated that MtCLR-2 has a similar role in the regulation of endoglucanase and total protein secretion in cellulose-induced cultures. Also, the considerably reduced endoglucanase activity was reported on A. nidulans ΔclrB [26] and P. oxalicum ΔclrB mutants [30]. Accordingly, significant decrease in expression levels of the major endoglucanase genes was observed in the ΔMtclr-2 strain, which was in accordance with findings from N. crassa, A. nidulans, A. niger, P. oxalicum, and A. oryzae, where the deletion of Mtclr-2 orthologs caused the downregulation of the essential endoglucanase genes under cellulosic conditions [26–28, 30, 33, 43]. These observations clearly indicate the conserved role of CLR-2 homologs in endoglucanase gene regulation. Similar to N. crassa CLR-2 and A. nidulans ClrB, several important LPMO-encoding genes were downregulated by the deletion of Mtclr-2 in response to Avicel, the majority of which have been found to exert oxidative roles toward cellulose [43]. Regarding β-glucosidase and cellobiohydrolase gene expression, differences in function of CLR-2 homologs have been reported. For example, the ΔclrB mutant of P. oxalicum exhibited reduced cellobiohydrolase activity [30] and enhanced β-glucosidase activity when exposed to Avicel [31], which was consistent with the downregulation of the main cellobiohydrolase genes and upregulation of the major β-glucosidase genes. In contrast, the present study showed that the disruption of Mtclr-2 decreased β-glucosidase activity while having no impact on cellobiohydrolase production, highlighting the complex regulatory networks in which CLR-2 orthologs are involved in regulating the expression of different cellulase enzymes. In addition, overexpression of TRIREDRAFT_26163, the T. reesei homolog of Ncclr-2, resulted in only a slight elevation in cellulase production [34]. Similarly, in T. cellulolyticus, the loss of tclB2, which is the homolog of Ncclr-2, exhibited minor effects on cellulase activity when cultivated on cellulose [35]. Therefore, the role of CLR-2 homologs in cellulose utilization appears to be partially conserved among filamentous fungi.

The production of cellulolytic enzymes in fungi is tightly regulated by transcription factors [18, 19]. According to our transcriptomic data analysis, transcription factors involved in the regulation of cellulase gene expression, including Mtclr-4 [23] and MtamyR [44], showed altered expression levels by the deletion of Mtclr-2, implying that MtCLR-2 regulates cellulase production by modulating transcript levels of regulatory proteins. These targeted transcription factors could function as secondary regulators, enabling more fine-tuned gene expression [38]. In N. crassa, xlr-1, which encodes a key transcription factor that regulates the expression of hemicellulolytic enzyme genes and is necessary for utilization of hemicellulose, was downregulated in the Δclr-2 strain [26]. However, this is not the case for M. thermophila, where the transcription of Mtxyr1, the homolog of xlr-1, remained unchanged in the mutant ΔMtclr-2 under conditions of cellulose exposure. In addition, CLR-1, another predominant regulator of cellulolytic enzymes in N. crassa, is essential for clr-2 expression, since the expression of clr-2 was abolished in the Δclr-1 mutant, while that of clr-1 was not influenced by the disruption of clr-2 during growth on Avicel [26]. Further DNA motif analysis of the bound genes revealed that CLR-1 binds to the promoter region of clr-2 under cellulosic conditions [38]. Similar to N. crassa clr-2, Mtclr-2 is not required for the expression of Mtclr-1 (MYCTH_2298863), the Ncclr-1 ortholog in M. thermophila, revealed by the transcriptome data showing the unaffected expression level of Mtclr-1 in the ΔMtclr-2 mutant. Whether Mtclr-2 is bound and regulated by MtCLR-1 needs to be further investigated. Unlike the results observed with Ncclr-2 and Mtclr-2, the expression of clrA, the ortholog of clr-1, which is involved in cellulose degradation, was reported to be (partially) dependent on ClrB in A. niger [29], suggesting divergences between the regulatory networks coordinating lignocellulose deconstruction and utilization among different filamentous fungi.

In N. crassa, CLR-2 binds to 5′-CGGN₁₁CCG-3′ motifs in the promoter regions of its regulon [38]. Combined ChIPseq and RNAseq analyses identified that 54 genes were both bound by CLR-2 and dependent on it for their expression [38]. Our data showed that egl2 and bgl1 were consistently downregulated at all examined time points in the Mtclr-2 disruptant, indicating the high dependence of MtCLR-2 for activation of their expression. Further EMSA analysis demonstrated that MtCLR-2 can directly bind to the promoter regions of egl2 and bgl1 in a zinc-dependent manner, although the lack of 14 aa in its DNA-binding domain compared to NcCLR-2. Like NcCLR-2, the 5′-CGGN₁₁CCG-3′ sequence was found in the upstream regions of both egl2 and bgl1, indicating that MtCLR-2 might also bind this motif to regulate its target genes. In addition, the 5′-CGGN₁₁CCG-3′ motif was identified in the promoter regions of Mtlpmo9B, Mtlpmo9C, and Mtlpmo9D, but was absent in those of Mtcre1 and Mtxyr1. This observation is consistent with the expression patterns of these genes in the ΔMtclr-2 mutant revealed by both RT-qPCR and/or transcriptomic analyses, further supporting the regulatory importance of this motif. It has been shown that ClrB in A. nidulans can bind to 5′-CGGN₈CCG-3′ motifs in the promoter region of the β-mannosidase gene mndB in the presence of zinc ions, and loss of either one of the CGG or CCG triplet completely abolished protein–DNA interaction [51]. Likewise, eglA and bgl4 were downregulated in the A. niger ΔclrB strain when grown in soybean hull or guar gum liquid media, and both gene possess predicted ClrB consensus binding sites (5′-CGGN₈CCG-3′) in their upstream regions [29]. These experimentally confirmed or putative binding sequences of ClrB in A. nidulans and A. niger are highly similar to the predicted binding site of MtCLR-2 identified in this study. Nevertheless, A. nidulans ClrB does not bind directly to the eglB promoter, which contains one 5′-CGGN₈CCG-3′ site. Instead, it is recruited to a CeRE-like (cellulose-responsive element-like) sequence upstream of eglB, which also contains a CCG triplet, through the assistance of McmA, a SRF (serum response factor)-type MADS box protein that regulates both cellulase production and asexual/ sexual development [51]. This implies a different type of ClrB-mediated regulatory system. Whether MtCLR-2 interacts with unknown cofactors to facilitate its binding and regulatory functions remains to be determined.

An unexpected finding of this study is that 68 genes encoding ribosomal proteins were downregulated in the absence of Mtclr-2, which may explain dramatically decreased protein secretion in the cultures of strain ΔMtclr-2 cultivated on cellulose. Studies in yeast indicate the activation of ribosomal protein genes (RPGs) transcription is accomplished by several partly specialized regulatory factors that are not unique to RPG promoters [52]. The most prominent transcription factors are Rap1 (Repressor/activator protein 1), Fhl1 (Forkhead-like 1), and Ifh1 (Interacts with forkhead 1), which form a regulatory complex at the majority of RPG promoters [53]. In addition, the high mobility group (HMG)-box protein Hmo1 binds to around half of the promoters of RPGs bound by Rap1-Fhl1-Ifh1 complex [54]. However, the homologs of these identified transcription factors regulating RPGs are absent in M. thermophila. On the other hand, the TOR (target of rapamycin) pathway has been demonstrated to be essential for the transcription of RPGs in S. cerevisiae, in which the key genes are Tor1 and Tor2 [55]. When the TOR pathway is inactivated by nutrient starvation or by treatment of cells with rapamycin, the transcript level of RPGs is downregulated [55, 56]. It has also been proposed that the transcription factor Sfp1, which acts downstream of the TOR pathway, modulates the transcription of RPGs in response to changes in nutrient availability [56, 57]. While orthologs of Tor1/Tor2 (MYCTH_2307294) and Sfp1 (MYCTH_2294631) are present in M. thermophila, their expression levels did not differ significantly between the ΔMtclr-2 and WT strains (log₂ fold change = − 0.58 and 0.03, respectively). In N. crassa, it has been reported that the disruption of cpc-1, which encodes a bZIP transcription factor known as the cross pathway control-1(CPC1), resulted in elevated expression of 37 ribosomal protein genes, suggesting that CPC1 acts as a repressor of ribosomal protein gene expression [58]. In our transcriptomic analysis, MYCTH_2315566, the homolog of cpc-1 in M. thermophila, was upregulated in the ΔMtclr-2 mutant (log₂ fold change = 1.63), which may partially be responsible for the observed downregulation of genes involved in ribosome biogenesis. It is also possible that, in the loss of Mtclr-2, fungal cells reduced the transcription of ribosomal protein genes as an adaptive response to nutrient limitation during cultivation on cellulose. Since the ΔMtclr-2 mutant is unable to secrete sufficient cellulases to efficiently degrade cellulose into monosaccharides and/or oligosaccharides, it likely experiences carbon scarcity. In such scenario, the downregulation of ribosome biogenesis may serve as a strategy to conserve energy for basic cellular functions, given that ribosome synthesis is one of the most energy-consuming processes in the cell [55, 57]. The growth defect of ΔMtclr-2 on cellulose, but not on glucose, suggests that the downregulation of ribosomal protein genes and the resulting secretion impairment, may be specifically associated with cellulose utilization. It has been shown that ribosomal protein gene expression is strongly induced when yeast cells are transferred from unfavorable carbon source to glucose-containing medium [55, 59, 60]. Moreover, in N. crassa, the rate of ribosomal protein synthesis increased considerably after a nutritional upshift from acetate to glucose, but was severely repressed upon a shift from glucose to glycerol [61]. Similar observation was reported in S. cerevisiae, where transcript levels of 90 ribosomal proteins were reduced in response to amino deficiency [62]. Likewise, in N. crassa, 67 genes encoding predicted ribosomal proteins were downregulated under amino acid starvation [58]. These studies indicate that the altered expression of ribosomal protein genes represents a common response to nutritional changes. However, the precise mechanism by which MtCLR-2 mediates the regulation of ribosomal protein gene expression under cellulolytic conditions merits further investigation.

Conclusions

MtCLR-2 was identified as a transcriptional activator that regulates cellulase production by directly binding to the promoter regions of egl2 and bgl1 to modulate their expression. Additionally, MtCLR-2 was found to be associated with ribosomal protein biosynthesis. This study not only provides insights into the regulatory network for cellulase gene expression but also highlights MtCLR-2 as a promising target for rational genetic engineering to enhance cellulolytic enzyme production in M. thermophila.

Materials and methods

Strains and culture conditions

Myceliophthora thermophila (ATCC 42464) was used as parental strain throughout this work. Conidial suspensions of M. thermophila strains were prepared by culturing them on potato dextrose agar (PDA) incubated at 45℃ for 7 days. The spores were harvested and suspended in 0.2% (v/v) Tween-80 solution and counted using a hemocytometer. For DNA extraction, the fungi were grown in Mandels medium containing 2% (w/v) glucose as carbon source, incubated at 45 °C with shaking at 150 rpm for 36 h [17]. For enzymatic activity assays, RT-qPCR, and comparative transcriptome analyses, mycelia were first pre-cultured from conidia in Mandels medium containing 2% (w/v) glucose for 36 h (final concentration, 1 × 10⁶ spores mL^− 1). The mycelia were then washed with carbon-free Mandels medium, and an equal amount (0.55 g) of wet mycelia was transferred into 50 mL of Mandels medium supplemented with 2% (w/v) Avicel (Sigma-Aldrich, Darmstadt, Germany), and incubated at 45 °C with shaking at 150 rpm for 48–96 h. For fungal colony morphology and conidiation assays, 1.5 µL of spore suspensions (1 × 10⁷ mL^− 1) from M. thermophila strains were inoculated onto the center of PDA solid plates containing 1% (w/v) Avicel and incubated at 45 °C for 3–7 days. For growth observation in liquid culture, conidia of fungal strains were added to 50 mL Mandels medium supplemented with 2% (w/v) glucose or 2% (w/v) Avicel (final concentration, 1 × 10⁶ conidia mL^− 1), and grown at 45 °C for 36 h. Fungal mycelia cultured in liquid shaking flasks were either subjected to macroscopic observation or harvested for microscopic analysis using light microscopy.

Escherichia coli DH5α was used for plasmid construction and propagation, cultured at 37℃ in Luria-Bertani (LB) medium supplemented with ampicillin (100 µg mL^–1).

Plasmid construction

All the primer sequences used in this study are listed in Table S1. For the knockout of Mtclr-2 (MYCTH_38704) using the CRISPR/Cas9 system, the Cas9-U6p-Mtclr-2-sgRNA expression cassette was constructed as described previously [17]. Specific sgRNA target site for Mtclr-2 was identified using the sgRNACas9 tool with minimum off-target effects [63]. The DNA fragments, including the U6 promoter of M. thermophila, and the target DNA sequence that fused with sgRNA scaffold were obtained by PCR using the pFC332-Cas9-U6p-sgRNA scaffold vector as the template [17] and cloned into the pFC332 vector to create the plasmid Cas9-U6p-Mtclr-2-sgRNA. To construct the donor DNA vector, the 5′ and 3′ flanking segments of Mtclr-2 were separately amplified from M. thermophila genomic DNA by PCR, fused with the selectable marker cassette Pgpd-neo from a pBC-neo plasmid [64], and inserted into the HindIII and EcoRI sites of pUC19 plasmid to generate Donor-Mtclr-2-neo using One Step Cloning Kit (Vazyme Biotech, Nanjing, China).

To generate Mtclr-2 overexpression vector, the gene-coding region of Mtclr-2 was amplified from genomic DNA and ligated into pUC19-Ppdc-TgpdA-hph plasmid at the NotI and XbaI sites, under the control of the strong constitutive Mtpdc (MYCTH_112121, pyruvate decarboxylase) promoter of M. thermophila. Similarly, for intracellular localization analysis of MtCLR-2, the PCR-amplified Mtclr-2 coding region without the stop codon and the gfp (green fluorescent protein) gene were assembled and cloned into the NotI and XbaI sites of pUC19-Ppdc-TgpdA-hph plasmid to generate the Mtclr-2-gfp cassette.

Transformation of Myceliophthora thermophila protoplasts

Protoplast preparation and transformation of M. thermophila was carried out following previously reported methods [17]. Possible transformants for gene deletion or overexpression in M. thermophila were screened for resistance to neomycin and hygromycin B (75 µg mL^− 1 hygromycin B combined with 100 µg mL^− 1 G418) or to hygromycin B alone (75 µg mL^− 1) after 4–5 days of incubation on PDA plates. Positive transformants were verified by PCR analysis with different specific primer pairs (Table S1), followed by sequencing.

Protein and enzyme activity assays

The extracellular protein concentration in the culture supernatant was measured using a Modified Bradford Protein Assay kit (Sangon Biotech, Shanghai, China) based on 595 nm absorbance. A 20 µL aliquot of culture supernatant was loaded on a 10% polyacrylamide gel (PAGE Gel Quick Preparation Kit, Yeasen Biotech, Nanjing, China) to analyze the proteins by SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis). Cellulase activities, including filter paper cellulase (FPase), endoglucanase (CMCase), β-glucosidase (pNPGase), and cellobiohydrolase (pNPCase), were determined as previously described [17]. One unit (U) of enzymatic activity was defined as the amount of enzyme that releases 1 µmol of either glucose or p-nitrophenol (pNP) from the substrate per minute under standard assay conditions.

Fluorescence microscopy examination

To determine subcellular localization of MtCLR-2 in M. thermophila, MtCLR-2-GFP positive transformants were inoculated into Mandels medium containing 2% (w/v) glucose or 2% (w/v) Avicel as the sole carbon source and incubated for 36 h at 45 °C. The mycelia were collected and incubated in darkness with 5 mg L^–1 4′,6-diamidino-2-phenylindole (DAPI) solution for 15 min, and washed with PBS solution three times. Fungal mycelia were observed under an Olympus BX53 fluorescence microscope, and the images were processed using Olympus cellSens Standard software.

Real‑time quantitative PCR

Total RNA was extracted from frozen mycelia using an RNA extraction kit TransZol Up (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. Reverse transcription was performed using the HiScript III RT SuperMix kit with gRNA wiper (Vazyme Biotech, Nanjing, China) following the manufacturer’s protocols. Quantitative PCR was performed with Hieff UNICON Universal Blue qPCR SYBR Green Master Mix (Yeasen Biotech, Shanghai, China), as described in our previous study [17]. The transcript level of each gene was estimated by the 2^−ΔΔCT method [65] with the actin gene (MYCTH_2314852) as an internal control. The relative transcript level was determined by calculating the radio of the transcript level in the Mtclr-2 deletion mutant to that in the WT control, which was set as 100%. Each reaction was conducted in triplicate.

Expression and purification of the MtCLR-2 DNA-binding domain

The three exon-coding sequences of Mtclr-2 were first obtained by amplifying from M. thermophila genomic DNA using the primer sets shown in Table S1. These fragments were then assembled and inserted into the HindIII site of pET-51b to generate pET-51b-MtCLR-2₁₋₇₉₈ plasmid. Due to the short length and limited expression of the MtCLR-2 DNA-binding domain (amino acids 44 to 75), an extended fragment comprising amino acids 30 to 89, which includes the core domain and 14 additional residues on both the N- and C-terminal sides, was selected for expression. To express the MtCLR-2 DNA-binding domain, the sequence encoding the amino acids 30 to 89 was amplified by PCR from the pET-51b-MtCLR-2₁₋₇₉₈ plasmid and was ligated into the pET-51b to create the pET-51b-MtCLR-2₃₀₋₈₉ recombinant plasmid. The codon optimized pUC57-MtCLR-2₁₋₂₇₂ vector, containing DNA sequence that encodes amino acids 1 to 272, was synthesized by IGE Biotechnology (Guangzhou, China). This plasmid includes an additional 14 amino acids that were absent in the MtCLR-2 DBD compared to the NcCLR-2 DBD. For the expression of the MtCLR-2 DBD that restores the missing 14 amino acids (residues 30 to 103), the corresponding DNA sequence was amplified by PCR using the pUC57-MtCLR-2₁₋₂₇₂ plasmid as the template and was inserted into HindIII-digested pET-51b to obtain the recombinant vector pET-51b-MtCLR-2_{(30−89)+14aa}. The recombinant plasmids were subsequently introduced into E. coli BL21(DE3) for protein expression, and the Strep II-tagged proteins were purified and verified as described previously [17].

Electrophoretic mobility shift assays (EMSAs)

EMSAs were carried out as previously reported [17]. Briefly, promoter regions of bgl1 (MYCTH_66804, P2, − 650 to − 300) and egl2 (MYCTH_86753, P2, − 650 to – 300; P3, − 1000 to − 600) were amplified from M. thermophila genomic DNA using specific primers shown in Table S1. For each EMSA reaction, varying amounts of purified recombinant MtCLR-2₃₀₋₈₉ (0–0.04 µg) or MtCLR-2_{(30−89)+14aa} (0–0.1 µg) were incubated with a fixed amount (100 ng) of the corresponding DNA probe in solution containing Gel-Shift Binding Buffer (Beyotime Biotech, Shanghai, China) with or without ZnSO₄ at a final concentration of 25 mM, at 25 °C for 30 min. After incubation, the protein-DNA complexes and free DNA were separated by electrophoresis on native 6% polyacrylamide gels with 0.5 ×TBE running buffer at 110 V for 70 min at 4 °C and finally stained using YeaRed Nucleic Acid Gel Stain (Yeasen Biotech, Shanghai, China). The promoter region of xyn1 (MYCTH_112050, P1, − 350 to − 1) was used as a negative control.

Analysis of transcriptomic data

The quality and quantity of total RNA extracted from M. thermophila wild-type strain, and its mutant were assessed using agarose gel electrophoresis and a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA). High-quality purified RNA was used for RNA sequencing (RNA-seq) on the Illumina NovaSeq 6000 platform, performed by Gene Denovo Biotechnology Corporation (Guangzhou, China). Clean reads were generated and mapped to the M. thermophila ATCC 42,464 genome using HISAT2 v2.4 [66]. Gene expression levels were calculated using RSEM software [67], with transcript abundance represented as fragments per kilobase of transcript per million mapped reads (FPKM). Differential gene expression analysis was conducted using DESeq2 [68], with criteria set at an absolute fold change > 2 and an adjusted P-value (padj) < 0.05. The detailed data are shown in Table S2. Gene Ontology (GO) enrichment analysis was performed using OmicShare tools (https://www.omicshare.com/tools). All differentially expressed genes (DEGs) were mapped to GO terms in the Gene Ontology database (http://www.geneontology.org/), and significantly enriched GO terms (P-value < 0.05) were identified via a hypergeometric test against the M. thermophila genome background. The raw transcriptome sequencing data have been deposited in the Gene Expression Omnibus (GEO) database under the accession number GSE305312 at the National Center for Biotechnology Information (NCBI).

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

G.L., D.L.B. and J.W. initiated, designed, supervised this study, edited and reviewed the manuscript. Y.L. planned and carried out experiments, interpreted experimental data, and drafted the manuscript. G.L., N.X., and J.W. supported the research funding. All authors read and approved the final manuscript.

Funding

This work was supported by the Natural Science Foundation of China (22078199), Natural Science Foundation of Guangdong Province (2024B1515020034), National Key R&D Program of China (2021YFA0910800), Shenzhen Municipal Science and Technology Basic Research Program (JCYJ20200109105210448).

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations

Ethics approval and consent to participate

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Competing interests

The authors declare no competing interests.