N, N-dimethylformamide induces cellulase production in the filamentous fungus Trichoderma reesei

Abstract

Background: The filamentous fungus Trichoderma reesei produces cellulase enzymes that are widely studied for lignocellulose bioconversion to biofuel. N,N-dimethylformamide (DMF) is a versatile organic solvent used in large quantities in industries.

Results: In this study, we serendipitously found that biologically relevant concentrations of extracellular DMF-induced cellulase production in the T. reesei hyper-cellulolytic mutant Rut-C30 and wild-type strain QM6a. Next, by transcriptome analysis, we determined that plc-e encoding phospholipase C was activated by DMF and revealed that cytosolic Ca²⁺ plays a vital role in the response of T. reesei to DMF. Using EGTA (a putative extracellular Ca²⁺ chelator) and LaCl₃ (a plasma membrane Ca²⁺ channel blocker), we demonstrated that DMF induced a cytosolic Ca²⁺ burst via extracellular Ca²⁺ and Ca²⁺ channels in T. reesei, and that the cytosolic Ca²⁺ burst induced by DMF-mediated overexpression of cellulase through calcium signaling. Deletion of crz1 confirmed that calcium signaling plays a dominant role in DMF-induced cellulase production. Additionally, 0.5-2% DMF increases the permeability of T. reesei mycelia for cellulase release. Simultaneous supplementation with 1% DMF and 10 mM Mn²⁺ to T. reesei Rut-C30 increased cellulase activity approximately fourfold compared to that without treatment and was also more than that observed in response to either treatment alone.

Conclusions: Our results reveal that DMF-induced cellulase production via calcium signaling and permeabilization. Our results also provide insight into the role of calcium signaling in enzyme production for enhanced cellulase production and the development of novel inducers of cellulase.

Keywords: Calcium signaling; Cellulase; DMF induced; Permeability; Phospholipase C; Trichoderma reesei; plc-e.

Fig. 1

Effects of DMF on hyphal growth and protein production in T. reesei QM6a. a Hyphae growth of T. reesei QM6a on plates. DMF was added at final concentrations of 0, 0.1, 0.5, 1, and 2% (v/v). b Colony diameters. c–f The effects of different concentrations of DMF (final concentration 0, 0.1, 0.5, 1, and 2%) on CMCase/biomass activity (c), pNPCase/biomass activity (d), total protein concentrations (e), and biomass (f) of T. reesei QM6a. Different letters indicate significant differences between the columns (p < 0.05, Duncan’s multiple-range test). Blue bar, adding 0% (v/v) DMF; purple bar, adding 0.1% (v/v) DMF; green bar, adding 0.5% (v/v) DMF; red bar, adding 1% (v/v) DMF; pink bar, adding 2% (v/v) DMF. Values are the mean ± SD of results from three independent experiments. Asterisks indicate a significant difference compared to the untreated strain (p < 0.05, Student’s t test)

Fig. 2

Effects of DMF on cell permeability of T. reesei mycelia. The extracellular electrical conductivity of mycelia was measured using an electrical conductivity meter after mycelia were treated with different concentrations of DMF (final concentrations of 0, 0.1, 0.5, 1, and 2%) and 1% (v/v) DMSO. Values are the mean ± SD of the results from three independent experiments. Filled round, adding 0% (v/v) DMF; blank square, adding 0.1% (v/v) DMF; five-pointed star, adding 0.5% (v/v) DMF; triangle, adding 1% (v/v) DMF; diamond, adding 2% (v/v) DMF; blank round, adding 1% (v/v) DMSO. Asterisks indicate a significant difference compared to the untreated strain at all time points examined (p < 0.05, Student’s t test)

Fig. 3

Cytosolic Ca²⁺ level and calcium signaling increases after DMF addition. a Cytosolic Ca²⁺ levels determined by Ca²⁺ fluorescent probe Fluo-3 AM. T. reesei QM6a was cultured in liquid Mandels’ medium for ~ 32 h, transformed to fresh MM with 0% or 1% DMF, and then cultivated for 48 to 60 h. For detection, 50 μM Fluo-3 AM was used, with the intensity of fluorescence monitored using automatic inverted fluorescence microscopy. Green fluorescence represents free cytosolic Ca²⁺. DIC, differential interference contrast. b Comparative fluorescence ratio of cytosolic Ca²⁺ levels. The x-axis represents the different DMF final concentrations, and the y-axis represents the Ca²⁺ fluorescence ratios measured by CLSM. c–e The effects of DMF on the transcriptional levels of calcium signaling-related genes cam (c), cna1 (d), and crz1 (e) in T. reesei QM6a. Blue bar, no DMF was added to the medium; red bar, 1% (v/v) DMF was added to the medium. Values are the mean ± SD of the results from three independent experiments. Asterisks indicate significant differences from untreated strains (p < 0.05, Student’s t test)

Fig. 4

Effect of Ca²⁺ inhibitors on cytosolic Ca²⁺ concentrations and cellulase production after DMF addition. A Fluorescence indicating LaCl₃ and EGTA effects on the cytosolic Ca²⁺ burst induced by DMF. T. reesei QM6a was cultured in liquid Mandels’ medium for ~ 32 h, transformed to fresh MM with 0% or 1% DMF and 0 or 5 mM LaCl₃/EGTA, and cultivated for 48 to 60 h. For detection, 50 μM Fluo-3 AM was used, and fluorescence intensity was monitored using automatic inverted fluorescence microscopy. Green fluorescence represents free cytosolic Ca²⁺. DIC, differential interference contrast. CK, not treated with inhibitors LaCl₃/EGTA. B Comparative fluorescence ratios indicating LaCl₃ and EGTA effects on the cytosolic Ca²⁺ burst induced by DMF. The x-axis represents different treatments with DMF and inhibitors, and the y-axis represents Ca²⁺ fluorescence ratios measured by CLSM. C, D CMCase/biomass activity (C) and pNPCase/biomass activity (D) were examined after T. reesei QM6a was cultured in medium with different DMF and inhibitor treatments. E, F The expression levels of cbh1 (E) and egl1 (F) in T. reesei QM6a were analyzed after culture in medium with various DMF and inhibitor treatments. Blue bar, no DMF was added to the medium; red bar, 1% (v/v) DMF was added to the medium. Values are the mean ± SD of the results from three independent experiments. Different letters indicate significant differences between the columns (p < 0.05, Duncan’s multiple-range test)

Fig. 5

Effect of CRZ1 on DMF-induced cellulase overexpression. a, b CMCase/biomass activity (a) and pNPCase/biomass activity (b) of T. reesei QM6a and Δcrz1 strains supplemented with 0% or 1% DMF. c, d The expression levels of cbh1 (c) and egl1 (d) in T. reesei QM6a and Δcrz1 strains cultured in medium supplemented with 0% or 1% DMF. Red bar, adding 1% (v/v) DMF in T. reesei QM6a; blue bar, adding 0% (v/v) DMF in T. reesei QM6a; purple bar, adding 1% (v/v) DMF in Δcrz1; green bar, adding 0% (v/v) DMF in Δcrz1. e SDS-PAGE of the secretomes in supernatants. T. reesei QM6a and Δcrz1 were cultured in liquid Mandels’ medium for ~ 32 h. Then, mycelia were transferred to fresh Mandels’ medium with 0% or 1% DMF and cultivated for 72 h. Two cultures of each strain with 0% or 1% DMF added were collected by centrifugation and diluted similar to the proper protein concentration for SDS-PAGE. Lines 1 and 2 represent supernatants from the QM6a culture supplemented with 0% or 1% DMF, respectively. Lines 3 and 4 represent supernatants from the Δcrz1 culture supplemented with 0% or 1% DMF, respectively. Values are the mean ± SD of the results from three independent experiments. Asterisks indicate significant differences (*p < 0.05, Student’s t test)

Fig. 6

Effects of combined DMF and Mn²⁺ on cellulase production in T. reesei Rut-C30. a–d CMCase activity (a), pNPCase activity (b), CMCase/biomass activity (c) and pNPCase/biomass activity (d) of T. reesei Rut-C30 supplemented with 1% DMF or (and) 10 mM MnCl₂. Blue bar, no addition; red bar, 1% (v/v) DMF was added to the medium; green bar, 10 mM MnCl₂ was added to the medium; purple bar, 1% (v/v) DMF and 10 mM MnCl₂ was added to the medium. Values are the mean ± SD of the results from three independent experiments. Asterisks indicate significant differences (*p < 0.05, Student’s t test)

Fig. 7

Mechanistic model of DMF induction of cellulase overexpression in T. reesei. DMF promotes a cytosolic Ca²⁺ burst that activates calcium signaling, which in turn is required for cellulase overexpression. The effects of adding EGTA and LaCl₃ suggest that extracellular Ca²⁺ and Ca²⁺ channels are responsible for the cytosolic Ca²⁺ burst and cellulase production induced by DMF. Deletion of crz1 confirmed that calcium signaling plays a dominant role in DMF-induced cellulase production. Deletion of plc-e implied that PLC-E is involved in Ca²⁺ signal transduction induced by DMF. Additionally, the cell permeability of T. reesei mycelia increased after treatment with DMF, which is beneficial for cellulase secretion. Solid arrows indicate data supported by our own experiments