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Mechanism of Zn2+ regulation of cellulase production in Trichoderma reesei Rut-C30

Biotechnology for Biofuels and Bioproducts volume 16, Article number: 73 (2023) Cite this article

Abstract

Background

Trichoderma reesei Rut-C30 is a hypercellulolytic mutant strain that degrades abundant sources of lignocellulosic plant biomass, yielding renewable biofuels. Although Zn2+ is an activator of enzymes in almost all organisms, its effects on cellulase activity in T. reesei have yet to be reported.

Results

Although high concentrations of Zn2+ severely suppressed the extension of T. reesei mycelia, the application of 1–4 mM Zn2+ enhanced cellulase and xylanase production in the high-yielding cellulase-producing Rut-C30 strain of T. reesei. Expression of the major cellulase, xylanase, and two essential transcription activator genes (xyr1 and ace3) increased in response to Zn2+ stimulation. Transcriptome analysis revealed that the mRNA levels of plc-e encoding phospholipase C, which is involved in the calcium signaling pathway, were enhanced by Zn2+ application. The disruption of plc-e abolished the cellulase-positive influence of Zn2+ in the early phase of induction, indicating that plc-e is involved in Zn2+-induced cellulase production. Furthermore, treatment with LaCl3 (a plasma membrane Ca2+ channel blocker) and deletion of crz1 (calcineurin-responsive zinc finger transcription factor 1) indicated that calcium signaling is partially involved in this process. Moreover, we identified the zinc-responsive transcription factor zafA, the transcriptional levels of which declined in response to Zn2+ stress. Deletion of zafA indicates that this factor plays a prominent role in mediating the Zn2+-induced excessive production of cellulase.

Conclusions

For the first time, we have demonstrated that Zn2+ is toxic to T. reesei, although promotes a marked increase in cellulase production. This positive influence of Zn2+ is facilitated by the plc-e gene and zafA transcription factor. These findings provide insights into the role of Zn2+ in T. reesei and the mechanisms underlying signal transduction in cellulase synthesis.

Background

The derivation of bioethanol from the degradation of lignocellulose is an important process [1, 2]. Saccharification, a vital step in the degradation of lignocellulose, is catalyzed by cellulases produced by filamentous fungi, such as Trichoderma reesei and Aspergillus nidulans [3,4,5]. Given its excellent extracellular enzyme production, safety, and reliability, T. reesei is used industrially as a model strain for cellulase production [6, 7]. Xyr1, a global transcriptional activator, regulates the expression of cellulase and xylanase genes [8], and a disruption of xyr1 has been demonstrated to result in abrogation of the expression of almost all cellulase- and hemicellulase-encoding genes [9]. In 2014, a further important transcriptional activator, ace3, was reported to regulate the production of cellulase and xylanase [10]. Although the loss of ace3 was found to result in slight reductions in the expression of hemicellulases [11] and xylanases [12], it completely abolished cellulase production. Microorganisms sense their surroundings and respond to external signals via a network of signal transduction pathways that provide strict control of cellulase production [13]. Among such pathways, the calcium signaling pathway is essential and highly conserved in filamentous fungi [13].

Ca2+ play vital cellular roles as a ubiquitous second messenger regulating cell growth, virulence, and stress resistance [14], and is a core component of the calcium signal transduction pathway in filamentous fungi [15]. In T. reesei, Chen et al. [16] demonstrated that Mn2+ regulates cellulase gene expression via calcium signaling, and Xu et al. [17, 18] have demonstrated that addition of Mn2+ and Na+ to liquid cultures of Ganoderma lucidum induces the biosynthesis of ganoderic acid via calcineurin signaling transduction. Subsequently, Gao et al. [19] revealed that reactive oxygen species (ROS) and Ca2+ cross-regulate hyphal branching and ganoderic acid biosynthesis induced by Cu2+ in G. lucidum. The complete Ca2+ signaling pathway includes free Ca2+, calmodulin (Cam), calcineurin (Cna), and calcineurin-responsive zinc-finger transcription factor 1 (Crz1/CrzA), and in response to an increase in cytoplasmic concentrations of Ca2+, activation of Cam and Cna promotes the dephosphorylation of Crz1/CrzA, which acts on downstream pathway genes [20].

Cytosolic Ca2+ levels increase via two pathways, in the first of which, Ca2+ from the external environment enters the cytoplasm via ion channels in the cell membrane [21,22,23], whereas in the second, Ca2+ within the intracellular Ca2+ pool enters the cytoplasm via a PI-PLC/IP3-mediated pathway [24]. In this latter pathway, phospholipase C (PLC) is activated in response to extracellular signals, promoting an increase in IP3 content, which subsequently leads to the release of Ca2+ from the intracellular Ca2+ pool [25, 26]. According to Chen et al. [15], plc-e can be activated by N, N-dimethylformamide, which promotes an increase in cytosolic Ca2+ in T. reesei.

Zn2+ serves as an important structural or catalytic cofactor for numerous transcription factors (TFs) and enzymes, and is accordingly essential for almost all organisms, including fungi [27, 28]. A number of studies on Zn2+ metabolism have focused on pathogenic fungi, such as Candida albicans [29], Cryptococcus gattii [28], and Aspergillus fumigatus [30], in which Zn2+ facilitates normal growth and plays important roles in a range physiological processes [31]. Zap1, a zinc-responsive TF, was first identified in Saccharomyces cerevisiae, in which it controls Zn2+ homeostasis and adaptive responses to Zn2+ deficiency [32]. Homologous to S. cerevisiae Zap1 is the transcriptional activator ZafA identified in A. fumigatus [33]. In a murine model of invasive aspergillosis, cells with loss of ZafA were found to be characterized by negligible virulence [33]. Schneider et al. demonstrated that Zap1 (an ortholog of S. cerevisiae Zap1) plays a vital role in the regulation of Zn2+ homeostasis and modulation of virulence in C. gattii [28]. Comparatively, however, there have been few studies that have examined the role of zinc in non-pathogenic filamentous fungi.

In this study, we used the high-yielding RUT-C30 strain of T. reesei as a parent strain to study the mechanisms whereby extracellular Zn2+ induces cellulase production. We discovered that the addition of intracellular Zn2+ causes a significant increase in cellulase production and has a strong inhibitory effect on hyphal growth. Transcriptome analysis and gene deletion were used to elucidate the molecular mechanisms underlying the Zn2+-induced cellulase production. In addition, we identified a zinc-responsive TF ZafA (an ortholog of S. cerevisiae Zap1 and A. fumigatus ZafA) in T. reesei and demonstrated its relevance with respect to cellulase production in response to Zn2+. The findings of this study provide important insights for further elucidation of the mechanisms whereby Zn2+ influences cellulase production in the filamentous fungi T. reesei.

Results

Effects of Zn2+ on hyphal growth and cellulase and xylanase production in T. reesei

To study the effect of Zn2+ on hyphal growth, the same amounts of fresh RUT-C30 conidia were inoculated onto minimal medium (MM) plates [supplemented with different concentrations of Zn2+ (0–5 mM final concentration) and 2% (w/v) glucose as the sole carbon source] for 4 days to compare colony growth. As shown in Fig. 1a, Zn2+ strongly inhibited hyphal growth. When the concentration of Zn2+ was increased to 1 mM, growth was inhibited to a certain extent compared with the untreated strain (by approximately 25.4%) (Fig. 1b). Compared with the untreated strain, treatment with 2 mM Zn2+ led to a 66.9% reduction in colony diameter, and treatment with ≥ 3 mM Zn2+ suppressed virtually all colony growth. These results accordingly revealed that at higher concentrations, Zn2+ represents a stressor that inhibits T. reesei growth.

Fig. 1
figure 1

Different concentrations of Zn2+ influence the hyphal growth of the Trichoderma reesei RUT-C30 strain. a RUT-C30 grown on MM plates supplemented with Zn2+ at final concentrations of 0–5 mM. b Colony diameter of T. reesei RUT-C30 cultured on MM plates under different concentrations of Zn2+. The final values are presented as the means ± standard deviation (SD) of three independent experimental results. Asterisks indicate significant differences compared with the control (*P < 0.05, according to Student’s t-test). MM, minimal medium

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Given the effects of Zn2+ on the growth of strains, we adopted the transfer method in this study. Equal masses of T. reesei RUT-C30 pre-cultured mycelia were transferred to MM supplemented with 1% (w/v) Avicel as the sole carbon and different concentrations of Zn2+ (0–5 mM) and incubated at 28℃ for 4 days to assess the effect of Zn2+ treatment on the production of cellulase, xylanase, and extracellular protein. As shown in Fig. 2, the addition of 1–4 mM Zn2+ promoted marked enhancements of cellulase, xylanase and extracellular protein per gram of T. reesei mycelium, annulling its negative effects on growth. Supplementation with 3 mM Zn2+ was found to have the most pronounced effects in this regard. Specifically, the addition of 3 mM Zn2+ stimulated pNPCase activity (representing exo-β-glucanase activity), which was enhanced by approximately 96.5% to 191.3% compared with the control without 3 mM Zn2+ supplementation (Fig. 2a). As shown in Fig. 2b, 3 mM Zn2+ also promoted a significant increase in CMCase activity (representing endo-β-glucanase activity), by approximately 67.9% to 77.7% compared with that of the control. Furthermore, compared with the control, supplementation with 3 mM Zn2+ resulted in approximate 46.9% and 82.6% increases in xylanase and filter paper hydrolase activities (FPase, representing total extracellular cellulase activity), respectively (Fig. 2c, d). As illustrated in Fig. 2e, we detected an approximate 92.8% increase in extracellular protein concentration in response to supplementation with 3 mM Zn2+ compared with the control strain. In contrast, exposure to 5 mM Zn2+ was found to have negative effects on CMCase, xylanase, and FPase activities, which we speculate could be attributed to the severe growth inhibition effects at high concentrations (Fig. 2f). Accordingly, in further studies, 3 mM Zn2+ was selected as the optimal concentration of for enhancing cellulase yields.

Fig. 2
figure 2

Different concentrations of Zn2+ influence the cellulase and xylanase synthesis of the Trichoderma reesei RUT-C30 strain. pNPCase activity (a), CMCase activity (b), xylanase activity (c), FPase activity (d), total protein concentrations (e), and biomass dry weight (f) of the RUT-C30 strain were determined after culturing for 2, 3, or 4 days in liquid MM containing different concentrations of Zn2+ (0–5 mM) and 1% (w/v) Avicel as the sole carbon source. The final values are presented as the means ± standard deviation (SD) of the three independent experimental results. Asterisks indicate significant differences compared with the control (*P < 0.05, according to Student’s t-test). MM, minimal medium

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Fig. 3
figure 3

Effects of 3 mM Zn2+ on cellulase-related gene transcription levels in the Trichoderma reesei RUT-C30 strain. Transcriptional levels of cbh1 (a), cbh2 (b), egl1 (c), egl2 (d), xyn1 (e), xyr1 (f), and ace3 (g) of the RUT-C30 strain were detected after culturing for 36, 48 or 60 h in liquid MM containing 0 or 3 mM Zn2+ with 1% (w/v) Avicel as the sole carbon source. The final values are presented as the means ± standard deviation (SD) of three independent experimental results. Asterisks indicate significant differences compared with the control (*P < 0.05, according to Student’s t-test). MM, minimal medium

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To further investigate the effects of Zn2+ on cellulase and xylanase production, we determined the transcription levels of the four main cellulase genes (cbh1, cbh2, egl1, and egl2), one major xylanase gene (xyn1), and two essential transcriptional activators of cellulase and xylanase (xyr1 and ace3) using real-time quantitative PCR (RT-qPCR). Consistent with the aforementioned cellulase and extracellular protein results, we found that the addition of 3 mM Zn2+ promoted marked increases in the expression levels of the four main cellulase genes and one major xylanase gene by approximately 0.54- to 2.33-fold compared with the control at 60 h (Fig. 3a–e). Consistently, we detected a marked up-regulation of the transcriptional levels of xyr1 and ace3 at all assessed time points (Fig. 3f, g).

To the best of our knowledge, this is the first time that Zn2+ supplementation has been demonstrated to promote a significant increase in the cellulase and xylanase production of T. reesei Rut-C30. However, the specific induction mechanisms have yet to be sufficiently established and accordingly warrants further investigation.

Transcriptomic changes in T. reesei following exposure to Zn2+

To further determine how Zn2+ influences T. reesei at the transcriptional level, we performed whole-transcriptome shotgun sequencing (RNA-seq) using RUT-C30 cultured for 48 h in MM containing 0 or 3 mM Zn2+, with 1% Avicel as the sole carbon source. We performed Illumina NovaSeq6000 RNA sequencing to analyze three independent biological samples from each of the assessed conditions. Following sequence quality control (41,388,676–54,546,188 reads, with no significant difference between parallel biological samples), the sequences of the total reads were mapped to the latest T. reesei reference genome (https://www.ncbi.nlm.nih.gov/assembly/GCA_002006585.1) with a coverage of 94.82–95.42%. The results of Pearson correlation analysis (r2 ≥ 0.718) revealed that there was a strong correlation between the three biological replicates of the strain with or without 3 mM Zn2+ (Additional file 1: Fig. S1). Subsequently, we screened genes for differential expression between the two conditions based on the following thresholds: a Log2fold change (Log2fc) ≥ 1 and an adjected p-value < 0.05.

As shown in the volcano plot presented in Fig. 4, in the presence of 3 mM Zn2+, 852 genes were differentially expressed, of which 520 were upregulated and 332 were downregulated (3 mM_vs._0 mM; Fig. 4a). Gene ontology (GO) annotation analysis of these differentially expressed genes (DEGs) revealed that the most enriched genes are associated with catalytic activity, membrane part, binding, and metabolic processes (Fig. 4b). A histogram representing the findings of Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the most enriched pathways affected by Zn2+ included “starch and sucrose metabolism”, “fructose and mannose metabolism,” “protein processing in endoplasmic reticulum,” “galactose metabolism,” “pentose and glucuronate interconversions,” and “amino sugar and nucleotide sugar metabolism” (Fig. 4c). Collectively, the data indicated that in the presence of Zn2+, significant changes occurred in multiple signaling pathways, which are worthy of further investigation.

Fig. 4
figure 4

RNA‑seq analysis of the Trichoderma reesei RUT-C30 strain treated with 0 or 3 mM Zn2+. Volcano analysis (a), Gene ontology (GO) annotation analysis (b), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis (c) of DEGs in the RUT-C30 strain treated with 0 and 3 mM Zn2+

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Among the 34 genes associated with cellulase and hemicellulose degradation in T. reesei, we detected upregulated transcriptional levels in 28 genes and downregulated levels in two (M419DRAFT_ 124931 and M419DRAFT_103113) (Table 1). Notably, we detected marked increases (Log2fc ≥ 2) in the mRNA levels of two main cellobiohydrolases (CEL7A and CEL6A), two endoglucanases (CEL7B and CEL45A), one cellulose-binding protein CIP1 (M419DRAFT_121449) [34], swollenin (M419DRAFT_104220) [35], one xylanase (XYN3), mannan endo-1,4-beta-mannosidase MAN1 (M419DRAFT_122377), and alpha-galactosidase AGL3 (M419DRAFT_39277). Additionally, 15 TFs are known to be associated with cellulase and hemicellulase expression, of which three are positive transcription regulators with increased mRNA levels (xyr1, ace3, and vib1 [37]) and one is a negative transcription regulator with reduced mRNA levels (rce1 [36]) (Table 2). These results are consistent with the marked increases in pNPCase, CMCase, xylanase, and FPase activities and RT-qPCR data for RUT-C30 cells treated with 3 mM Zn2+.

Table 1 Comparison of cellulase and hemicellulase gene expression levels in the Trichoderma reesei RUT-C30 strain grown with or without 3 mM Zn2+
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Table 2 Comparison of transcription factor gene expression levels in the Trichoderma reesei RUT-C30 strain grown with or without 3 mM Zn2+
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PLC-E is required for Zn2+ induction of cellulase production

Metal ions have been reported to regulate cellulase gene expression via calcium signaling in T. reesei [16], and consequently, we speculated as to whether calcium signaling would be involved in Zn2+-induced cellulase expression. To verify this conjecture, we determined the mRNA levels of four major calcium signal pathway-related genes based on transcriptional profiling (Table 3). Notably, we detected a significant increase in the mRNA levels of plc-e encoding a phospholipase C protein, which can be activated by extracellular receptors, and induces the release of calcium from internal stores via the generation of inositol-1,4,5-trisphosphate (IP3) [37].

Table 3 Comparison of calcium signal transduction-related genes expression levels in Trichoderma reesei RUT-C30 strain with or without 3 mM Zn2+
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Furthermore, to determine whether plc-e plays an important role in Zn2+-induced excessive production of cellulase, plc-e was deleted in T. reesei RUT-C30 to obtain the mutant strain Δplc-e. Interestingly, the facilitation effect of Zn2+ on cellulase synthesis in Δplc-e was initially suppressed during the early phase (36 h for qPCR and 2 days for activities) and effectively attenuated in the latter phase (Fig. 5a–f), thereby providing evidence to indicate that plc-e is involved in the Zn2+ induction process. In the absence of Zn2+, the loss of plc-e promoted a slight enhancement of pNPCase and CMCase activities, whereas in the presence of Zn2+, we detected reductions in pNPCase and CMCase activities in Δplc-e compared with those in the parental RUT-C30 (Fig. 5a, b). Collectively, these findings indicate that the deletion of plc-e effectively attenuates the induction effect of Zn2+ on cellulase production. Similar findings were obtained with respect to the transcriptional levels of cbh1, cbh2, egl1, and egl2 (Fig. 5c–f).

Fig. 5
figure 5

Effects of plc-e on cellulase production after Zn2+ addition. pNPCase activity (a), CMCase activity (b) of Trichoderma reesei RUT-C30 and Δplc-e cultured for 2, 3, or 4 days in liquid MM with or without 3 mM Zn2+ and 1% (w/v) Avicel as the sole carbon source, respectively. The mRNA levels of cbh1 (c), cbh2 (d), egl1 (e), and egl2 (f) were also detected. The final values are presented as the means ± standard deviation (SD) of three independent experimental results. Asterisks indicate significant differences compared with the control (*P < 0.05, according to Student’s t-test). MM, minimal medium

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On the basis of transcription profiling, we established that the transcriptional levels of the calcium signaling genes (cam and crz1) were slightly enhanced in the presence of Zn2+ (Table 3). RT-qPCR was used to quantitatively determine differences in the expression levels of these genes in the presence and absence of 3 mM Zn2+. As shown in Additional file 2: Fig. S2, the transcriptional levels of cam were slightly enhanced after 36 h of treatment with Zn2+.

To evaluate whether calcium signal transduction plays a role in the Zn2+ induction process, we used LaCl3 (a plasma membrane Ca2+ channel blocker) to inhibit the influx of external Ca2+ [38], and we generated the crz1 deletion strain in RUT-C30 to inhibit the calcium signal transduction pathway. Analyses of cellulase activities (pNPCase and CMCase activities) and transcriptional levels of key cellulase genes (cbh1 and egl1) revealed that treatment with LaCl3 led to a marked reduction in cellulase activity and the transcriptional levels of cbh1 and egl1 compared with those in the no-LaCl3 control, regardless of the presence of Zn2+ (Additional file 3: Fig. S3). Furthermore, in the presence of LaCl3, the facilitation effect of Zn2+ on cellulase synthesis was significantly attenuated during the early phase of induction (2 to 3 days) (Additional file 3: Fig. S3a, b). Quantitative analysis using RT-qPCR revealed that exposure to LaCl3 had a slightly negative effect on Zn2+-induced cbh1 and egl1 expression (Additional file 3: Fig. S3c, d). Collectively, these findings wound tend to indicate that calcium signal transduction is partially involved during the early phase of the Zn2+ induction process.

Although the calcium signaling pathway was blocked in the crz1 deletion strain, we found that supplementation with 3 mM Zn2+ could still enhance the pNPCase and CMCase activities in this strain compared to those observed in the control (without 3 mM Zn2+) on day 4 (Additional file 4: Fig. S4a, b). Similar results were obtained in the RT-qPCR analysis (Additional file 4: Fig. S4c, d), thus indicating that in addition to the calcium signaling pathway, other pathways are involved in Zn2+-induced cellulase synthesis.

Identification of the zinc-responsive transcription factor zafA

In fungi, zinc-responsive TFs play important roles in regulating zinc homeostasis [32, 33]. In this regard, the TF zafA has been extensively studied in S. cerevisiae [39], C. gattii [28], and A. fumigatus [33]. NCBI BLAST analysis revealed that M419DRAFT_96242 detected in T. reesei in the present study is a homolog of the zafA in S. cerevisiae and A. fumigatus. Furthermore, RNA‑seq analysis revealed a slight, although significant, reduction in the mRNA levels of M419DRAFT_96242 (zafA) following exposure to 3 mM Zn2+ compared with the control group (0 mM Zn2+) (Additional file 5: Table S1). As revealed by RT-qPCR, compared with the control, the transcription levels of M419DRAFT_96242 (zafA) were downregulated by 48.99% in response to 3 mM Zn2+ supplementation (Additional file 6: Fig. S5).

As shown in Fig. 6a, six zinc-finger C2H2 domains containing 702 amino acids were predicted in zafA using the Pfam database (http://pfam.xfam.org). Phylogenetic analysis (Fig. 6b) based on the zafA protein sequence revealed that zafA homologs are widely distributed in a range of Ascomycota, including Sordariomycetes, Pezizomycetes, Leotiomycetes, and Eurotiomycetes, with high amino acid similarity. In addition, zafA homologs have been identified in numerous Trichoderma species, indicating that this protein may have a conserved function. However, the roles of zafA in strain growth and cellulase production (in either T. reesei or other cellulose-degrading species) under conditions of zinc stress have yet to be evaluated.

Fig. 6
figure 6

The identification of ZafA. Functional domain prediction (a) and phylogenetic tree analysis (b) of zafA

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ZafA mediates Zn2+‑stimulated excessive production of cellulase in the RUT-C30 strain

To determine whether the reduced expression of zafA is associated with the Zn2+ induction process, we generated a zafA deletion strain (∆zafA) in which the signal transduction pathway was blocked. We investigated the effects of the parental strain RUT-C30 and ∆zafA mutants on cellulase production in response to 0 and 3 mM Zn2+ treatments. As shown in Fig. 7a, b, the deletion of zafA resulted in a marked reduction or complete inhibition of Zn2+-induced cellulase production compared with the control. In the absence of 3 mM Zn2+ supplementation, we detected no clear differences between the two strains with respect to pNPCase and CMCase activities. Compared with the parental strain RUT-C30 in the absence of 3 mM Zn2+, we detected marked enhancements of approximately 160.4% and 70.4% in pNPCase and CMCase activities, respectively, following exposure to 3 mM Zn2+. However, with the deletion of zafA, we detected notably less pronounced increases in pNPCase and CMCase activities of 46.4% and 23.4%, respectively, following exposure to Zn2+ pressure compared with no Zn2+ supplementation. Additionally, RT-qPCR was performed to determine the transcription levels of four major cellulase-encoding genes (cbh1, cbh2, egl1, and egl2) in T. reesei RUT-C30 and ∆zafA, and we accordingly found the transcript levels to be consistent with the cellulase activity data. The significant enhancement of the transcriptional levels of these four cellulase genes induced by Zn2+ was effectively attenuated by the deletion of zafA (Fig. 7c–f).

Fig. 7
figure 7

Effect of zafA on cellulase production after Zn2+ addition. pNPCase activity (a), CMCase activity (b) of Trichoderma reesei RUT-C30 and ΔzafA cultured for 2, 3, or 4 days in liquid MM with or without 3 mM Zn2+ and 1% (w/v) Avicel as the sole carbon source, respectively. The expression levels of cbh1 (c), cbh2 (d), egl1 (e), and egl2 (f) were also determined. The final values are presented as the means ± standard deviation (SD) of three independent experimental results. Asterisks indicate significant differences compared to the control (*P < 0.05, according to Student’s t-test). MM, minimal medium

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In summary, these findings indicate that Zn2+ induces an enhancement of cellulase production in T. reesei Rut-C30 primarily via zafA, and this enhancement effect induced by Zn2+ was effectively prevented in the ∆zafA mutant, in which the associated signaling pathway was blocked.

DEGs associated with G-protein-coupled receptors and transporters

The transfer of extracellular signals to intracellular sites generally requires transport mediated by G-protein-coupled receptors (GPCRs) [40] and transporters [41]. To date 58 GPCRs have been reported in T. reesei [42], of which we detected the up- and downregulation of 14 and three, respectively, in response to treatments in the present study (Additional file 7: Table S2). These 17 differentially expressed GPCRs included one class III GPCR, one class V GPCR, two class VI GPCR, one class VII GPCR, one class XI GPCR, and 11 PTH11-like GPCRs, among which, 10 of the 11 PTH11-like GPCRs were downregulated, the exception being M419DRAFT_76201. In addition, among 152 transporters, we detected 44 differentially expressed DEGs, among which 31 and 13 were up- and downregulated genes, respectively (Additional file 8: Table S3). The stp1 (M419DRAFT_136988) gene, which was notably upregulated by 21.18 (2.27)-fold, has been established to be associated with cellobiose transport [43]. In addition, we identified one Fe2+/Zn2+-regulated transporter (M419DRAFT_91910) that was markedly downregulated by 24.29 (19.56)-fold.

Collectively, these findings provide convincing evidence to indicate that 3 mM Zn2+ promotes notable changes in transcription and signal transduction in T. reesei.

Discussion

In this study, we sought to examine the effects of Zn2+ on the growth, enzyme production, and regulatory signaling pathways of T. reesei. Although Zn2+, a key cofactor of numerous TFs and enzymes, has been widely studies in filamentous fungi [30], yeasts (S. cerevisiae) [39, 44], plants, and animals [45], to the best of our knowledge, there have been no published studies on the properties of Zn2+ in T. reesei. In this study, we found that strain growth on solid medium was substantially inhibited in the presence of > 2 mM Zn2+ (Fig. 1), thereby indicating that T. reesei was subjected to heightened stress. In S. cerevisiae, excessive Zn2+ has been found to cause oxidative damage to cells by regulating the expression of antioxidant defense genes [27]. In T. reesei, it has been found that in response increases in the concentrations of Ca2+, Mn2+, and Sr2+ to 100 mM, 40 mM, and 120 mM, respectively, the strain growth is generally slow and sparse [16, 20, 46]. In Ganoderma lucidum, hyphal branch length and growth have been observed to be significantly reduced in the presence of 5 mM Cu2+ [19], whereas treatment with 17 mM Ca2+ was found to result in a substantial reduction in the diameter of Penicillium brevicompactum colonies [49]. The findings of these studies indicate that fungi have specific tolerance to metal ions.

We found that exposure of T. reesei to Zn2+ promoted increases in production of the enzymes cellulase and xylanase, with maximal enhancement being recorded in those fungi treated with 3 mM Zn2+, a finding which would be beneficial from the perspective of industrial production (Fig. 2). In this regard, recent studies have demonstrated that extracellular supplementation with metal ions can promote the production of primary and secondary metabolites in filamentous fungi [16,17,18,19, 46]. On the basis of transcriptome analyses, we discovered that 852 genes were differentially expressed in T. reesei treated with 3 mM Zn2+, including a number of upregulated cellulase- and hemicellulose-associated genes (3 mM_vs._0 mM; Fig. 4a and Table 1), which is consistent with our RT-qPCR data (Fig. 3). Importantly, we found that the transcription levels of intracellular plc-e were significantly elevated in response to stimulation with Zn2+, whereas the deletion of plc-e from the parental strain (Rut-C30) resulted in an attenuation of Zn2+-induced cellulase production during the early phase of the Zn2+ induction process (Fig. 5). Previous studies have shown that plc-e is involved in regulating the expression of cellulase and Ca2+ signaling genes in T. reesei under the stimulation of extracellular signals [6, 15]. In the present study, we detected a slight enhancement in the mRNA levels of genes associated with the Ca2+ signaling pathway in response to treatment with Zn2+, (Table 3), thereby indicating that plc-e might play a role in the release of Ca2+ from intracellular Ca2+ pools. Treatment with LaCl3 and deletion of crz1 to block cytosolic Ca2+ signaling indicated that calcium signal transduction is partially involved in the early phase of Zn2+ induction, thereby implying the involvement of other pathways in the Zn2+-induced excessive production of cellulase (Additional file 3: Fig. S3, Additional file 4: Fig. S4), which warrants further investigation.

ZafA, an important transcription factor, plays a key role in maintaining Zn2+ homeostasis, which has been shown to be associated with gliotoxin biosynthesis in A. fumigatus [30]. In the present study, RNA-seq analysis revealed that the expression of one zinc-finger protein was significantly downregulated in response to treatment with Zn2+ (Additional file 5: Table S1). On the basis of an NCBI blastx search, we established that this protein is homologous to zap1 in S. cerevisiae [39] and zafA in A. fumigatus [33], and we accordingly designated the protein zafA. Phylogenetic tree analysis revealed that zafA is expressed in a range of fungi, particularly species in the genus Trichoderma (Fig. 6b). With respect to fungi, transcription factors have been studied primarily in pathogenic species such as A. fumigatus [47] and C. albicans [48]. To date, however, the role of zafA in Zn2+ homeostasis has yet to be reported in cellulase-producing strains. In the present study, we demonstrated that following the deletion of zafA, the efficacy of Zn2+ in promoting cellulase production and the expression of cellulase-related genes was markedly attenuated, and in some case was completely inhibited (Fig. 7). Collectively, these observations would thus tend to indicate that zafA plays a prominent role in T. reesei when subjected to Zn2+ stress.

Exposure of T. reesei Rut-C30 to sufficiently high concentrations of Zn2+ was found induce stress-like responses in the fungi, and consequently, we examined the expression levels of two major antioxidant enzymes (sod1 and cat1 [46]) in response to 3 mM Zn2+ treatment based on RT-qPCR. We accordingly found that the mRNA levels of sod1 were markedly enhanced at 36 h and 60 h, which was not substantially altered by the addition of Zn2+ at 48 h (Additional file 9: Fig. S6). Contrastingly, the levels of cat1 were observed to undergo a gradual, albeit marked, decline with time (Additional file 9: Fig. S6). These findings thus provide evidence indicating that cells were subjected to oxidative stress in response to treatment with Zn2+. Similar findings have been reported for S. cerevisiae, in which elevated levels of intracellular ROS have been detected in zinc-sensitive mutants exposed to high Zn2+ stress [27]. In our previous study, we established that exposure to 70 mM Sr2+ promoted a ROS burst and associated increases in the expression of sod1 and cat1 in T. reesei [46]. Similarly, Chen et al. [49] demonstrated that the mRNA levels of antioxidant enzyme genes in P. brevicompactum were markedly upregulated in response to Ca2+. Treatment with metal ions exposes T. reesei Rut-C30 to stress, in response to which there is an upregulated expression of antioxidant genes to cope with the danger, and in our aforementioned previous study, we demonstrated that a ROS scavenger can alleviate intracellular ROS promoted by exposure to Sr2+ to enhance cellulase production [46]. In further studies, we accordingly intend to examine the effects of simultaneous treatment with Zn2+ and ROS scavengers on cellulase hyperproduction in RUT-C30.

In the presence of Zn2+, we detected a significant enhancement in the transcription levels of xyr1 and ace3 (Fig. 3f, g and Table 2), which are vital positive transcription activators that regulate cellulase gene expression in T. reesei [9, 12]. It is reasonable to speculate that zafA enhances the transcription of cellulase genes or associated activators. However, further studies will be necessary to establish the mechanisms underlying zafA recognition of the promoter sequences of these genes. Furthermore, we also detected the prominently enhanced expression of vib1 (Table 2), the deletion of which had the effect of reducing the expression of almost all cellulase and hemicellulase genes, vital sugar transporter genes, and the primary transcriptional activators xyr1 and ace3 in T. reesei [49]. In this regard, it has been found that in Neurospora crassa, Vib1 modulates cellulase synthesis by regulating the expression of the essential cellulase regulator CLR2 [50]. In the present study, we found the upregulation of vib1 to be consistent with the activation of xyr1 and ace3 in response to Zn2+. Furthermore, Zn2+ stimulation was found to be associated with the differential expression of 17 GPCRs and 44 transporter genes, which accordingly warrant further investigation with respect to their role in altered membrane signaling.

Conclusions

In this study, we discovered that supplementation of medium with 3 mM Zn2+ markedly inhibited the hyphal growth of T. reesei Rut-C30, whereas this treatment promoted a substantial enhancement in cellulase and xylanase production. Furthermore, we detected a significant upregulation of the transcription of major cellulase and xylanase genes, as well as that of two vital transcriptional activator genes (ace3 and xyr1). Transcriptional analysis revealed that the expression of plc-e was similarly significantly upregulated. We established that Zn2+ modulates the expression of cellulase genes, partly via the plc-e gene and Ca2+ signal transduction. In addition, we identified a transcription factor, zafA, associated with Zn2+ homeostasis, which was found to play a prominent role in Zn2+-induced excessive production of cellulase. On the basis of our findings, we thus elucidated a putative mechanism whereby Zn2+ regulates cellulase production in T. reesei (Fig. 8). Our identification of a novel inducer that promotes cellulase synthesis and the mechanisms underlying induction provide a valuable basis for further research on Zn2+ signal transduction in T. reesei.

Fig. 8
figure 8

Putative mechanism of Zn2+ induction of cellulase production in Trichoderma reesei. Supplementation of medium with 3 mM Zn2+ enhanced cellulase production and the expression of plc-e and calcium signaling genes. Disruption of plc-e, treatment with LaCl3 (a plasma membrane Ca2+ channel blocker), and the deletion of crz1 (calcineurin-responsive zinc-finger transcription factor 1) revealed that calcium signaling is partially involved in the induction process. Moreover, there were marked reduction in mRNA levels of the zinc-responsive transcription factor zafA in response to Zn2+ stimulation. Deletion of zafA indicated that this transcription factor plays a prominent role in mediating Zn2+-induced excessive production of cellulase. The solid arrows indicate data supported by our results, and the dashed arrows indicate undefined regulation

Full size image

Methods

Strains and growth conditions

Escherichia coli DH5α cells were used as the host for plasmid amplification. Fungal transformation was based on infection using the GV3101 strain of Agrobacterium tumefaciens [51]. T. reesei Rut-C30 (ATCC 56765) [52] was used as the host strain for genetic transformation. Luria broth (LB) was used to culture E. coli and A. tumefaciens, and MA medium [12] containing 2% (w/v) glucose and MM [46] containing 1% (w/v) Avicel or 2% (w/v) glucose were used for general fungal culture. All strains of T. reesei were maintained on potato dextrose agar (PDA) plates in the dark at 28 °C. Fresh conidia were collected using glycerin for subsequent analyses. MM, which was used to assess the effect of Zn2+ on cellulase production and hyphal growth, contained (g/L) (NH4)2SO4 (5), urea (0.3), KH2PO4 (15), CaCl2 (0.6), MgSO4 (0.6), MnSO4·H2O (0.0016), FeSO4·7H2O (0.005), and CoCl2·6H2O (0.002), pH 5.5, with 1% (w/v) Avicel or 2% (w/v) glucose as the sole carbon source. Conidia (2 × 106) were grown for 36 h at 28 °C in 100 mL of MA medium supplemented with 2% (w/v) glucose as the sole carbon source (220 rpm). Equal amounts of mycelia (approximately 0.1 g) were collected and washed thoroughly using 20 mL of fresh MM lacking a carbon source, and then transferred to 50 mL of fresh MM containing 1% (w/v) Avicel, with the addition of 0–5 mM ZnCl2. After 2–4 days of culture, samples were collected for enzymatic activity and protein concentration determinations. Mycelia induced for 36, 48, or 60 h were collected and maintained frozen at − 80 °C for subsequent RNA isolation and RT‑qPCR analyses.

Construction of T. reesei Δplc-e, ΔzafA, and Δcrz1 mutants

In this study, RUT-C30 was used as the parent strain for gene knockout. A pEASY®-Uni Seamless Cloning and Assembly Kit (TransGen, Shanghai, China) was used to construct deletion cassettes for deletion of the plc-e, zafA, and crz1 genes. The primers used for plasmid construction and diagnostic PCR are listed in Additional file 10: Table S4. Specifically, 722-bp upstream and 757-bp downstream fragments of plc-e were generated from the RUT-C30 genome using KOD-Plus-Neo (TOYOBO, Osaka, Japan). Initially, the upstream fragment obtained by PCR was ligated into PacI- and XbaI-linearized LML2.1 [53] to generate pFplc-e. Subsequently, the downstream fragment was inserted into the SwaI sites of pFplc-e to the generate deletion binary pDplc-e (Additional file 11: Fig. S7). Similarly, the deletion cassettes of zafA and crz1 were amplified and inserted into LML2.1 to generate the deletion vectors pDzafA and pDcrz1, respectively. Deletion cassettes were used to transform T. reesei RUT-C30 to facilitate the knockout of plc-e (Δplc-e mutant), zafA (ΔzafA mutant), and crz1 (Δcrz1 mutant) using Agrobacterium-mediated transformation [54]. The xylose-induced Cre recombinase system was used to self-excise the hygromycin-resistant cassette [55]. Primers XX-CF, XX-CR, XX-OF, and XX-OR (XX represents the gene name) were used in diagnostic PCR to verify the putative gene disruption mutants generated by double crossover.

Fungal growth and cellulase, xylanase, protein, and biomass production

For fungal hyphal growth assays, glycerin was used to collect fresh conidia, which were diluted to 2.5 × 106 mL−1 in sterile water. An equal volume of diluted conidia (2 μL) was inoculated onto the center of MM plates containing 2% (w/v) glucose and incubated for 4 days at 28 °C. Cellulase, xylanase, protein, and biomass production assays were performed as previously described [46, 56].

RNA isolation and quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR)

The mRNA levels of specific gene mRNAs were assessed using RT-qPCR, as described by Cai et al. [52]. Briefly, total RNA from 100 mg of T. reesei mycelia was extracted using a FastRNA Pro Red Kit (MPbio, Irvine, CA, USA) according to the manufacturer’s instructions. For reverse transcription, we used TransScript® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) (TransGen Biotech, Beijing, China) was used to reverse-transcribe 800 ng of total RNA to produce cDNA. RT-qPCR was performed using PerfectStart™ Green qPCR SuperMix (TransGen Biotech) with 1 μL of cDNA and 200 nM of the forward and reverse primers in a final volume of 20 μL. The sequences of the primers used in the RT-qPCR analysis are shown in Additional file 10: Table S4. For gene transcription analysis, the sar1 gene was used as the reliable reference in SYBR Green assays as previously described [57]. For RT-qPCR analysis, thermocycling was performed using an ABI StepOne thermocycler (Applied Biosystems, Foster City, CA, USA).

Chemical reagent treatments

For an assessment of Zn2+ stress, we added different concentrations of ZnCl2 to fungal growth medium. For plate growth studies, T. reesei was cultured on solid MM for 4 days. Different concentrations of ZnCl2 were added immediately after the mycelia had been transferred to the medium. For enzyme production analysis, conidia were germinated in MA medium to yield mycelia, which were subsequently transferred to MM supplemented with different concentrations of ZnCl2. To examine the roles of cytosolic Ca2+ in response to Zn2+ stress, mycelia were also treated with LaCl3 (a plasma membrane Ca2+ channel blocker), which was used at a final concentration of 5 mM after 1 day in T. reesei culture.

Whole-transcriptome shotgun sequencing (RNA‑seq) analysis

T. reesei RUT-C30 mycelia, treated with 0 or 3 mM Zn2+ in liquid MM containing 1% (w/v) Avicel as the sole carbon source, were harvested after 48 h for RNA-seq. For sequencing, all treated and untreated strains were sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China) in triplicate. The latest reference genome of T. reesei (https://www.ncbi.nlm.nih.gov/assembly/GCA_002006585.1) was used in this study for bioinformatic analysis. The whole-transcriptome data have been submitted to the NCBI SRA website (https://www.ncbi.nlm.nih.gov/sra/PRJNA923496) with the Accession Number PRJNA923496.

Statistical analysis

In this study, at least three independent experiments were performed to obtain reliable data with identical or similar results. The standard deviations (SDs) from the mean of triplicate determinations are indicated by error values. Student’s t test or Duncan’s multiple-range test was used for bicomponent or multiple comparisons, respectively. Within each set of experiments, p < 0.05 was considered to indicate significant differences between the data.

Availability of data and materials

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

Abbreviations

CMCase:

endo-β-Glucanase activity

crz1 :

Calcineurin-responsive zinc-finger transcription factor 1

DEG:

Differentially expressed gene

FPase:

Total extracellular cellulase activity

GPCR:

G-protein-coupled receptor

MM:

Minimal medium

PDA:

Potato dextrose agar

pNPCase:

Exo-β-glucanase activity

RNA‑seq:

Whole-transcriptome shotgun sequencing

ROS:

Reactive oxygen species

RT-qPCR:

Quantitative real-time reverse transcription polymerase chain reaction

TF:

Transcription factor

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Acknowledgements

We would like to thank Editage (www.editage.cn) for English language editing.

Funding

This research was supported by the National Key Research and Development Program of China (2022YFA0912300), the Natural Science Foundation of Shanghai (No. 22ZR1417600), the National Natural Science Foundation of China (32000050), and the Chenguang Program of the Shanghai Education Development Foundation and Shanghai Municipal Education Commission (21CGA34).

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Authors and Affiliations

  1. The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, P. O. Box 311, Shanghai, 200237, China

    Ni Li, Jing Li, Yumeng Chen, Yaling Shen, Dongzhi Wei & Wei Wang

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  1. Ni Li
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  2. Jing Li
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  3. Yumeng Chen
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  4. Yaling Shen
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  5. Dongzhi Wei
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  6. Wei Wang
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Contributions

WW directed and coordinated the study and reviewed the manuscript. NL planned and conducted experiments and wrote the manuscript. JL and YC analyzed the data and supported the research funding. YS reviewed the manuscript. DW assisted with revision of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Wei Wang.

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Supplementary Information

Additional file 1: Figure S1.

The heat map showed the correlation between the biological replicates of each sample in RNA‑seq analysis. The value in the square is the correlation coefficient between the two samples. The larger the value, the greater the correlation between the two samples and the closer they are. These results mean that the RNA‑seq data is very reliable.

Additional file 2: Figure S2.

Effect of 3 mM Zn2+ on calcium signal transduction related gene transcription levels in RUT-C30 strain. Transcriptional levels of cam (a), cna1 (b), and crz1 (c) of the RUT-C30 strain were detected after culturing in liquid MM for 36, 48 or 60 h containing 0 or 3 mM Zn2+ with 1% (w/v) Avicel as the sole carbon source. The final values are presented as the mean±standard deviation (SD) of three independent experimental results. Asterisks indicate significant differences compared to the control (*P <0.05, according to Student’s t-test).

Additional file 3: Figure S3.

Effect of LaCl3 on cellulase production after Zn2+ treatment. pNPCase (a) and CMCase (b) activity were measured in the RUT-C30 strain after exposed to Zn2+ or LaCl3. The transcriptional levels of cbh1 (c) and egl1 (d) were detected after culturing the RUT-C30 strain in medium supplemented with 0 or 3 mM Zn2+ and with (+) or without (−) 5 mM LaCl3. The final values are presented as the mean±standard deviation (SD) of three independent experimental results. Asterisks indicate significant differences compared to the control (*P <0.05, according to Student’s t-test).

Additional file 4: Figure S4.

Effect of crz1 on cellulase production after Zn2+ addition. pNPCase activity (a), CMCase activity (b) of RUT-C30 and Δcrz1 cultured in liquid MM for 2, 3, or 4 days with or without 3 mM Zn2+ and 1% (w/v) Avicel as the sole carbon source, respectively. The expression levels of cbh1 (c) and egl1 (d) were also determined. The final values are presented as the mean±standard deviation (SD) of three independent experimental results. Asterisks indicate significant differences compared to the control (*P <0.05, according to Student’s t-test).

Additional file 5: Table S1.

The mRNA level of zafA (M419DRAFT_96242) was detected by RNA‑seq analysis.

Additional file 6: Figure S5.

Effect of 3 mM Zn2+ on zafA transcription level in RUT-C30 strain. Transcriptional level of zafA of the RUT-C30 strain were detected after culturing in liquid MM for 36, 48 or 60 h containing 0 or 3 mM Zn2+ with 1% (w/v) Avicel as the sole carbon source. The final values are presented as the mean±standard deviation (SD) of three independent experimental results. Asterisks indicate significant differences compared to the control (*P <0.05, according to Student’s t-test).

Additional file 7:

Table S2. The changes of 58 GPCR genes [1] in response to Zn2+ stimulus, NS represented not significant, p adjust>0.05

Additional file 8: Table S3.

The changes of 152 transporter genes in response to Zn2+ stimulus, NS represented not significant, p adjust>0.05

Additional file 9: Figure S6.

Effect of 3 mM Zn2+ on two major antioxidant enzyme gene transcription levels in RUT-C30 strain. Transcriptional levels of sod1 (a) and cat1 (b) of the RUT-C30 strain were determined after culturing in liquid MM for 36, 48 or 60 h containing 0 or 3 mM Zn2+ with 1% (w/v) Avicel as the sole carbon source. The final values are presented as the mean±standard deviation (SD) of three independent experimental results. Asterisks indicate significant differences compared to the control (*P <0.05, according to Student’s t-test).

Additional file 10: Table S4.

Primers used in this study.

Additional file 11: Figure S7.

Construction and verification of T. reesei deletion mutant, which were performed as described in our previous study [1]. a: The skeleton schematic diagram to delete plc-e in the parent strain RUT-C30. b: The skeleton schematic diagram to delete zafA in the parent strain RUT-C30. c: The skeleton schematic diagram to delete crz1 in the parent strain RUT-C30. d: Validated electrophoretic diagram to verify the knockout of plc-e, zafA, and crz1 in the parent strain RUT-C30, respectively; M: marker.

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Li, N., Li, J., Chen, Y. et al. Mechanism of Zn2+ regulation of cellulase production in Trichoderma reesei Rut-C30. Biotechnol Biofuels 16, 73 (2023). https://doi.org/10.1186/s13068-023-02323-1

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Keywords

Biotechnology for Biofuels and Bioproducts

ISSN: 2731-3654

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