- Open Access
Induction of cellulase production by Sr2+ in Trichoderma reesei via calcium signaling transduction
Bioresources and Bioprocessing volume 9, Article number: 96 (2022)
Trichoderma reesei RUT-C30 is a well-known high-yielding cellulase-producing fungal strain that converts lignocellulose into cellulosic sugar for resource regeneration. Calcium is a ubiquitous secondary messenger that regulates growth and cellulase production in T. reesei. We serendipitously found that adding Sr2+ to the medium significantly increased cellulase activity in the T. reesei RUT-C30 strain and upregulated the expression of cellulase-related genes. Further studies showed that Sr2+ supplementation increased the cytosolic calcium concentration and activated the calcium-responsive signal transduction pathway of Ca2+–calcineurin-responsive zinc finger transcription factor 1 (CRZ1). Using the plasma membrane Ca2+ channel blocker, LaCl3, we demonstrated that Sr2+ induces cellulase production via the calcium signaling pathway. Supplementation with the corresponding concentrations of Sr2+ also inhibited colony growth. Sr2+ supplementation led to an increase in intracellular reactive oxygen species (ROS) and upregulated the transcriptional levels of intracellular superoxide dismutase (sod1) and catalase (cat1). We further demonstrated that ROS content was detrimental to cellulase production, which was alleviated by the ROS scavenger N-acetyl cysteine (NAC). This study demonstrated for the first time that Sr2+ supplementation stimulates cellulase production and upregulates cellulase genes via the calcium signaling transduction pathway. Sr2+ leads to an increase in intracellular ROS, which is detrimental to cellulase production and can be alleviated by the ROS scavenger NAC. Our results provide insights into the mechanistic study of cellulase synthesis and the discovery of novel inducers of cellulase.
Lignocellulose is a well-known renewable biomass energy source that can be degraded to bioethanol (Baldrian and Valášková 2008; Somerville et al. 2010). The economic and practical degradation of cellulose has been widely recognized and accepted (Li et al. 2018). Cellulose degradation is dependent on Trichoderma reesei, a strain that efficiently produces cellulase and hemicellulase (Fischer et al. 2021; Li et al. 2016, 2021; Martzy et al. 2021; Yan et al. 2021). Trichoderma reesei can produce large amounts of cellulases and hemicellulases using inducible carbon sources (such as Avicel). In contrast, negligible amounts of cellulases and hemicellulases are produced using fast-utilizing carbon sources (such as glucose) (Antoniêto et al. 2016). The production of cellulases is an extremely complex process regulated by many factors, such as transcription factors (Xin et al. 2010) and the second messenger Ca2+ (Chen et al. 2019).
Cellulase synthesis in T. reesei is regulated by several transcription factors (Zhang et al. 2018). Among these factors, xylanase regulator 1 (XYR1) is involved in activation of the expression of cellulases and hemicellulases (Carle-Urioste et al. 1997; Furukawa et al. 2009; Zeilinger et al. 1996), as well as the regulation of d-xylose metabolism (Stricker et al. 2006), which is considered as a key transactivator (Cao et al. 2019). Deletion of xyr1 results in a substantial reduction in cellulase and hemicellulase production (Stricker et al. 2006). ACE3, another crucial transcriptional activator, is essential for cellulase and xylanase production (Häkkinen et al. 2014). Deletion of ace3 is fatal for cellulase gene expression, whereas this deletion slightly reduces the expression of hemicellulases (Luo et al. 2020) and xylanase (Zhang et al. 2019).
Ca2+ signaling is a well-known secondary signaling pathway that broadly regulates primary and secondary metabolism in microorganisms (Roy et al. 2021). The intracellular calcium signaling components include free Ca2+, calmodulin (CaM), calcineurin (CnA), and calcineurin-responsive zinc finger transcription factor 1 (Crz1) (Bootman et al. 2002). Increases in cytosolic Ca2+ levels lead to the activation of Crz1 via Ca2+/CaM/CnA, which activates downstream genes with different functions by binding to their promoter regions (Martins-Santana et al. 2020). Chen et al. (2016) reported that the cytosolic Ca2+ concentration activates the binding of the transcription factor Crz1 to the cellulase gene cbh1 and main transcriptional activator of cellulase genes xyr1, thereby upregulating cellulase gene expression. Notably, Chen et al. (2018) demonstrated that Mn2+ induces a cytosolic Ca2+ burst to enhance the expression of cellulase genes through the Mn2+/Ca2+ exchanger in T. reesei. In Penicillium brevicompactum, addition of Ca2+ increases the production of the secondary metabolite mycophenolic acid (Chen et al. 2021a).
Reactive oxygen species (ROS) are chemically reactive chemicals that contain oxygen and are considered as harmful by-products of aerobic metabolism (Ren et al. 2017). ROS play a critical role in the development of many filamentous fungi, including Aspergillus nidulans (Mendoza-Martínez et al. 2017), Podospora anserina (Malagnac et al. 2004), and Neurospora crassa (Takemoto et al. 2007). Chen et al. (2022) demonstrated that a higher concentration of Mn2+ increased ROS production, ultimately leading to increased mycophenolic acid production in P. brevicompactum. Liu et al. (2018) observed that the ROS producer H2O2 and ROS scavenger N-acetyl cysteine (NAC) altered intracellular ROS levels in Ganoderma lucidum, which is an efficient approach for enhancing the production of ganoderic acid.
Ca2+ can promote the transcription of cellulase genes in the hypercellulolytic T. reesei strain RUT-C30. Therefore, we investigated whether other ions of group IIA metals can also increase cellulase production. High Mg2+ concentrations significantly inhibit growth (Groisman et al. 2013), which was not considered in this study. Furthermore, a previous study on Ca2+ was conducted in T. reesei RUT-C30. Therefore, in the current study, the effect of Sr2+ on cellulase production was investigated using RUT-C30. The effects of Sr2+ on mycelial growth and cellulase production were measured, and the transcript levels of crz1 and intracellular Ca2+ levels were evaluated to determine the role of calcium signaling in response to Sr2+. This study provides new perspectives for improving cellulase production and insights into the mechanism of cellulase regulation in T. reesei.
Materials and methods
Strains and growth conditions
Trichoderma reesei RUT-C30 (ATCC 56765) and QM6a (ATCC 13631) were used for the experiments. All T. reesei strains were cultured on potato dextrose agar (PDA) plates at 28 °C in the dark. Fresh conidia were washed with PDA plates and inoculated. MA medium (Zhang et al. 2019) was used for the general fungal culture. Minimal medium (MM; (NH4)2SO4 5 g/L; KH2PO4 15 g/L; urea 0.3 g/L; MgSO4 0.6 g/L; CaCl2 0.6 g/L; FeSO4·7H2O 5 mg/L; CoCl2·6H2O 2 mg/L; MnSO4·H2O 1.6 mg/L; ZnSO4·7H2O 1.4 mg/L; pH 5.5) (Chen et al. 2018) with 2% (w/v) glucose or 1% (w/v) Avicel was used to assess the effect of Sr2+ on hyphal growth and cellulase production. Transfer experiments were performed to study the effects of Sr2+ on cellulase production. The conidia were first cultured in 100 mL MA medium containing 2% glucose at 28 °C for 36 h to form mycelia. Mycelia (1 mL) were collected by centrifugation, washed with MM without a carbon source, and transferred into 50 mL of fresh MM containing 1% (w/v) Avicel (PH-101; Sigma-Aldrich, St. Louis, MO, USA) with the addition of SrCl2 to final concentrations of 0–120 mM.
Enzyme activity analysis and fungal growth
The culture (1 mL) was collected and centrifuged at 14,000×g at 4 °C for 5 min. The resulting supernatant was used to determine cellulase activity. pNPCase, CMCase, FPase, and xylanase activities were measured as previously reported (Zhang et al. 2016; Liu et al. 2019). The Detergent Compatible Bradford Protein Assay Kit (Beyotime, Shanghai, China) was used to determine protein concentrations. Biomass was measured indirectly using the intracellular protein method (Bischof et al. 2013) with some modifications. Three parallel samples (1 mL each) was collected, and the mycelia were collected by filtration, lysed with 1 M NaOH for 2 h, and centrifuged to obtain the intracellular proteins. The BCA Protein Concentration Assay Kit (Beyotime, Shanghai, China) was used to determine the total protein concentration. The biomass dry weight was calculated as 0.32 g intracellular protein per gram of dry cell weight (Chen et al. 2018).
For fungal growth experiments, fresh conidia were diluted to 2.5 × 106/mL with sterile water, inoculated an equal volume of conidia fluid (2 μL) into the center of MM plates, and incubated at 28 °C in the dark for 4 days.
RNA extraction and RT‑qPCR
RNA isolation and reverse transcription quantitative polymerase chain reaction (RT‑qPCR) analyses were performed as described by Zhang et al. (2018) with some modifications. Briefly, total RNA from mycelia was extracted using the FastRNA Pro Red Kit (MPbio, Irvine, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized from total RNA using the TransScript Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen Biotech, Beijing, China). For RT-qPCR, the transcriptional levels of cbh1 (encoding cellobiohydrolase I), egl1 (encoding endoglucanase I), xyr1 (encoding the main factor XYR1), ace3 (encoding the main factor ACE3), crz1 (calcineurin-responsive zinc finger transcription factor 1, Trire2:36391), sod1 (copper/zinc superoxide dismutase, Trire2:123029), and cat1 (catalase, Trire2:70600) were analyzed using PerfectStart™ Green qPCR SuperMix (TransGen Biotech). The 2−ΔΔCt method was used for calculations (Livak and Schmittgen 2001). The sar1 gene was used as an internal reference to normalize the data. The primers used for RT-qPCR are described in Additional file 2: Table S1.
Labeling and detection of cytosolic Ca2+ and ROS
Cytosolic Ca2+ and ROS levels were assessed according to the manufacturer’s instructions, with some modifications. For fluorescent detection of Ca2+ and ROS, the mycelia were incubated with the fluorescent probe Fluo-4 AM/DCHF-DA (Beyotime, Shanghai, China) at 28 °C for 30 min. The mycelia were washed three times with phosphate-buffered saline (PBS) (pH 5.0) to remove excess fluorophores and avoid excessive background noise. Images of the DCF- and Fluo-4 AM-labeled mycelia were visualized using an S Plan Fluor ELWD 20×, 0.5 numerical aperture (NA) objective and a digital sight camera on an Eclipse Ti inverted microscope system (Ti-E; Nikon, Tokyo, Japan), comprising a FITC filter (420–490 nm band-pass excitation filter and an emission filter of 535 nm). The average fluorescence intensities were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
To evaluate the effect of Sr2+ on cellulase production, different concentrations of SrCl2 were added immediately after transferring the mycelia to fresh MM. To evaluate the roles of cytosolic Ca2+ and ROS under Sr2+ stress, the mycelia were treated with the ROS scavenger NAC, hydrogen peroxide (H2O2), and plasma membrane Ca2+ channel blocker (LaCl3). NAC was added immediately after the mycelia were transferred to fresh MM. LaCl3 and H2O2 were added at 24 h after the mycelia were transferred to fresh MM.
All experimental data were obtained with at least three parallel samples with similar or identical results. Error values represent the standard deviation (SD) from the mean of three replicates. Student’s t-test was used to compare two samples to detect statistical significance. Duncan’s multiple-range test was used for multiple comparisons. Differences were considered as significant at p < 0.05.
Effects of Sr2+ on T. reesei growth
To investigate the effect of Sr2+ on colony growth, different concentrations of Sr2+ (0–120 mM) were added to solid MM containing 20 g/L glucose as the sole carbon source. An equal amount of fresh RUT-C30 conidia was spotted onto poured plates. The growth status of mycelia after Sr2+ supplementation is shown in Fig. 1a. The T. reesei strains grew more slowly as the Sr2+ concentration was increased. As shown in Fig. 1b, hyphal growth significantly differed between the 50 mM Sr2+ and no Sr2+ supplementation groups. Treatment with 70 and 100 mM Sr2+ caused a 31% and 35.2% reduction in colony diameter, respectively, compared to that of untreated strains. When the Sr2+ concentration was increased to 120 mM, the treated strains exhibited the most severe reduction in colony diameter (approximately 46.9%). This result was consistent with the biomass production data for MM liquid culture with 1% (w/v) Avicel as the sole carbon source (Fig. 1c). These results indicate that supplementation with large amounts of Sr2+ inhibited the growth of T. reesei strains under both glucose and Avicel carbon sources.
Cellulase production increased under Sr2+ pressure in T. reesei
To evaluate the effect of Sr2+ on cellulase production, the same amount of RUT-C30 mycelia was transferred into liquid MM with 1% (w/v) Avicel as the carbon source and different concentrations of Sr2+ (0–120 mM). As shown in Fig. 2a–e, addition of different concentrations of Sr2+ significantly improved cellulase activity and total protein secretion. Addition of 70 mM Sr2+ resulted in the largest improvement and significantly increased the pNPCase, CMCase, FPase, and xylanase activities, as well as the total protein concentration by 176%, 59.6%, 69.6%, 60%, and 87.2%, respectively, compared to that in the control (without Sr2+) on day 3 of cultivation. These results indicate that addition of 50–120 mM Sr2+ significantly improved the cellulase yield. The optimal concentration of Sr2+ for enhancing cellulase production was 70 mM in RUT-C30, which was used in subsequent analyses.
To further investigate the effect of Sr2+ on cellulase synthesis, the expression levels of two vital cellulase genes (cbh1 and egl1) and essential cellulase transcription activators xyr1 and ace3 were detected using RT-qPCR, as shown in Fig. 3. The transcription levels of cbh1 and egl1 increased by 546% and 520%, respectively, at 48 h, which was consistent with the upregulation of enzyme activities after addition of 70 mM Sr2+ (Fig. 2a–e). Addition of Sr2+ upregulated the expression of cellulase activator ace3 by approximately 72.3% at 48 h (Fig. 3). At 72 h, the expression level of ace3 increased to 272% compared to that in the control. These results indicate that Sr2+ supplementation significantly increased the transcription levels of cellulase genes and the essential cellulase transcription activator ace3. However, addition of Sr2+ led to downregulation of xyr1 expression (approximately 46%) at 48 h (Fig. 3).
Cytosolic Ca2+ accumulation in T. reesei was induced by high Sr2+ concentrations
Cytosolic concentrations of Ca2+ were detected using the fluorescent probe Fluo-4 AM, which emits green fluorescence after crossing the cell membrane and binding to cytosolic Ca2+ (Chen et al. 2021b). The fluorescence intensity represents the relative amount of free intracellular Ca2+. As shown in Fig. 4a, following addition of 70 mM Sr2+, the green fluorescence intensity in RUT-C30 cells was stronger than that in the control (without Sr2+ treatment). Analysis using ImageJ software showed that the fluorescence intensity of the experimental group increased by twofold after addition of Sr2+ (Fig. 4b), suggesting that the cytosolic Ca2+ content increased after Sr2+ treatment.
Increased concentrations of the second messenger Ca2+ in the cytosol lead to a response in the downstream calcium pathway (Li et al. 2019; Liu et al. 2019). Crz1, a critical activator of the calcium signal transduction terminal, plays an essential role in calcium signal transduction pathways (Chen et al. 2016; Martins-Santana et al. 2020). The transcription level of the transcriptional regulator crz1 detected using RT-qPCR was upregulated after Sr2+ induction (Fig. 4c).
A plasma membrane Ca2+ channel blocker, LaCl3 (Zhang et al. 2022), which prevents the influx of extracellular calcium ions, was used to examine the effect of Sr2+ on the cytosolic Ca2+ burst. As illustrated in Fig. 4a, b, the significant increase in intracellular Ca2+ induced by Sr2+ was markedly weakened after supplementation with LaCl3. At 5 mM LaCl3, the fluorescence intensity of the mycelia decreased by 40.6% compared with that in the absence of LaCl3. However, LaCl3 supplementation only negligibly reduced the fluorescence intensity compared with that in the control without Sr2+ (Fig. 4b).
These results indicate that Sr2+ induces a cytosolic calcium burst and results in calcium signal transduction, and that adding a plasma membrane Ca2+ channel blocker can block the Sr2+-induced calcium burst (Fig. 4b).
Sr2+ induced cytosolic Ca2+ burst and substantially increased cellulase production
LaCl3 (5 mM) can block the Sr2+-induced calcium burst (Fig. 4b). To explore whether blocking the cytosolic Ca2+ burst attenuates Sr2+-induced cellulase overexpression, the activities of pNPCase (Fig. 5a) and CMCase (Fig. 5b) and the transcriptional levels of the vital cellulase genes cbh1 (Fig. 5c) and egl1 (Fig. 5d) were measured.
Under 5 mM LaCl3 treatment, 70 mM Sr2+ did not induce cellulase overproduction in T. reesei. The pNPCase and CMCase activities decreased by 1.85- to 2.97-fold and 1.17- to 2.22-fold, respectively, when LaCl3 was added compared to the sample without LaCl3 addition at 2–4 days of cultivation (Fig. 5a, b). The transcript levels of cbh1 and egl1 were consistent with the cellulase activity data. After LaCl3 addition, the expression levels of cbh1 and egl1 decreased to 20–30% (Fig. 5c, d). Sr2+-induced cellulase overexpression was significantly attenuated by LaCl3 treatment.
These results indicate that blocking the burst of cytosolic Ca2+ by LaCl3 substantially reduced and even eliminated the induction of cellulase production by 70 mM Sr2+. The cytosolic Ca2+ and calcium signaling pathways participate in Sr2+-induced cellulase overproduction in T. reesei. High cellulase expression induced by Sr2+ was due to a Ca2+ burst in the cytoplasm.
Cytosolic ROS levels are significantly upregulated
The ROS content in mycelia exposed to Sr2+ was detected using the fluorescent probe dichlorodihydrofluorescein diacetate (DCFH-DA) (Gao et al. 2018). As shown in Fig. 6a, the green fluorescence intensity of the strain exposed to 70 mM Sr2+ was enhanced compared to that of the control (without Sr2+ supplementation), indicating that Sr2+-induced stress increased cytosolic ROS levels. To further evaluate the influence of Sr2+ on the cytosolic ROS content, the transcript levels of two major antioxidant enzymes, catalase (CAT) and superoxide dismutase (SOD), were detected using RT-qPCR (Fig. 6b). After addition of 70 mM Sr2+, the expression of sod1 increased by approximately 78–150% at 48–60 h. The transcript levels of cat1 increased by 165–487% at 60–72 h, indicating that the strain was under high ROS pressure.
NAC and H2O2 were used to alter the intracellular ROS content. As shown in Fig. 7a, b, adding 4 mM H2O2 reduced the levels of pNPCase and CMCase activity (approximately 33.1% and 16.8%, respectively). When the T. reesei strains were treated with 3 mM NAC, pNPCase and CMCase activities were markedly increased compared to those without NAC treatment (Fig. 7a–d). Under 70 mM Sr2+ treatment, pNPCase and CMCase activities also slightly increased in the presence of 3 mM NAC (approximately 11.5% and 8.4%, respectively). As shown in Fig. 7e, biomass production was measured following different chemical treatments. Addition of NAC slightly increased biomass production (approximately 11.9%), whereas treatment with H2O2 decreased biomass production (approximately 18.6%). Combined treatment with Sr2+ and H2O2 resulted in a large decrease in biomass (approximately 31.7%).
These results indicate that supplementation with 70 mM Sr2+ markedly increased ROS levels during T. reesei cultivation. Addition of the ROS scavenger NAC also decreased the ROS levels produced by Sr2+ and increased cellulase activity.
Metal ions are essential for primary and secondary cellular metabolic processes. Ca2+ (Schmoll 2011) and Mg2+ (Blaszczyk and Duda-Chodak 2013) are critical environmental factors that affect primary and secondary metabolism in various organisms. There are few reports on the effects of Sr2+, another group IIA metal ion. To the best of our knowledge, this study provides the first report that Sr2+ positively regulates cellulase production in T. reesei (Fig. 2a–e). Addition of 70 mM Sr2+ increased pNPCase and CMCase activity in the T. reesei wild-type strain QM6a by 53.3% and 51.6%, respectively, compared to those in the untreated control after 3 days of cultivation (Additional file 1: Figure S1a, b).
Addition of 70 mM Sr2+ had the most significant effect on cellulase activity. The upregulated expression levels of two major cellulase genes (cbh1 and egl1) and the cellulase transcription factor (ace3) (Fig. 3) were consistent with the cellulase activity data. Overexpression of ace3 enables a high level of cellulase production in T. reesei (Luo et al. 2020). This suggests that upregulated ACE3 may mediate Sr2+-induced cellulase overexpression (Fig. 8). Addition of Sr2+ resulted in downregulation of xyr1 expression (Fig. 3), suggesting that xyr1 is not involved in this process.
Under Sr2+ pressure, treatment with Sr2+ led to a cytosolic Ca2+ burst and increased crz1 transcription (Fig. 4c). According to previous studies, Ca2+ is an essential secondary messenger that can cooperate with intracellular cAMP (Benčina et al. 2005; Chen et al. 2021b), high‐osmolarity glycerol (de Castro et al. 2014) and ROS (Chen et al. 2021a; Gao et al. 2018) to regulate life activities in filamentous fungi, which is worthy of further study. However, cytosolic Ca2+ burst (Fig. 4a, b) and cellulase overproduction (Fig. 5a–d) were eliminated by adding the plasma membrane Ca2+ channel blocker LaCl3. These results indicate that calcium signal transduction is essential for Sr2+-induced cellulase production. This finding is similar to that of Chen et al. (2018), who reported that Mn2+ upregulates cellulase genes via calcium channels and signaling. Both Sr2+ and Mn2+ can induce cellulase overproduction; therefore, the process of Sr2+ induction is similar to that of the Mn2+/Ca2+ transport system in cells. In Thermosynechococcus elongatus, Ca2+/Sr2+ exchange affects photosystem II oxygen-evolving enzymes (Boussac et al. 2004) and electron transfer pathways from the S2 to S3 state (Boussac et al. 2015). Our results also demonstrate that Ca2+ plays a dominant role in Sr2+-induced cellulase overproduction.
According to the periodic table of elements, the number of electrons in the outermost unpaired orbital of Sr2+ is the same as that of Ca2+. This may partially explain their interaction and is worthy of further evaluated. Further studies are also required to investigate whether other metal ions interact with Ca2+ to regulate cellulase expression in T. reesei.
In this study, 70 mM Sr2+ inhibited growth of the strain (Fig. 1a–c), indicating that the strain was under pressure. Chen et al. (2016) reported that 100 mM Ca2+ repressed hyphal growth compared to that in the control. In addition, large amounts of ROS were produced after treatment with 70 mM Sr2+ based on analysis using an ROS probe (Fig. 6a). Gao et al. (2018) reported similar results, showing that treatment with Cu2+ significantly increased the intracellular ROS content in G. lucidum. Sr2+ was found to induce an increase in ROS levels, which affected cellulase production and was eliminated by adding the ROS scavenger NAC (Fig. 7a–d). In P. brevicompactum, treatment with high concentrations of Ca2+ and Mn2+ results in a surge in ROS levels, which regulate the synthesis of the secondary metabolite mycophenolic acid (Chen et al. 2021a, 2022). In G. lucidum, ROS were previously found to induce overproduction of the secondary metabolite ganoderic acid (Liu et al. 2018). These results suggest that ROS acts as a signal to regulate cellulase expression in T. reesei; however, this prediction requires further study. NAC can relieve ROS pressure and improve cell growth and cellulase yield, and its supplementation can improve industrial enzyme production. Large amounts of renewable lignocellulose can be hydrolyzed into sugars by cellulase to produce biofuels and chemicals. Therefore, it is crucial to decrease the price of cellulase. In industrial cellulase production, supplementation with Sr2+ and an ROS scavenger can increase cellulase production and improve economic efficiency.
Our findings indicate that adding extracellular Sr2+ can rapidly and easily improve cellulase production. A putative mechanism was characterized to explain the effect of Sr2+ treatment on cellulase expression in T. reesei RUT-C30 (Fig. 8). When the strain was exposed to high concentrations of Sr2+ (70 mM), calcium signal transduction played a crucial role in Sr2+-induced cellulase production. However, the ROS content in the cytoplasm was significantly increased, thereby reducing cellulase expression. This study provides an effective approach for increasing cellulase production and insights into the effects of divalent metal ions on the life activities of filamentous fungi.
Availability of data and materials
All data generated or analyzed during this study are included and available in this published article and its additional files.
- crz1 :
Calcineurin-responsive zinc finger transcription factor 1
Filter paper activity, representing total extracellular cellulase activity
Reactive oxygen species
Real-time quantitative PCR
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This research was funded by the Shanghai Agriculture Applied Technology Development Program, China (Grant No. 2021-02-08-00-12-F00758), Natural Science Foundation of Shanghai (22ZR1417600), and National Natural Science Foundation of China (32000050).
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Effects of Sr2+ on cellulase production in the parental strain QM6a. The pNPCase activity (a), CMCase activity (b) of T. reesei QM6a were measured after culture in MM medium for 2, 3, or 4 days with (or without) 70 mM Sr2+. 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).
The primers used for RT-qPCR.
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Li, N., Zeng, Y., Chen, Y. et al. Induction of cellulase production by Sr2+ in Trichoderma reesei via calcium signaling transduction. Bioresour. Bioprocess. 9, 96 (2022). https://doi.org/10.1186/s40643-022-00587-3
- Trichoderma reesei
- Calcium signaling
- Signal transduction