Involvement of the Methyltransferase CcLaeA in Regulating Laccase Production in Curvularia clavata J1

Abstract

Laccases are synthesized by a diverse range of fungi. Nevertheless, despite the industrial significance of laccases, the regulatory mechanism governing laccase production has been relatively understudied. This research aims to explore the regulatory function of the methyltransferase CcLaeA in laccase biosynthesis using the newly isolated fungal strain Curvularia clavata J1. Through CRISPR-Cas9-mediated gene disruption, the deletion of CclaeA led to a 1.5-fold increase in extracellular laccase activity in the ΔCclaeA mutant when compared to the wild-type strain. This finding indicates that CcLaeA functions as a transcriptional repressor of laccase biosynthesis. Transcriptomic analysis demonstrated that CcLaeA does not directly regulate the expression of laccase genes. Instead, it modulates genes associated with hydrolases and peptidases. This modulation potentially reduces the enzymatic degradation of laccase at the protein level. This study significantly enhances our understanding of fungal laccase regulation. By establishing a connection between the deletion of CclaeA and the improvement of enzyme stability and activity, this research offers practical insights for engineering fungal strains to optimize laccase yields for bioremediation and biofuel applications. Furthermore, the integration of targeted gene knockout with multi-omics validation sets up a methodological framework for investigating regulatory networks in non-model fungi. This framework is expected to accelerate the development of sustainable biocatalysts, thereby contributing to the advancement of biotechnology in various industrial sectors.

1. Introduction

As a crucial structural polymer in lignocellulosic biomass, lignin constitutes 20–35% of the dry weight of plant matter. This makes it one of the most abundant renewable organic materials on Earth. This aromatic biopolymer functions as an essential carbon reservoir in terrestrial ecosystems and holds great promise for sustainable biorefinery applications [1]. Lignin is a complex aromatic polymer formed by phenylpropane units, which are connected through a diverse range of carbon–carbon and carbon–oxygen bonds. Its structure is highly elaborate and varies significantly [2]. From an environmental standpoint, efficient lignin degradation is a vital part of the ecosystem’s material cycle. In the context of resource utilization, the products resulting from lignin degradation hold significant application potential. These products can be utilized as raw materials for biofuels, chemicals, and other value-added materials. This not only aids in alleviating the energy crisis but also reduces reliance on fossil fuels [3,4].

Filamentous fungi, as pivotal lignin-degrading microorganisms in nature, present distinct advantages [5,6]. A multitude of filamentous fungi have the ability to secrete a diverse array of crucial enzymes for lignin degradation. These enzymes include laccase, manganese peroxidase, and lignin peroxidase as well as other auxiliary enzymes. Through their synergistic interplay, they efficiently break the intricate chemical bonds of lignin, thereby facilitating its depolymerization and degradation [7]. Among the various key enzymes secreted by filamentous fungi for lignin degradation, laccase plays a particularly prominent role. It belongs to the family of multicopper oxidases and is capable of catalyzing the oxidation of a wide spectrum of phenolic and non-phenolic substrates [8]. Specifically, it targets the relatively labile chemical bonds within the lignin structure. By breaking these bonds, laccase initiates the depolymerization process of lignin. Studies have shown that the expression of laccase is stringently regulated at the transcriptional level. A variety of transcription factors from different fungi are intricately involved in this complex regulatory mechanism, including Cu-binding transcription factors (TFs) Cuf1/Ace1, pH-responsive TF PacC and oxidative stress-responsive TF ATF1 [9,10,11,12]. In addition, a recent study has identified two novel Zn2Cys6-type transcription factors in the white-rot fungus Trametes hirsute that respond to aromatic compounds and regulate the expression of laccases and intracellular enzymes by forming heterodimers [13], providing new insights into the regulatory mechanism of laccase production. Furthermore, certain heat shock transcription factors precisely regulate laccase gene expression through post-transcriptional mechanisms, such as alternative splicing [14]. These TFs regulate the expression of laccase genes by binding to their promoter regions, which contain various cis-elements that contribute to diverse expression patterns in response to different environmental conditions. Despite the identification of several regulatory factors associated with laccase expression, our understanding of the regulatory elements that govern laccase expression in filamentous fungi remains limited. Thus, further in-depth investigation and analysis of the specific transcription factors or other novel regulators and their underlying regulatory mechanisms are essential.

In filamentous fungi, the laeA gene assumes a pivotal physiological role. Initially isolated from Aspergillus nidulans in 2004, laeA acts as a global regulatory gene for secondary metabolism in filamentous fungi. The protein encoded by laeA belongs to the S-adenosylmethionine (SAM)-dependent methyltransferase family and harbors a conserved SAM-binding domain. This implies that LaeA might exert epigenetic regulatory functions via methylation modifications [15,16,17]. Regarding growth and development, the laeA gene is of great significance in the morphological differentiation of filamentous fungi. Disruption of this gene can cause changes in morphology and cell structure during asexual development, thereby influencing the normal growth and developmental processes of the fungi [18]. Simultaneously, the laeA gene plays a central regulatory part in the synthesis of secondary metabolites. It mainly regulates the enzymes involved in the production of fungal secondary metabolites through transcriptional activation [17,19]. Moreover, the LaeA factor demonstrates a certain preference in gene regulation [20]. Furthermore, LaeA can regulate not only endogenous secondary metabolic gene clusters but also heterologously expressed secondary metabolic gene clusters. This heterologous regulatory effect underscores the functional conservation of the laeA gene throughout biological evolution [19].

Current research regarding the regulation of laccase activity by LaeA-like proteins in filamentous fungi is scarce. Although several studies have hinted at a potential association, the underlying molecular mechanisms remain largely uncharted. For example, the interaction patterns between LaeA and laccase-encoding genes, along with the signal transduction pathways involved, have not been clearly elucidated. In this study, we investigated the role of LaeA homolog of C. clavata in laccase production, and showed that the disruption of CclaeA in C. clavata J1 led to a 49.2% increase in laccase activity. Transcriptome analysis uncovered substantial alterations in the expression levels of genes associated with laccase synthesis and secretion pathways. These differentially expressed genes might be directly or indirectly regulated by CcLaeA, thereby providing valuable clues regarding the molecular mechanisms of laccase regulation. This research identifies a new target for filamentous fungi fermentation engineering, optimizes laccase production, and paves the way for future gene research in filamentous fungi and the development of biotransformation technologies.

2. Materials and Methods

2.1. Fungal Strain and Culture Media

Curvularia clavata J1 (CGMCC 41301) was isolated from soil and used as the parental wild-type (WT) strain. Escherichia coli Trans1-T1 (TransGen, Beijing, China) was used for plasmid construction and propagation, and was cultured in Luria–Bertani (LB) medium supplemented with 100 µg/mL ampicillin. Potato dextrose agar (PDA) was used for plate culture of the fungus. For flask culture, mycelia from the C. clavata strains were placed into 100 mL of Vogel’s minimal medium (MM) [21] supplemented with 2% (w/v) glucose (MMG) or 1% lignosulfonate (catalog no. S4340, Solarbio, Beijing, China) and 0.25% glucose (MML). The culture was incubated at 30 °C with continuous shaking at 200 rpm for 4 days. The mycelia were then harvested for RNA sequencing and the supernatant was assayed for enzyme activity. For the preparation of protoplasts, MMGY medium (1% (w/v) Vogel’s salts, 2% (w/v) glucose, 0.5% (w/v) yeast extract) was used to culture mycelia. MMG medium supplemented with 1 M sorbitol was used to screen transformants after protoplast transformation.

2.2. Search for the CclaeA Gene in C. clavata J1 and Sequence Analyses

The LaeA protein from the closely related species Cochliobolus heterostrophus (Bipolaris maydis) (ChLae1, GenBank accession no. AEP40318; [22]) was used to search for the LaeA homolog of C. clavata YC1106 [23]. The target protein was named CcLaeA and submitted to sequence analyses. Analysis of conserved domains was conducted using Conserved Domain Database (CDD) at NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 5 May 2024) [24]. Multiple sequence alignments were performed using ClustalX 2.1 software [25]. The neighbor-joining phylogenetic tree was constructed with 1000 bootstrap replicates by MEGA7 software [26].

2.3. Construction of Cassettes for CRISPR/Cas9-Mediated Genome Editing

For the deletion of CclaeA, the 5′- and 3′-flanking regions of CclaeA were amplified using the primers laeA-up-F/laeA-up-R and laeA-down-F/laeA-down-R, listed in Table S1. The PtrpC-neo cassette was amplified using the primer pair PtrpC-F and neo-T-R. After assembly into the pCE2 TA/Blunt-Zero cloning vector (Vazyme Biotech, Nanjing, China), the donor fragment 5′-PtrpC-neo-3′ was amplified using the primer pair laeA-up-F/laeA-down-R and used for protoplast transformation of the WT strain of C. clavata J1.

To select specific single guide RNAs (sgRNAs) targeting the corresponding gene, the sgRNAcas9 tool [27] was used to identify sgRNA target sites with high scores. To construct the gRNA expression cassette for CclaeA gene, the target DNA sequence in the plasmid U6p-Mtalp1-sgRNA [28] was replaced with that of CclaeA by PCR with paired primers laeA-gRNA-F and U6p-laeA-R, followed by self-recombination using the ClonExpress Ultra One Step Cloning Kit (Vazyme Biotech, Nanjing, China), resulting in the plasmid U6p-CclaeA-sgRNA. The gRNA expression cassette was then amplified using the primer pair U6p-F and gRNA-R. The Cas9-expression PCR cassette Ptef1-Cas9-TtrpC was amplified from the plasmid p0380-bar-Ptef1-Cas9-TtrpC [29] using the primer pair Ptef-cas-F and TtrpC-cas-R. All constructed plasmids were verified by sequencing.

2.4. PEG-Mediated Transformation of C. clavata J1 and Selection of Transformants

Mycelia were crushed into small pieces using sterile glass beads (5 mm diameter) and placed into 100 mL of MMGY medium. After incubation at 30 °C and 200 rpm for 16 h, the actively growing mycelia were harvested and used to prepare the protoplasts as previously described [30] with some modification. Briefly, 5 mg/mL of lysing enzymes from Trichoderma harzianum (catalog no. L1412; Sigma-Aldrich, St. Louis, MO, USA) and 1 mg/mL of Driselase from Basidiomycetes sp. (catalog no. D9515; LABLEAD, Beijing, China) dissolved in 1 M KCl was added to release the protoplasts from the mycelia. Protoplasts were collected by centrifugation at 2000 rpm for 5 min and then washed with solution B (50 mM CaCl₂, 1 M sorbitol, 10 mM Tris-HCl [pH 7.5]). After being collected by centrifugation, protoplasts were resuspended to a final concentration of approximately 2 × 10⁶ protoplasts/mL in solution B. Then, a total of 10 µg PCR products of the Cas9-expression cassette Ptef1-Cas9-TtrpC, the gRNA expression cassette U6p-CclaeA-sgRNA, and the corresponding donor fragment 5′-PtrpC-neo-3′ were mixed at the same an equimolar ratio and used to co-transform protoplasts of the WT strain. Transformants were screened using selection medium supplemented with 100 mg/L geneticin (G418) and incubated at 30 °C for 4 days.

The disruption of CclaeA in transformants was confirmed by PCR analysis. For this purpose, DNA was extracted from the selected transformants using the method described by Li et al. [5] and used as a template for amplification of the target region of CclaeA using primers laeA-in-F and laeA-in-R (Table S1).

2.5. Enzyme Activity Assays

Laccase activity was determined using the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) oxidation method as described previously [28], with minor modifications as follows. The assay system consisted of 20 µL of supernatant and 180 µL of 100 mM sodium acetate buffer (pH 5.0) containing 1 mM ABTS. After incubation at 45 °C for 10 min, oxidation of ABTS was monitored by determining the increase in absorbance at 420 nm. One unit (U) of laccase activity was defined as the amount of enzyme that oxidizes 1 µmol of ABTS per minute.

Manganese peroxidase (MnP) and lignin peroxidase (LiP) activities were determined using commercially available assay kits (Solarbio, Beijing, China) following the manufacturer’s instructions. Specifically, MnP activity was measured by monitoring the oxidation rate of guaiacol using the MnP activity assay kit (catalog no. BC1620), while LiP activity was assessed through the oxidation rate of veratryl alcohol using the LiP activity assay kit (catalog no. BC1610). Enzyme activities were expressed in units (U), where one unit corresponds to the amount of enzyme required to oxidize 1 nmol of substrate (guaiacol for MnP or veratryl alcohol for LiP) per minute under the assay conditions.

2.6. Growth Test

For solid culture, the WT and ΔCclaeA strains were inoculated on PDA plates and incubated at 30 °C for 6 days. The colony diameter was measured to assess growth, and conidia production was quantified using a hemacytometer. For liquid culture, mycelia of the WT and ΔCclaeA strains were inoculated into 100 mL of MML medium and cultivated at 30 °C with shaking at 200 rpm for 4 days. After cultivation, the dry cell weight was determined to evaluate growth under liquid conditions.

2.7. RNA Sequencing and mRNA Expression Analysis

After harvesting via vacuum filtration, mycelia were immediately homogenized in liquid nitrogen for total RNA extraction. Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), treated with DNase I and purified using a Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA integrity was confirmed by agarose gel electrophoresis and an Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). Qualified RNA with A260/A280 > 2.0 and RNA integrity number (RIN) > 8.0 was used for RNA-Seq, which was performed by Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China), using a DNBSEQ-T7 platform. Prior to read mapping, adaptors and low-quality reads were trimmed using fastp v0.19.5 [31]. The RNA-seq experiments were conducted on three biological duplicates for each condition. Filtered clean reads were aligned against predicted transcripts from the C. clavata YC1106 genome v1.0 [23] using HISAT2 v2.1.0 [32]. The read counts were determined using RSEM v1.3.3 [33]. The abundance of each transcript was calculated based on the fragments per kilobase of transcript per million mapped reads (FPKM) values (Table S2). Differential gene expression analysis was performed in R v3.6.3, using DESeq2 (https://bioconductor.org/packages/release/bioc/html/DESeq2.html, accessed on 12 October 2024) [34]. Genes with a fold-change of >2.0 (|log2 ratio| > 1) and a Q value (adjusted p value) of <0.05 were considered significantly differentially expressed between different conditions or strains. To discover significantly up- and downregulated genes, only genes with relatively high transcript abundance (FPKM > 20 in at least one strain or condition) were considered for further analysis.

RNA-Seq data are available at the Gene Expression Omnibus under accession numbers GSM8550458, GSM8550459, GSM8550460, GSM8550461, GSM8550462, and GSM8550463.

3. Results

3.1. Isolation and Identification of C. clavata Strain J1

Multiple fungal strains were isolated from forest soil samples collected in Guizhou Province, China. These strains were then assessed for their growth capacity on sodium lignosulfonate as the substrate. Among them, a strain demonstrating significant growth, designated as J1, was chosen for further investigation. The partial 18S rRNA gene sequence of strain J1 was determined and utilized to construct a phylogenetic tree (Figure 1A). The 18S rRNA sequence of strain J1 (GenBank accession no. PQ252867.1) shared 99.64% identity (99.3% coverage) with that of C. clavata strain YC1106 (CP089277.1: 4,183,531–4,184,630), indicating that strain J1 is a strain belonging to C. clavata species. In prior research, C. clavata strains have been shown to possess the ability to degrade azo dyes and agro-industrial residues from the palm oil industry [35,36]. During these processes, lignin peroxidase and laccase were the main ligninolytic enzymes involved in the degradation of color. Nevertheless, to date, there has been no report on the lignin-degrading ability of these strains. Notably, C. clavata strain J1, which was screened in this study, displayed relatively high laccase activity (34 U/L) but low manganese peroxidase (MnP) activity (<2 U/L) and lignin peroxidase (LiP) activity (8.2 U/L) (Figure 1B) when grown on lignosulfonate. This finding suggests that it might be capable of degrading lignin.

3.2. Analysis of the CclaeA Gene and Its Deduced Protein

To identify the CclaeA gene in C. clavata YC1106, homology searches were conducted using the Cochliobolus heterostrophus (Bipolaris maydis) ChLae1 as a query. These searches led to the identification of the CclaeA gene, designated as yc1106_06579. This gene encodes a 322-amino-acid protein that exhibits 95.9% identity with ChLae1, but a relatively low identity (<53%) with those from genera Aspergillus, Penicillium, Fusarium and Trichoderma (Figure 2A). Analysis of the conserved domains of CcLaeA revealed the presence of a methyltransferase domain, along with four appropriately spaced sequence motifs (Motif I, post-Motif I, Motif II, and Motif III) (Figure 2B). These features are characteristic of LaeA proteins in filamentous fungi.

3.3. Disruption of CclaeA Gene in C. clavata J1 Using CRISPR-Cas9 System

To investigate the function of the CclaeA gene, we initiated the development of a transformation system for C. clavata J1. Although a transformation system had been previously established for C. clavata strain BAUA-2787 [37], it is relatively time-consuming. This system requires 40 h of cultivation and 3 h for protoplast formation. In this study, we optimized this process. We added an enzyme solution containing Driselase and Lysing Enzymes from Trichoderma harzianum to the fresh mycelia. These mycelia were collected from cultures grown in 100 mL of MMGY medium at 30 °C and 200 rpm for 16 h. By using this method, protoplasts could be efficiently obtained within 1.5 h.

Subsequently, we generated a single-knockout mutant at the CclaeA locus using the CRISPR/Cas9 system (Figure 3A). The disruption of CclaeA was verified by diagnostic PCR. Amplification of a clear band for CclaeA (463 bp) was observed in the WT strain, while amplification of an approximately 1610 bp band was observed for the transformants with CclaeA disruption (Figure 3B). No evident morphological differences were observed when comparing the transformants and WT strains. We then assayed the laccase production activity of the wild-type (WT) strain and the ΔCclaeA mutant. As depicted in Figure 3C, the laccase activity of the ΔCclaeA mutant increased by 49.2% compared to that of the WT strain.

3.4. Comparative Transcriptional Analysis of the WT Strain and the CclaeA Mutant

To gain a comprehensive understanding of the expression patterns, we conducted a transcriptional analysis of the wild-type (WT) and ΔCclaeA strains (ΔCclaeA) grown on lignosulfonate (Table S2). The percentage of reads mapped to the reference genome ranged from 92.34% to 93.9% (93.33% on average). Pearson correlation analysis demonstrated that the biological replicates were reliable for all tested samples (>0.95 for WT strain and >0.99 for the ΔCclaeA mutant). When compared to the WT strain, the transcript levels of 876 genes were significantly changed in the ΔCclaeA mutant. Specifically, 366 genes were significantly upregulated, and 510 genes showed significantly reduced transcription (Figure 4A and Table S3). We first investigated the expression profiles of the laccase-encoding genes in C. clavata J1. Genome annotation indicated that there are ten genes that encodes laccase in the genome of C. clavata YC1106. Among them, the gene yc1106_07441 (designated as lac1 here) had the highest expression level in the WT strain (Figure 4B and Table S3). This finding implies that lac1 is likely the main contributor to laccase activity in this organism. However, contrary to the increased laccase activity observed in the ΔCclaeA mutant, the expression of lac1 was significantly downregulated in the mutant (Figure 4B and Table S3). Considering the pleiotropic regulatory role of LaeA as a global transcriptional modulator [38,39], this result suggests its potential involvement in biological processes beyond the direct regulation of laccase expression.

Notably, transcriptional analysis revealed significant downregulation of multiple genes associated with pyruvate metabolism in the ΔCclaeA mutant. These genes include those encoding phosphoenolpyruvate synthase (yc1106_09606), lactate dehydrogenase isoforms (yc1106_07039 and yc1106_09605), phosphoenolpyruvate carboxykinase (yc1106_08972), and pyruvate decarboxylase (yc1106_00375) (Table S3). These coordinated transcriptional changes suggest that CcLaeA may play a regulatory role in directing carbon flux through central metabolic pathways.

A Gene Ontology (GO) analysis of the upregulated genes showed that they were enriched only in the categories of “regulation of cellular processes” and “regulation of biological processes” (Figure 4C and Table S4). In contrast, the downregulated genes were significantly enriched in multiple GO terms that were mainly associated with hydrolase and peptidase activity (Figure 4D and Table S4). Consistent with this observation, a gene (yc1106_04001) encoding a peptidase with signal peptide showed the most significant downregulation (Table S3). This suggests that the increase in laccase activity of the ΔCclaeA mutant may be attributed to a decrease in the proteolytic degradation of laccase.

4. Discussion

In response to the global trend of low-carbon energy and industrial development, bio-based manufacturing has received increasing attention, but a key challenge is the accessibility of cost-effective, abundant and renewable feedstocks. While sugar-based feedstocks have traditionally been the main source for the bio-industry, their high price and limited availability due to arable land constraints remain significant barriers. However, the use of lignin represents a potential breakthrough, providing an alternative feedstock to sugar-based feedstocks for bio-based manufacturing in the future. The complex structure of lignin is the origin of its valorization in terms of degradation and utilization, resulting in limited practical application in the field of lignin biodegradation. The relatively low enzymatic activity of laccase further limits its catalytic capacity, which ultimately limits the efficiency of enzymatic hydrolysis processes.

Rational engineering strategies have primarily focused on increasing the laccase yield through gene manipulation. For example, heterologous expression of laccase genes in Pichia pastoris has been optimized using high-density fermentation techniques to reach titers of up to >10,000 U/L [40,41]. In addition, the RNAi-mediated knockdown of specific laccase isoforms has provided valuable insights into their functional diversity and regulatory mechanisms [42]. Recent advances have also included the development of constitutive expression systems by modifying promoter regions, exchanging nucleotide sequence of signal peptide and manipulating a certain TF, PacC [43,44,45,46]. This approach reduces the dependence on inducers and streamlines industrial fermentation processes. In addition to rational engineering strategies, fermentation optimization factors such as pH, temperature and oxygen concentration play a crucial role in laccase production [47]. Optimal conditions are generally in the range of pH 5–7 and temperature 28–30 °C. However, there are few reports on exploring the laccase regulatory network to increase laccase production in filamentous fungi. Although transcriptomic and co-expression network analysis conducted by Chen et al. [48] identified potential candidate genes associated with laccase activity in Trametes gibbosa, the study did not provide direct experimental evidence demonstrating the involvement of specific regulatory elements in the modulation of laccase activity.

In this study, we isolated Curvularia clavata strain J1, a filamentous fungus capable of using lignosulfonate as a substrate. Significantly, when this fungus was cultured on lignosulfonate, an increase in laccase activity was observed. Research on C. clavata species is still limited, and no previous studies have explored its gene regulatory mechanisms. Via functional genomics, we identified the global regulatory protein methyltransferase CcLaeA as a negative regulator of laccase activity. This was evidenced by the significantly increased laccase production in the ΔCclaeA mutant strain. Using the genomic sequence of C. clavata strain YC1106 as a reference, a high percentage of reads (93.33%) mapped to the reference genome was obtained. Additionally, we confirmed the presence of all ten laccase-encoding genes in strain J1 through PCR amplification using primers designed based on the reference genome. All cloned sequences from strain J1 exhibit >99% identity with the corresponding sequences from strain YC1106, indicating a close evolutionary relationship and suggesting similar biological characteristics between the two strains. Despite their close phylogenetic relationship, the two fungal strains may exhibit strain-specific variations in gene content, including differences in the number of strain-specific genes, as well as variations in genomic organization, as observed in other fungal species [30].

Even in well-studied fungal systems, our understanding of laccase expression regulators is limited. Therefore, this work enriches the fundamental knowledge of the transcriptional control mechanisms of laccase genes across different fungal taxa. Our findings in the ΔCclaeA mutant strain uncover a paradoxical relationship: although laccase activity was significantly enhanced, transcriptional analysis indicated a downregulation of the putative dominant laccase gene (lac1). This suggests that CcLaeA-mediated regulation of laccase activity may not rely on transcriptional control mechanisms. A previous study demonstrated that Penicillium oxalicum LaeA methylated histone H2B at lysine 122 (H2BK122) and lysine 130 (H2BK130) to regulate gene expression [17]. Additionally, Myceliophthora thermophila LaeA was found to modulate the methylation levels of histone H3 lysine 9 (H3K9) [16]. It is well known that altered histone methylation levels can lead to changes in gene expression. Hence, the differentially expressed genes in the ΔCclaeA mutant may be partly due to the changed methylation levels of histones in their promoter regions.

Transcriptomic profiling further identified substantial suppression of hydrolase- and peptidase-associated genes in the ΔCclaeA mutant compared to the WT strain. The discordance between reduced lac1 expression and elevated enzymatic activity suggests that compensatory regulatory pathways may exist. Potential post-transcriptional mechanisms, such as enhanced enzyme stability, translational efficiency, or post-translational modifications, could reconcile this apparent contradiction. These findings underscore the complexity of fungal enzymatic regulation and highlight the need for multi-omics approaches to elucidate non-canonical regulatory networks. Elucidation of these regulatory interconnections will be critical for engineering robust fungal strains with enhanced lignocellulose-degrading capacities, thereby accelerating biotechnological innovations in lignin valorization for sustainable biorefinery applications.

The discovery of LaeA’s direct upregulation of laccase activity represents a critical advancement in lignin degradation, offering fundamental insights into microbial metabolic regulation and lignocellulosic bioprocessing optimization. Going forward, genome-scale metabolic network analyses will be employed to map regulatory networks within complex lignin-degrading enzymatic systems, while machine learning-driven predictive models will enable precise characterization of enzyme interactions and synergistic effects. Advanced optimization algorithms will be systematically applied for temporal-spatial control of multi-enzyme expression patterns, maximizing enzymatic synergy and enhancing product yields from lignin-derived aromatic compounds. These efforts collectively establish a synthetic biology framework enabling targeted depolymerization of lignin into value-added monomers in high-performance microbial chassis. Notably, current metabolic pathways exhibit limited diversity in upstream bioconversion processes, necessitating directed evolution of monoenzyme systems and rational design of enzyme cocktails to achieve selective lignin degradation. By integrating lignin degradation intermediates into the tricarboxylic acid (TCA) cycle, we aim to boost carbon flux efficiency and amplify metabolic pathway capacities. Ultimately, this research trajectory will pave the way for next-generation cell factories capable of producing food-grade functional compounds and protein-rich biomass from lignocellulose through circular production schemes. Such innovations hold transformative potential to address global food security challenges, advance sustainable waste valorization, and contribute to the development of carbon-neutral bioeconomies.

5. Conclusions

This study investigated the regulatory role of the methyltransferase CcLaeA in laccase production in C. clavata J1. Disruption of CclaeA resulted in a significant 1.5-fold increase in laccase activity. Transcriptomic analysis revealed that the ΔCclaeA mutant exhibited downregulation of multiple genes encoding hydrolases and peptidases, suggesting that reduced enzymatic degradation of laccase may contribute to the observed increase in laccase activity. These findings identify CcLaeA as a novel regulator of laccase activity in C. clavata J1 and expand our understanding of the biological processes regulated by LaeA-like proteins. Further studies are required to elucidate the precise molecular mechanisms underlying CcLaeA-mediated regulation of laccase activity.

6. Patent

This fungal strain has been patented in China (patent no. 202411022370.5).