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Metformin regulates cellulase production in Trichoderma reesei via calcium signaling and mitochondrial function

Microbial Cell Factories volume 23, Article number: 314 (2024) Cite this article

Abstract

Background

Trichoderma reesei is renowned for its cellulase-producing ability and is used for biofuel production from lignocellulose. In plants and fungi, cellulase production is induced by cellulose and suppressed by glucose; however, whether metformin can enhance cellulase production and mitochondrial function in T. reesei remains unclear. Metformin reduces blood glucose levels by inhibiting hepatic gluconeogenesis; therefore, it is worth investigating whether metformin transmission modulates cellulase biosynthesis in T. reesei.

Results

Metformin increased cellulase production and the transcription of cellulase-related genes. It also enhanced the concentrations of Ca2+ in the cytosol and mitochondria and regulated the transcription levels of cellulase-related genes by modulating calcium homeostasis in T. reesei QM6a. In addition, metformin was identified as an antioxidant that can enhance cellulase activity by reducing reactive oxygen species (ROS). Our results demonstrated that metformin influences the state of mitochondria by enhancing mitochondrial activity and membrane potential to promote cellulase production.

Conclusion

Collectively, these results indicate that metformin enhances cellulase production, scavenges ROS, and protects mitochondrial activity in T. reesei.

Graphical Abstract

Background

Naturally occurring cellulose is abundant in various waste materials originating from wood and corn stover, households, and other agricultural byproducts. Through the action of cellulase, an extracellular enzyme, cellulose can be converted into valuable resources, such as biofuels and other bio-based products, using cost-effective and environmentally friendly bioprocesses [1]. Cellulase also has extensive applications in the detergent, papermaking, fruit juice, and beverage industries [2]. Trichoderma reesei can produce substantial amounts of cellulase and thus could be used to facilitate the conversion of biowaste into biofuels [3, 4]. Exploiting the cellulase expression mechanism in T. reesei has long been a topic of great interest. This study provides valuable insights for discovering new strategies to enhance cellulase production and to develop T. reesei as a microbial host for heterologous gene expression.

In recent years, microbial cell research has focused on identifying novel strategies that promote cellulase activity in T. reesei and elucidating its underlying induction mechanisms. Previous studies have shown a relationship between Mn2+ and cellulase activity in T. reesei and demonstrated that N, N-dimethylformamide can enhance cellulase production in T. reesei by triggering Ca2+ signaling [5, 6]. Furthermore, Sr+, Zn2+, and PEG8000 may enhance cellulase activity in T. reesei [7, 8]. These findings highlight the significance and necessity of selecting appropriate strategies that promote cellulase activity and may improve our understanding of the cellulase production mechanism of T. reesei.

Cellulase production is modulated by several factors [9], including transcription, secondary-messenger-mediated signaling pathways involving Ca2+, and reactive oxygen species (ROS). Ca2+ participates in many different intracellular signaling pathways [10, 11], and calcium signaling components, such as free Ca2+, calmodulin (CAM), calcineurin (CNA), and calcineurin-responsive zinc finger transcription factor 1 (CRZ1), which coalesce to form a signaling cascade [11]. For example, intracellular Ca2+ can trigger CRZ1, which regulates its downstream target gene XYR1, to enhance cellulase activity [12].

ROS originate from physiological processes such as aerobic respiration and are indispensable signaling molecules that can regulate developmental and physiological processes in fungi [13, 14] and the induction of exogenous substances [15]. Mitochondria are important organelles that contribute to cellular energy supply metabolism [16] and signal transduction pathway regulation and act as a major source of ROS [17]. In fungi, ROS have many biological functions; however, high ROS concentrations are harmful [18]. Wu et al. suggested that putrescine may indirectly affect the production of ganoderic acid (GA) biosynthesis by regulating ROS concentrations, which may influence the expression of key genes associated with GA biosynthesis [19]. Gao et al. also reported that mycelial branching and secondary metabolism were dependent on intracellular ROS [20]. ROS have an essential regulatory role during the treatment of mammalian diseases and in plant responses to environmental stress; however, ROS have been relatively poorly studied in filamentous fungi.

Metformin, also called metformin hydrochloride, is used to treat type 2 diabetes owing to its potent hypoglycemic effects [21]. In patients with type 2 diabetes, metformin can reduce blood glucose levels by inhibiting hepatic gluconeogenesis in a redox-dependent manner [22]. Metformin not only influences glucose metabolism, but is also beneficial in treating other underlying conditions, such as aging, cardiovascular diseases, cancer, and neurodegenerative diseases [23]. Studies have demonstrated that metformin can stimulate mitochondrial fission, enhance mitochondrial respiration, aid in the recovery of the mitochondrial life cycle, and stabilize mitochondrial function [24]. Moreover, metformin reduces oxidative stress, inhibits chronic inflammation, and boosts antioxidant defenses [25]. In plants and fungi, cellulase production is induced by cellulose and suppressed by glucose [9]; however, whether metformin can enhance cellulase expression in T. reesei remains unclear.

Therefore, this study aimed to investigate the effect of metformin on cellulase production in T. reesei QM6a. Changes in cellulase activity and cellulase regulatory mechanisms were analyzed using 60 mM of metformin. In addition, calcium signaling, the direction of calcium ion flow, antioxidant capacity, and mitochondrial function after metformin treatment were examined, and a potential mechanism to elucidate how metformin enhances cellulase production in T. reesei QM6a was proposed. This study offers a new perspective on cellulase production and the underlying regulatory mechanisms influenced by metformin.

Materials and methods

Strains and culture conditions

The experiments were conducted using Trichoderma reesei QM6a (ATCC 13631) purchased from ATCC. Fresh conidia of T. reesei were inoculated on potato dextrose agar (PDA) medium and incubated at 28 °C in the dark. Thereafter, the universal fungal medium, Mandels (MA), was used to germinate conidia in all subsequent experiments [26]. Metformin’s effect on cellulase production was assessed using minimal medium (MM) with 1% (w/v) Avicel (#C104842, Aladdin, Shanghai, China) [7]. Conidia were grown in MA medium (100 mL) with 2% glucose (28 °C for 36 h), then, the mycelia were centrifuged at 12,000 × g for 10 min, washed with carbon-free MM, and transferred to 50 mL of fresh MM containing 1% (w/v) Avicel (pH-101; Sigma-Aldrich, St. Louis, MO, USA) supplemented with different concentrations of metformin (0, 20, 40, 60, and 80 mM).

Enzyme activity analysis

To assess enzymatic activity, we performed cultivations (1 mL) at various time intervals and centrifuged the biomass at 12,000 × g at 4 °C for 5 min. Cellulase activity was determined using the supernatant. The activities of p NPCase, FPase, and CMCase were determined as previously described [27].

cDNA extraction and RT-qPCR

Gene target expression levels were tested using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses. Mycelial RNA was extracted using the Fast-Pure Plant DNA Isolation Mini Kit (Vazyme Biotech Co., Ltd., Nanjing, China) according to the manufacturer’s instructions. RT-qPCR was performed using an ABI Step One thermocycler (Applied Biosystems, Foster City, CA, USA). Data was normalized using sar1 as an internal reference, and the 2 − ΔΔCt method was used for analysis. The RT-qPCR primers are listed in Table S1.

Detection of cytosolic Ca2+ and ROS

The levels of cytosolic Ca2+ and ROS were evaluated using the fluorescent probes Fluo-4 AM and DCHF-DA, respectively (Beyotime, Shanghai, China). The assays were conducted according to the manufacturer’s instructions, with minor modifications. Briefly, the hyphae and fluorescent probes were maintained at 28 °C for 30 min and then the hyphae were rinsed in Hank’s Balanced Salt Solution (HBSS; no calcium, magnesium, and phenol red) to reduce noise [28]. In this study, all fluorescent detection was performed using an inverted microscope (Eclipse Ti-E, Nikon, Tokyo, Japan). The ImageJ-win 64 software (National Institutes of Health, Bethesda, MD, USA) was used to analyze the average fluorescence intensity of all probes in mycelia.

Detection of mitochondrial Ca2+

Rhod-2 AM is a calcium indicator with high affinity and mitochondrial localization. Rhod-2 AM was used to determine the source of metformin-activated Ca2+ in mitochondria [29] according to the manufacturer’s instructions (#40776ES50, YEASEN, Shanghai, China), with minor modifications. The mycelium was incubated with fluorescent probes for 30 min at 28 °C in the dark and then washed with HBSS to remove the dye and reduce background noise.

Mitochondrial activity and membrane potential detection

MitoTracker® Red CMXRos was used to visualize the number of active mitochondria in cells according to the manufacturer’s instructions (#40741ES50, YEASEN, Shanghai, China), with slight modifications. JC-1 is a commonly used fluorescent probe for detecting mitochondrial membrane potential (ΔΨm) [30]. To explore the changes in mitochondrial function after metformin treatment, mycelia were stained with JC-1, and the ratio of red/green fluorescence intensity was calculated to determine mitochondrial health. MitoTracker® Red CMXRos was pre-warmed at 37 °C before incubation and JC-1 labeling was increased by adding 1% Tween-20 during dissolution. The labeling compounds were subsequently transferred to the mycelia and cultivated at 28 °C before the hyphae were washed thrice with HBSS.

Chemical treatments

Different concentrations of metformin were added during the transfer experiments. N-Acetyl-L-cysteine (NAC) and L-ascorbic acid (VC) were used as ROS scavengers, H2O2 was used as an ROS generator, and LaCl3 was used as a plasma membrane Ca2+ channel repressor. In the transfer experiment, fresh MM was added directly to NAC and VC (mycelium transferred) at the time of mycelial transfer, and LaCl3 and H2O2 were added 24 h after mycelial transfer.

Statistical analysis

To ensure reproducibility, experimental data was obtained from three independent experiments and independent sets of experiments. The error bars represent the standard deviation of the mean of the samples taken in triplicate. Statistical significance was determined using the Student’s t-test. Significance was set at *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Results

Cellulase production increases after metformin treatment in T. reesei QM6a

To determine the effect of metformin on cellulase production, the mycelia of T. reesei QM6a were precultured to equal biomass before being transferred into liquid MM containing 1% (w/v) Avicel and different concentrations of metformin (0, 20, 40, 60, and 80 mM). The addition of metformin at different concentrations significantly improved cellulase activity compared to the control group; in particular, 60 mM metformin markedly increased the activities of p NPCase, CMCase, and FPase by 90, 67, and 95%, respectively, after 4 days of cultivation (Fig. 1a–c). Metformin addition did not significantly alter the strain biomass quantity (Fig. S1); thus, enzyme activity is reported in units per milliliter (U/mL). These findings indicated that a range of 20–80 mM metformin promoted cellulase activity after 4 days of cultivation, with 60 mM being the optimal concentration for augmenting cellulase production in T. reesei QM6a. Results of an SDS-PAGE analysis (Fig. S2) of the extracellular proteins secreted by QM6a supplemented with 0 and 60 mM metformin agreed with the cellulase activity results presented above.

Fig. 1
figure 1

Effects of different metformin concentrations on cellulase production in T. reesei. (a) p NPCase, (b) CMCase, (c) FPase activity and (d) protein concentration in T. reesei QM6a detected 2, 3, or 4 days after treatment with 0–80 mM metformin. The effects of 0 mM Met (not metformin-treated) and 60 mM Met (medium included 60 mM Met) on the expression of different cellulase-related genes (e) cbh1, (f) cbh2, (g) egl1, (h) ace3, and (i) xyr1 in T. reesei QM6a (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

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We analyzed the expression levels of three key cellulase genes (cbh1, cbh2, and egl1) to investigate the effect of metformin on cellulase synthesis. Moreover, after 72 and 84 h of culture supplemented with 0 and 60 mM metformin, we assessed the expression of xyr1 and ace3 (two critical cellulase transcription activators) using RT-qPCR [26]. Of note, 60 mM metformin significantly increased the transcription levels of cbh1, cbh2, and egl1 after 72 and 84 h, which is consistent with the observed increases in cellulase activity shown in Fig. 1a–c. Metformin supplementation increased the transcription of ace3, which is a pivotal transcription factor, by 180.06% (Fig. 1g). In contrast, the expression of another cellulase activator, xyr1, was upregulated by 22.18% at 84 h after metformin supplementation (Fig. 1h). These findings indicate that ace3 has a more pronounced influence than xyr1 in regulating cellulase-related gene expression after metformin treatment.

Calcium signaling pathways in T. reesei are induced by metformin treatment

We evaluated how the addition of metformin could upregulate cellulase-related gene expression. The fluorescent probe Fluo-4 AM was used to evaluate the concentrations of Ca2+ in the cytosol; the substance emits green fluorescence upon crossing the cell membrane and interacting with cellular Ca2+ [31]. The relative quantity of free Ca2+ within the cells was characterized by the green fluorescence intensity. Fluorescence intensity in the experimental group (with 60 mM metformin) increased by 2.36 times compared to the control group (Fig. 2b), indicating that metformin elevated the level of cytosolic Ca2+ in T. reesei QM6a.

Fig. 2
figure 2

Effects of different metformin concentrations on cytosolic Ca2+ concentration and calcium signaling. (a) Cytosolic Ca2+ levels were assessed using Fluo-4 AM. Trichoderma reesei QM6a was cultivated in an MM medium for 2 days and subsequently treated with LaCl3, 60 mM metformin, or 0 mM metformin. Hyphae were stained with 4 Fluo-4 AM to monitor the Ca2+ concentrations. (b) Comparative fluorescence ratios demonstrating the effect of metformin and LaCl3 on cytosolic Ca2+. The x-axis depicts the various treatments with metformin or LaCl3, while the y-axis depicts the Ca2+ fluorescence ratio as detected by CLSM. (c) Transcriptional levels of crz1 after 48, 60, or 72 h of 0 mM or 60 mM metformin treatment (**p < 0.01, ***p < 0.001, ****p < 0.0001)

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Calcium serves as a pivotal secondary messenger in the activation of calcium signaling pathways. Variations in calcium levels can stimulate the production of transcription factors, such as crz1, which orchestrates the regulation of downstream genes. To explore the potential link between metformin-induced elevation of intracellular calcium levels and augmentation of cellulase activity via crz1 activation, the expression levels of genes associated with the regulation of calcium signaling were analyzed using RT-qPCR. Metformin supplementation markedly increased the expression of crz1 after 60 and 72 h of treatment (Fig. 2c), indicating that metformin may enhance cellulase activity by modulating calcium signaling and upregulating crz1 expression.

LaCl3 is an antagonist of plasma membrane calcium channels that inhibits the influx of extracellular Ca2+, and Ca2+ serves as a critical checkpoint at the boundary between life and death in cells [32]. Thus, the effect of metformin on cytoplasmic Ca2+ spikes with LaCl3 as a modulatory agent was investigated. The metformin-induced increase in intracellular Ca2+ levels was significantly attenuated by the addition of LaCl3 (Fig. 2a). The addition of 5 mM LaCl3 resulted in a reduction in the fluorescence intensity of metformin-induced mycelia compared with samples without LaCl3. These findings suggest that metformin rapidly increases cytosolic calcium levels and subsequently activates the calcium signaling pathway (Fig. 2a, b).

Furthermore, the impact of this intervention on metformin-induced cellulase overexpression was evaluated, including p NPCase and CMCase activity, and the expression levels of key cellulase-related genes, such as cbh1 and egl1.

At 60 mM, metformin did not induce cellulase over-production in T. reesei QM6a (with 5 mM LaCl3). The addition of LaCl3 led to a reduction in p NPCase and CMCase activity, respectively, after 3 days of incubation compared with samples without LaCl3, and the expression levels of cbh1 and egl1 reflected these findings (Fig. 3a, b). Metformin-induced upregulation of cbh1 and egl1 was inhibited following the introduction of LaCl3 (Fig. 3c).

Fig. 3
figure 3

Effect of LaCl3 on the production of cellulase after metformin treatment. The T. reesei QM6a strain, which was grown in a medium enriched with and without 60 mM metformin and in a medium enriched with (+) or without (−) 5 mM LaCl3, exhibited (a) increased p NPCase and (b) CMCase activity. Transcription levels of cbh1 and egl1 (c) were measured 72 h after treatment (*p < 0.05, **p < 0.01, ****p < 0.0001). Figure 3c shows the relative expression ratio of 60 mM metformin compared to no metformin addition (set as 1)

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Treatment with LaCl3 significantly attenuated metformin-induced cellulase overexpression, indicating that LaCl3 obstructs the surge of cytosolic Ca2+ that is essential for metformin-induced cellulase production. These results demonstrate that metformin increases cellulase activity by activating Ca2+ signaling pathways.

Metformin increases mitochondrial calcium

Our findings revealed that metformin promoted the accumulation of cytoplasmic calcium ions; however, after the addition of LaCl3, cytosolic Ca2+ was significantly but not completely inhibited after metformin treatment. Metformin has been reported to target mitochondria, thus playing a crucial role in maintaining calcium homeostasis and regulating normal cellular metabolism [33]. In addition, other potential mechanisms for Ca2+ activation by metformin have been reported, such as the modulation of the calcium ion pool within mitochondria. Rhod-2 AM, a positively charged fluorescent probe, was employed to specifically accumulate in mitochondria through potential-driven uptake, producing a distinct red, dot-like staining pattern under fluorescence microscopy [34]. To test our hypothesis, Rhod-2 AM was used to identify Ca2+ changes within mitochondria. The intensity of Rhod-2 AM fluorescence was markedly higher in metformin-treated cells than in untreated cells (Fig. 4a). Quantitative analysis revealed an 80.7% increase in fluorescence intensity in the experimental group (60 mM metformin) than in the control group (Fig. 4b). In conclusion, metformin not only increased the intracellular Ca2+ concentration but also induced mitochondrial calcium ion uptake, which may have contributed to the upregulation of cellulase-related genes in T. reesei QM6a.

Fig. 4
figure 4

Mitochondrial Ca2+ levels increase after metformin treatment. (a) Mitochondrial Ca2+ levels were measured using the specific fluorescent probe Rhod-2 AM. The T. reesei QM6a strain was cultured in MM for 2 days with 0 mM or 60 mM metformin. Hyphae were treated with Rhod-2 AM and the relative fluorescence was microscopically monitored, where stronger red fluorescence indicated a higher mitochondrial Ca2+ concentration. (b) Comparative fluorescence ratios demonstrating the effects of metformin on mitochondrial Ca2+. The x-axis represents the different metformin treatments, while the y-axis represents the Ca2+ fluorescence ratio as detected by CLSM (**p < 0.01)

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Metformin reduces ROS generation

ROS are natural byproducts of aerobic respiration primarily generated in the mitochondria. ROS accumulation can have harmful effects on cells, and metformin is reported to possess antioxidant properties [35]. Li et al. reported the negative effect of ROS on cellulase production in T. reesei RUT-C30 [7]; therefore, we aimed to investigate the effect of metformin on the cellular redox system under ROS stress and its impact on cellulase production. To measure the cellulase activity in T. reesei QM6a, the intracellular ROS concentration was manipulated using NAC and VC as ROS scavengers and H2O2 as a ROS-generating agent. Treatment with 4 mM H2O2 led to a reduction in both p NPCase and CMCase activity (by approximately 17.27% and 18.41%, respectively), compared with control (QM6a without 4 mM H2O2) (Fig. 5). Conversely, when treated with 1 mM NAC, there was a noticeable increase in p NPCase and CMCase activity (21.56% and 16.14%, respectively), indicating the positive effect of ROS scavenging on cellulase production (Fig. 5). Enzyme activity was enhanced after the treatment of T. reesei QM6a with VC, which was consistent with previous findings and suggests that high ROS concentrations negatively affect cellulase production by T. reesei QM6a (Fig. 5) Additionally, the cellulase activity was higher after treatment with 60 mM metformin than that in the control (no treatment), although it was slightly reduced by the addition of 4 mM H2O2. Notably, p NPCase and CMCase activity were 92% and 70.03% higher in the presence of 60 mM metformin than those with NAC or VC treatments, respectively. NAC and VC are widely used antioxidants, and VC is commonly used in the nutraceutical, medical, and beauty industries. Several studies have demonstrated that metformin has antioxidant properties and scavenges ROS to reduce inflammation in plants and animals [36]. Our data suggest that metformin may enhance cellulase activity by scavenging ROS and is a suitable antioxidant for T. reesei QM6a.

Fig. 5
figure 5

Effect of reactive oxygen species (ROS) on metformin-induced cellulase production. Activities of p NPCase (a) and CMCase (b) in T. reesei QM6a strain with or without 60 mM metformin treatment, 4 mM H2O2, 1 mM NAC, or 1 mM VC in MM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

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To validate this hypothesis, the ROS levels in T. reesei mycelia were assessed using DCFH-DA (S0033S, Beyotime, Beijing, China) [37]. The findings indicated a marked increase in ROS levels upon 4 mM H2O2 treatment in the control group, whereas the metformin-treated strains exhibited significantly lower ROS levels, indicating metformin’s ROS-scavenging capabilities and its role in enhancing cellulase production (Fig. 6).

Fig. 6
figure 6

Metformin reduces ROS levels. To measure intracellular ROS levels, the fluorescent probe DCFH-DA was utilized. Trichoderma reesei QM6a was grown in liquid MM with or without metformin and H2O2. (a) Hyphae were treated with DCFH-DA, and the fluorescence intensity was continuously monitored using automatic inverted fluorescence microscopy. (b) Higher green fluorescence intensity indicates increased levels of intracellular ROS. Differential interference contrast (DIC) microscopy was also employed (**p < 0.01)

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In summary, high ROS concentrations have a detrimental effect on cellulase production in T. reesei QM6a. However, metformin functions as an antioxidant in QM6a cells by scavenging ROS, thereby enhancing cellulase activity.

Metformin promotes cellulase enzyme activity by regulating mitochondrial health

Mitochondria are colloquially referred to as the “powerhouse of the cell” and their biological activities play an important role in several different signal transduction pathways, including biosynthetic, calcium homeostasis, and redox control-related pathways [38]. Bharath et al. found that metformin enhanced mitochondrial activity and functional integrity in humans [29]. To explore the relationship between increased cellulase activity in metformin-treated T. reesei QM6a and mitochondria, MitoTracker® Red CMXRos dye was used to characterize the mycelial mitochondrial activity [39]. Fluorescence intensity notably increased following supplementation with 60 mM metformin, exhibiting a 46.35% increase in the treated strain compared with that of the untreated control (Fig. 7b). These results suggested that the heightened mitochondrial activity induced by metformin was linked to the elevated cellulase activity in T. reesei QM6a.

Fig. 7
figure 7

Impact of metformin on cellulase production in relation to mitochondrial health. (a) Mitochondrial activity in T. reesei QM6a with or without metformin treatment at a concentration of 100 nM was assessed using MitoTracker® Red CMXRos. (b) Higher levels of red fluorescence intensity are associated with improved mitochondrial health, followed by (c) measurement of mitochondrial membrane potential (ΔΨm) with JC-1 dyes. Fluorescence intensity was monitored using automatic inverted fluorescence microscopy. (d) Ratio of red to green fluorescence intensity is indicative of mitochondrial function integrity (**p < 0.01)

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Mitochondrial membrane potential (ΔΨm) is a universal selective indicator used to evaluate the health of mitochondrial functions; when mitochondria are damaged, the membrane potential decreases, and mitochondrial activity is reduced [40]. To further investigate the integrity of mitochondrial function after the addition of metformin, a JC-1 fluorescent probe was used to label the strains before and after metformin induction to detect mitochondrial membrane potential. The red fluorescence intensity of the control group (without metformin) was markedly weaker than that of the group treated with 60 mM metformin. The green fluorescence intensity was considerably higher, while the orange fluorescence intensity was also significantly enhanced in the merged images of red–green fluorescence (Fig. 7c). Quantification of the fluorescence signals revealed a 26% increase in red fluorescence intensity, a 10.23% decrease in green fluorescence intensity, and a 40% increase in red/green fluorescence intensity in metformin-treated strains (Fig. 7d). These results suggest that 60 mM metformin effectively enhanced the mitochondrial membrane potential (ΔΨm) of T. reesei QM6a, which promoted mitochondrial function and increased cellulase activity.

Discussion

Cellular metabolism is significantly affected by the induction of metal ions and exogenous compounds, such as Mn2+, Sr2+, and Zn2+ [5, 7, 41], which can trigger the calcium signaling pathway to increase cellulase production. In the present study, cellulase production and the transcription of cellulase-related genes (e.g., cbh1, cbh2, egl1, and ace3) were enhanced by metformin application. We also found that the concentrations of extracellular metformin were almost constant in the fermentation broth of T. reesei, which is worthy of further investigation.

Of note, the transcription factor ACE3 is essential in cellulase production in T. reesei. Ca2+ is a crucial secondary messenger that plays a pivotal role in the biological functionality of cells and is activated by several external stimuli, including changes in temperature, pH, light conditions, and drug application [42]. Our findings demonstrate that metformin boosts cellulase production by activating the calcium signaling pathway. Fungi can regulate various cellular metabolic processes by sensing changes in intracellular Ca2+ concentrations, which activate the expression of target genes [43]. Our study demonstrated that crz1 responds to rapid increases in Ca2+ induced by metformin and regulates cellulase production. The plasma membrane Ca2+ inhibitor LaCl3 was used to further demonstrate that metformin activates the calcium signaling pathway to promote cellulase activity.

Maintenance of intracellular Ca2+ homeostasis depends on various intracellular stores, including the mitochondria, endoplasmic reticulum, and Golgi apparatus; however, the regulation of Ca2+ homeostasis in T. reesei QM6a remains unclear. Besides being the “powerhouse of the cell,” mitochondria also regulate Ca2+ homeostasis [33]. Mitochondrial response signals rely on increased concentrations of intracellular Ca2+ and store Ca2+, stabilizing the new homeostasis of intracellular Ca2+ [44]. In addition, increased mitochondrial Ca2+ partly compensates for impaired carbohydrate-mediated mitochondrial respiration [45]. Our study found that mitochondrial Ca2+ is released into the cytoplasm when intracellular Ca2+ is blocked (Fig. 2). Previous reports on calcium transport between mitochondria and the cytoplasm are consistent with our data, indicating that mitochondria regulate intracellular Ca2+ homeostasis.

We labeled intracellular calcium ions with a fluorescent probe and found that 60 mM metformin increased both cytoplasmic and mitochondrial Ca2+ concentrations. The calcium signaling transcription factor, CRZ1, was also upregulated in response to metformin treatment, suggesting that metformin affects cytoplasmic and mitochondrial Ca2+ concentrations by regulating Ca2+ homeostasis in the cytoplasm and mitochondria. Calcium signaling also regulates the transcriptional level of downstream CRZ1, increasing the transcriptional level of the cellulase synthesis gene in T. reesei.

ROS are produced during various physiological processes, such as exposure to drugs, xenobiotics, and metal ions. They can impair mitochondrial function and directly or indirectly damage DNA and proteins [46]. High levels of ROS negatively impact cellulase activity in T. reesei RUC-30, and significant increases in ROS induced by PEG8000 inhibited mycelial growth and cellulase expression [7, 8]. Our results indicate that high concentrations of ROS negatively affect cellulase activity and that metformin acts as a ROS scavenger. Our findings suggest that metformin has anti-inflammatory properties, which may be due to the inhibition of ROS production. These results are consistent with those of previous studies, which have demonstrated that metformin treatment can decrease blood oxidative stress and cholesterol levels [47]. Metformin may also inhibit mitochondrial dysfunction and modulate antioxidant protection to promote the clearance of damaged mitochondria [48]. Remarkably, the increase in cellulase activity induced by metformin was equally effective to that induced by NAC or VC, further suggesting that metformin acts as an antioxidant, promoting cellulase activity and protecting T. reesei QM6a from cellular damage.

Metformin promotes mitochondrial activity and health, which may alleviate age-associated inflammation [29]. Yao et al. demonstrated that healthy mitochondrial transplantation is an effective strategy for engineering stem cells for tissue regeneration [49]. Gorospe et al. demonstrated that experimentally increasing mitochondrial membrane potential is sufficient to restore timely cell cycle progression in Saccharomyces cerevisiae [50]. These reports indicate that the physiological health of mitochondria is closely associated with cell health. In this study, we indicated that the mitochondrial activity of T. reesei QM6a was promoted by 60 mM metformin, which significantly improved mitochondrial membrane potential. Metformin enhances mitochondrial respiration, membrane potential, and ATP in liver cells [25]. Our results are similar to those previously reported, and we hypothesized that metformin increases membrane potential and promotes ATP production to enhance cellulase production in T. reesei QM6a. Additionally, we hypothesized that metformin improves mitochondrial function and enhances the expression levels of cellulase-related genes.

Conclusions

In this study, we identified that metformin enhances cellulase expression in T. reesei QM6a and proposed a potential mechanism by which metformin modulates cellulase production (see Graphical abstract). Metformin induces rapid increases in intracellular Ca2+ and increases mitochondrial Ca2+ levels. These elevated Ca2+ levels activate the signal transduction pathway of the crz1 transcription factor, which in turn induces cellulase production. Moreover, high ROS stress negatively affects cellulose production in T. reesei, while metformin scavenges ROS to promote cellulase production. This research reveals metformin’s ability to target mitochondria to increase cellulase gene transcription by boosting mitochondrial activity and function, providing new insights into signal transduction mechanisms during cellulase synthesis.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

CMCase:

Endo-β-glucanase activity

crz1:

Calcineurin-responsive zinc finger transcription factor 1

FPase:

Filter paper activity, representing total extracellular cellulase activity

PNPCase:

Exo-β-glucanase activity

ROS:

Reactive oxygen species

RT:

Qpcr-Real-time quantitative PCR

References

  1. Pang A-P, Luo Y, Hu X, Zhang F, Wang H, Gao Y, et al. Transmembrane transport process and endoplasmic reticulum function facilitate the role of gene cel1b in cellulase production of Trichoderma reesei. Microb Cell Factories. 2022;21:90.

    Article  CAS  Google Scholar 

  2. Saini JK, Patel AK, Adsul M, Singhania RR. Cellulase adsorption on lignin: a roadblock for economic hydrolysis of biomass. Renew Energy. 2016;98:29–42.

    Article  CAS  Google Scholar 

  3. Areeshi MY. Microbial cellulase production using fruit wastes and its applications in biofuels production. Int J Food Microbiol. 2022;378:109814.

    Article  CAS  PubMed  Google Scholar 

  4. Wang Y, Liu R, Liu H, Li X, Shen L, Zhang W, et al. Development of a powerful synthetic hybrid promoter to improve the cellulase system of Trichoderma reesei for efficient saccharification of corncob residues. Microb Cell Factories. 2022;21:5.

    Article  Google Scholar 

  5. Chen M, Shen Y, Lin L, Wei W, Wei D. Mn2+ modulates the production of mycophenolic acid in Penicillium Brevicompactum NRRL864 via reactive oxygen species signaling and the investigation of pb-pho. Fungal Biol. 2022;126:461–70.

    Article  CAS  PubMed  Google Scholar 

  6. Chen Y, Wu C, Shen Y, Ma Y, Wei D, Wang W. N, N-dimethylformamide induces cellulase production in the filamentous fungus Trichoderma reesei. Biotechnol Biofuels. 2019;12:36.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Li N, Zeng Y, Chen Y, Shen Y, Wang W. Induction of cellulase production by Sr2+ in Trichoderma reesei via calcium signaling transduction. Bioresour Bioprocess. 2022;9:96.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Liu S, Quan L, Yang M, Wang D, Wang Y-Z. Regulation of cellulase production via calcium signaling in Trichoderma reesei under PEG8000 stress. Appl Microbiol Biotechnol. 2024;108:178.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Zhang J, Zhang G, Wang W, Wang W, Wei D. Enhanced cellulase production in Trichoderma reesei RUT C30 via constitution of minimal transcriptional activators. Microb Cell Factories. 2018;17:75.

    Article  Google Scholar 

  10. Petersen OH, Michalak M, Verkhratsky A. Calcium signalling: past, present and future. Cell Calcium. 2005;38:161–9.

    Article  CAS  PubMed  Google Scholar 

  11. Berridge MJ, Bootman MD, Roderick HL. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol. 2003;4:517–29.

    Article  CAS  PubMed  Google Scholar 

  12. Chen L, Zou G, Wang J, Wang J, Liu R, Jiang Y, et al. Characterization of the Ca2+ -responsive signaling pathway in regulating the expression and secretion of cellulases in Trichoderma reesei Rut-C30: Ca2+ signaling in cellulase biosynthesis. Mol Microbiol. 2016;100:560–75.

    Article  CAS  PubMed  Google Scholar 

  13. Gessler NN, Aver’yanov AA, Belozerskaya TA. Reactive oxygen species in regulation of fungal development. Biochem Mosc. 2007;72:1091–109.

    Article  CAS  Google Scholar 

  14. Kalyanaraman B, Cheng G, Hardy M, Ouari O, Bennett B, Zielonka J. Teaching the basics of reactive oxygen species and their relevance to cancer biology: mitochondrial reactive oxygen species detection, redox signaling, and targeted therapies. Redox Biol. 2018;15:347–62.

    Article  CAS  PubMed  Google Scholar 

  15. Van Acker H, Coenye T. The role of reactive oxygen species in antibiotic-mediated killing of bacteria. Trends Microbiol. 2017;25:456–66.

    Article  PubMed  Google Scholar 

  16. Nakayama H, Otsu K. Mitochondrial DNA as an inflammatory mediator in cardiovascular diseases. Biochem J. 2018;475:839–52.

    Article  CAS  PubMed  Google Scholar 

  17. Foo J, Bellot G, Pervaiz S, Alonso S. Mitochondria-mediated oxidative stress during viral infection. Trends Microbiol. 2022;30:679–92.

    Article  CAS  PubMed  Google Scholar 

  18. Spinelli JB, Haigis MC. The multifaceted contributions of mitochondria to cellular metabolism. Nat Cell Biol. 2018;20:745–54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wu C-G, Tian J-L, Liu R, Cao P-F, Zhang T-J, Ren A et al. Ornithine decarboxylase-mediated production of putrescine influences ganoderic acid biosynthesis by regulating reactive oxygen species in Ganoderma lucidum. Cullen D, editor. Appl Environ Microbiol. 2017;83:e01289–17.

  20. Gao T, Shi L, Zhang T, Ren A, Jiang A, Yu H et al. Cross talk between calcium and reactive oxygen species regulates hyphal branching and ganoderic acid biosynthesis in ganoderma iucidum under copper stress. Atomi H, editor. Appl Environ Microbiol. 2018;84:e00438-18.

  21. TeSlaa T. Metformin induces a Lac-Phe gut-brain signalling axis. Nat Metab. 2024;6(4):603–5.

    Article  CAS  PubMed  Google Scholar 

  22. Horakova O, Kroupova P, Bardova K, Buresova J, Janovska P, Kopecky J, et al. Metformin acutely lowers blood glucose levels by inhibition of intestinal glucose transport. Sci Rep. 2019;9:6156.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Nesti L, Natali A. Metformin effects on the heart and the cardiovascular system: a review of experimental and clinical data. Nutr Metab Cardiovasc Dis. 2017;27:657–69.

    Article  CAS  PubMed  Google Scholar 

  24. Portela LV, Gnoatto J, Wigner Brochier A, Haas CB, De Assis AM, De Carvalho AK, et al. Intracerebroventricular metformin decreases body weight but has pro-oxidant effects and decreases survival. Neurochem Res. 2015;40:514–23.

    Article  CAS  PubMed  Google Scholar 

  25. Wang Y, An H, Liu T, Qin C, Sesaki H, Guo S, et al. Metformin improves mitochondrial respiratory activity through activation of AMPK. Cell Rep. 2019;29:1511–e15235.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Zhang J, Chen Y, Wu C, Liu P, Wang W, Wei D. The transcription factor ACE3 controls cellulase activities and lactose metabolism via two additional regulators in the fungus Trichoderma reesei. J Biol Chem. 2019;294:18435–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Wang M, Zhao Q, Yang J, Jiang B, Wang F, Liu K, et al. A mitogen-activated protein kinase Tmk3 participates in high osmolarity resistance, cell wall integrity maintenance and cellulase production regulation in Trichoderma reesei. PLoS ONE. 2013;8:e72189.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Avci FY, Li X, Tsuji M, Kasper DL. Isolation of carbohydrate-specific CD4(+) T cell clones from mice after stimulation by two model glycoconjugate vaccines. Nat Protoc. 2012;7:2180–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Shen B, Wang S, Bharathi G, Li Y, Lin F, Hu R, et al. Rapid and targeted photoactivation of Ca2+ channels mediated by squaraine to regulate intracellular and intercellular signaling processes. Anal Chem. 2020;92:8497–505.

    Article  CAS  PubMed  Google Scholar 

  30. Gao J, Wang Z, Guo Q, Tang H, Wang Z, Yang C, et al. Mitochondrion-targeted supramolecular nano-boat simultaneously inhibiting dual energy metabolism for tumor selective and synergistic chemo-radiotherapy. Theranostics. 2022;12:1286–302.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Yang J, Gong Y, Liu Q, Cai J, Zhang B, Zhang Z. Thioredoxin silencing-induced cardiac supercontraction occurs through endoplasmic reticulum stress and calcium overload in chicken. Metallomics. 2018;10:1667–77.

    Article  PubMed  Google Scholar 

  32. Schwarze J, Carolan JC, Stewart GS, McCabe PF, Kacprzyk J. The boundary of life and death: changes in mitochondrial and cytosolic proteomes associated with programmed cell death of Arabidopsis thaliana suspension culture cells. Front Plant Sci. 2023;14:1194866.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Arduino DM, Perocchi F. Mitochondrial dysfunctions trigger. J Physiol. 2018;596:2717–33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Brisac C, Téoulé F, Autret A, Pelletier I, Colbère-Garapin F, Brenner C, et al. Calcium flux between the endoplasmic reticulum and mitochondrion contributes to poliovirus-induced apoptosis. J Virol. 2010;84:12226–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Chandel NS, Schumacker PT. Cellular oxygen sensing by mitochondria: old questions, new insight. J Appl Physiol. 2000;88:1880–9.

    Article  CAS  PubMed  Google Scholar 

  36. Fei Q, Ma H, Zou J, Wang W, Zhu L, Deng H, et al. Metformin protects against ischaemic myocardial injury by alleviating autophagy-ROS-NLRP3-mediated inflammatory response in macrophages. J Mol Cell Cardiol. 2020;145:1–13.

    Article  CAS  PubMed  Google Scholar 

  37. Kessler A, Huang P, Blomberg E, Odnevall I. Unravelling the mechanistic understanding of metal nanoparticle-induced reactive oxygen species formation: insights from a Cu Nanoparticle Study. Chem Res Toxicol. 2023;36:1891–900.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Scatena R, Bottoni P, Giardina B. Advances in mitochondrial medicine. Springer Dordrecht. 2012. pp. 461–462.

  39. Liu P, Zhang G, Chen Y, Zhao J, Wang W, Wei D. Enhanced cellulase production by decreasing intercellular pH through H+-ATPase gene deletion in Trichoderma reesei RUT-C30. Biotechnol Biofuels. 2019;12:195.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Bazhin AA, Sinisi R, De Marchi U, Hermant A, Sambiagio N, Maric T, et al. A bioluminescent probe for longitudinal monitoring of mitochondrial membrane potential. Nat Chem Biol. 2020;16:1385–93.

    Article  CAS  PubMed  Google Scholar 

  41. Li N, Li J, Chen Y, Shen Y, Wei D, Wang W. Mechanism of Zn2+ regulation of cellulase production in Trichoderma reesei Rut-C30. Biotechnol Biofuels Bioprod. 2023;16:73.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Yang Y, Xie P, Li Y, Bi Y, Prusky DB. Updating insights into the regulatory mechanisms of calcineurin-activated transcription factor Crz1 in pathogenic fungi. J Fungi. 2022;8:1082.

    Article  CAS  Google Scholar 

  43. Gohain D, Tamuli R. Calcineurin responsive zinc-finger-1 binds to a unique promoter sequence to upregulate neuronal calcium sensor-1, whose interaction with MID-1 increases tolerance to calcium stress in Neurospora Crassa. Mol Microbiol. 2019;111:1510–28.

    Article  CAS  PubMed  Google Scholar 

  44. Robb-Gaspers LD, Burnett P, Rutter GA, Denton RM, Rizzuto R, Thomas AP. Integrating cytosolic calcium signals into mitochondrial metabolic responses. EMBO J. 1998;17:4987–5000.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Angebault C, Panel M, Lacôte M, Rieusset J, Lacampagne A, Fauconnier J. Metformin reverses the enhanced myocardial SR/ER–mitochondria interaction and impaired complex I-driven respiration in dystrophin-deficient mice. Front Cell Dev Biol. 2021;8:609493.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Li J, Sun Y, Liu F, Zhou Y, Yan Y, Zhou Z, et al. Increasing NADPH impairs fungal H2O2 resistance by perturbing transcriptional regulation of peroxiredoxin. Bioresour Bioprocess. 2022;9:1.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Chakraborty A, Chowdhury S, Bhattacharyya M. Effect of metformin on oxidative stress, nitrosative stress and inflammatory biomarkers in type 2 diabetes patients. Diabetes Res Clin Pract. 2011;93:56–62.

    Article  CAS  PubMed  Google Scholar 

  48. Paudel YN, Angelopoulou E, Piperi C, Shaikh MF, Othman I. Emerging neuroprotective effect of metformin in Parkinson’s disease: a molecular crosstalk. Pharmacol Res. 2020;152:104593.

    Article  CAS  PubMed  Google Scholar 

  49. Yao X, Ma Y, Zhou W, Liao Y, Jiang Z, Lin J, et al. In-cytoplasm mitochondrial transplantation for mesenchymal stem cells engineering and tissue regeneration. Bioeng Transl Med. 2021;7:e10250.

    Article  PubMed  PubMed Central  Google Scholar 

  50. Gorospe CM, Carvalho G, Herrera Curbelo A, Marchhart L, Mendes IC, Niedźwiecka K, et al. Mitochondrial membrane potential acts as a retrograde signal to regulate cell cycle progression. Life Sci Alliance. 2023;6:e202302091.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Funding

This research was supported by the National Natural Science Foundation of China (32370093), National Key Research and Development Program of China (2022YFA0912300), Natural Science Foundation of Shanghai (22ZR1417600 and 23ZR1414800), Chenguang Program of the Shanghai Education Development Foundation, Shanghai Municipal Education Commission (21CGA34), and the Open Funding Project of the State Key Laboratory of Bioreactor Engineering (2022).

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  1. Jiajia Wang and Yumeng Chen contributed equally to this work.

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  1. The State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China

    Jiajia Wang, Yumeng Chen, Jiawei Cong & Wei Wang

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JW planned and conducted the experiments, interpreted the experimental data, and wrote the manuscript. YC analyzed the data, reviewed the manuscript, and supported the research funding. JC analyzed partial data. WW directed and coordinated the study and reviewed the manuscript. All authors have read and approved the final manuscript.

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Correspondence to Wei Wang.

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Wang, J., Chen, Y., Cong, J. et al. Metformin regulates cellulase production in Trichoderma reesei via calcium signaling and mitochondrial function. Microb Cell Fact 23, 314 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12934-024-02593-w

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Keywords

Microbial Cell Factories

ISSN: 1475-2859

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