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Background: Knowledge with respect to regulatory systems for cellulase production is prerequisite for exploitation of such regulatory networks to increase cellulase production, improve fermentation efficiency and reduce the relevant production cost. The TOR (Target of Rapamycin) signaling pathway is considered as a central signaling hub coordinating eukaryotic cell growth and metabolism with environmental inputs. However, how and to what extent the TOR signaling pathway and rapamycin are involved in cellulase production remains elusive.
Result: At the early fermentation stage, high-dose rapamycin (100 μM) caused a temporary inhibition effect on cellulase production, cell growth and sporulation of Trichoderma reesei independently of the carbon sources, and specifically caused a tentative morphology defect in RUT-C30 grown on cellulose. On the contrary, the lipid content of T. reesei was not affected by rapamycin. Accordingly, the transcriptional levels of genes involved in the cellulase production were downregulated notably with the addition of rapamycin. Although the mRNA levels of the putative rapamycin receptor trFKBP12 was upregulated significantly by rapamycin, gene trTOR (the downstream effector of the rapamycin-FKBP12 complex) and genes associated with the TOR signaling pathways were not changed markedly. With the deletion of gene trFKBP12, there is no impact of rapamycin on cellulase production, indicating that trFKBP12 mediates the observed temporary inhibition effect of rapamycin.
Conclusion: Our study shows for the first time that only high-concentration rapamycin induced a transient impact on T. reesei at its early cultivation stage, demonstrating T. reesei is highly resistant to rapamycin, probably due to that trTOR and its related signaling pathways were not that sensitive to rapamycin. This temporary influence of rapamycin was facilitated by gene trFKBP12. These findings add to our knowledge on the roles of rapamycin and the TOR signaling pathways play in T. reesei.
Keywords
rapamycin, T. reesei, trFKBP12
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
- SupportingmaterialFigS20201106FL1.docx
Figure S1 Effects of 100 μM rapamycin on (hemi)cellulase activities and protein secretion of T. reesei RUT-C30 cultured in TMM+2% glucose (A) and TMM+2% lactose (B) at 24 h, 72 h, 120 h, and 168 h, respectively. FPase: the filter paper activity; pNPCase: the CBH activity; CMCase: the CMC activity; pNPGase: the β-glucosidase activity; Secreted protein: secreted protein concentration; pNPXase: the β-xylosidase activity. Data are represented as the mean of three independent experiments and error bars express the standard. Figure S2 Hyphal morphology of T. reesei RUT-C30 cultured in TMM+2% glucose (A) or lactose (B) with different concentrations of rapamycin at 24, 72, 120, and 168 h, which was observed under CLSM. Scale bar =10 μm. Figure S3 The lipid content of T. reesei RUT-C30 on TMM+2% cellulose, lactose, or glucose with various concentrations of rapamycin. The blue circles in (A) represented mycelial radius at 120 h. The lipid content of RUT-C30 at 24 h stained with Nile Red was observed under CLSM. Scale bar =10 μm. Figure S4 Hyphal morphology of T. reesei △FKBP12 and KU70 cultured in TMM+2% cellulose with/ without 100 μM rapamycin at 24, 72, 120, and 168 h, which was observed under CLSM. Scale bar =20 μm. Figure S5 FKBP12 (A) and TOR (B) sequence alignments with those of Saccharomyces cerevisiae, Homo sapiens, Arabidopsis thaliana, Schizosaccharomyces pombe, and Oryza sativa. Amino acid residues that form the hydrophobic rapamycin-binding pocket of FKBP12 (A) are in red. Mutation of amino acid residues of TOR (B) in red confers rapamycin resistance.
- TableS1TotalDEGsinT.reeseiRUTC30.xlsx
Table S1 Transcriptional level of total DEGs in T. reesei RUT-C30 with or without 100 μM rapamycin.
- TableS2DEGsrelatedtotransportersinT.reeseiRUTC30.xlsx
Table S2 Transcriptional level of DEGs related to transporters in T. reesei RUT-C30.
- TableS3Genesinvolvedinhemicellulaseproduction.xlsx
Table S3 Transcriptional level of genes involved in (hemi)cellulase production in T. reesei RUT-C30.
- TableS4DEGsinBiosynthesisofsecondarymetabolites.xlsx
Table S4 Transcriptional level of DEGs related to “Biosynthesis of secondary metabolites” in T. reesei RUT-C30.
- TableS5GenesinvolvedinTORsignalpathways.xlsx
Table S5 Transcriptional level of genes involved in TOR signal pathways in T. reesei RUT-C30.
- TableS6DEGsrelatedtogeneexpression.xlsx
Table S6 Transcription level of genes associated with gene expression in T. reesei RUT-C30.
- TableS7PrimersfortrFKBPdeletionandconfirmation.docx
Table S7 Primers for trFKBP deletion and confirmation.
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View the published article at Biotechnology for Biofuels and Bioproducts.
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Received 21 Dec, 2020
Review #1 received
Received 21 Dec, 2020
Reviewer #2 agreed
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Reviewer #1 agreed
On 04 Dec, 2020
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
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Complete author information
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Peer Review Timeline
CURRENT STATUS: Published
View the published article at Biotechnology for Biofuels and Bioproducts.
Version 1
Posted 19 Nov, 2020
No community comments so far
Editorial decision: Major revision
On 07 Feb, 2021
Review #3 received
Received 06 Feb, 2021
Reviewer #3 agreed
On 02 Jan, 2021
Review #2 received
Received 21 Dec, 2020
Review #1 received
Received 21 Dec, 2020
Reviewer #2 agreed
On 08 Dec, 2020
Reviewers invited
Invitations sent on 04 Dec, 2020
Reviewer #1 agreed
On 04 Dec, 2020
First submitted
On 16 Nov, 2020
Editor assigned
On 16 Nov, 2020
Submission checks complete
On 16 Nov, 2020
Editor invited
On 16 Nov, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Biotechnology and Bioengineering
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Biotechnology for Biofuels and Bioproducts.
Background: Knowledge with respect to regulatory systems for cellulase production is prerequisite for exploitation of such regulatory networks to increase cellulase production, improve fermentation efficiency and reduce the relevant production cost. The TOR (Target of Rapamycin) signaling pathway is considered as a central signaling hub coordinating eukaryotic cell growth and metabolism with environmental inputs. However, how and to what extent the TOR signaling pathway and rapamycin are involved in cellulase production remains elusive.
Result: At the early fermentation stage, high-dose rapamycin (100 μM) caused a temporary inhibition effect on cellulase production, cell growth and sporulation of Trichoderma reesei independently of the carbon sources, and specifically caused a tentative morphology defect in RUT-C30 grown on cellulose. On the contrary, the lipid content of T. reesei was not affected by rapamycin. Accordingly, the transcriptional levels of genes involved in the cellulase production were downregulated notably with the addition of rapamycin. Although the mRNA levels of the putative rapamycin receptor trFKBP12 was upregulated significantly by rapamycin, gene trTOR (the downstream effector of the rapamycin-FKBP12 complex) and genes associated with the TOR signaling pathways were not changed markedly. With the deletion of gene trFKBP12, there is no impact of rapamycin on cellulase production, indicating that trFKBP12 mediates the observed temporary inhibition effect of rapamycin.
Conclusion: Our study shows for the first time that only high-concentration rapamycin induced a transient impact on T. reesei at its early cultivation stage, demonstrating T. reesei is highly resistant to rapamycin, probably due to that trTOR and its related signaling pathways were not that sensitive to rapamycin. This temporary influence of rapamycin was facilitated by gene trFKBP12. These findings add to our knowledge on the roles of rapamycin and the TOR signaling pathways play in T. reesei.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
- SupportingmaterialFigS20201106FL1.docx
Figure S1 Effects of 100 μM rapamycin on (hemi)cellulase activities and protein secretion of T. reesei RUT-C30 cultured in TMM+2% glucose (A) and TMM+2% lactose (B) at 24 h, 72 h, 120 h, and 168 h, respectively. FPase: the filter paper activity; pNPCase: the CBH activity; CMCase: the CMC activity; pNPGase: the β-glucosidase activity; Secreted protein: secreted protein concentration; pNPXase: the β-xylosidase activity. Data are represented as the mean of three independent experiments and error bars express the standard. Figure S2 Hyphal morphology of T. reesei RUT-C30 cultured in TMM+2% glucose (A) or lactose (B) with different concentrations of rapamycin at 24, 72, 120, and 168 h, which was observed under CLSM. Scale bar =10 μm. Figure S3 The lipid content of T. reesei RUT-C30 on TMM+2% cellulose, lactose, or glucose with various concentrations of rapamycin. The blue circles in (A) represented mycelial radius at 120 h. The lipid content of RUT-C30 at 24 h stained with Nile Red was observed under CLSM. Scale bar =10 μm. Figure S4 Hyphal morphology of T. reesei △FKBP12 and KU70 cultured in TMM+2% cellulose with/ without 100 μM rapamycin at 24, 72, 120, and 168 h, which was observed under CLSM. Scale bar =20 μm. Figure S5 FKBP12 (A) and TOR (B) sequence alignments with those of Saccharomyces cerevisiae, Homo sapiens, Arabidopsis thaliana, Schizosaccharomyces pombe, and Oryza sativa. Amino acid residues that form the hydrophobic rapamycin-binding pocket of FKBP12 (A) are in red. Mutation of amino acid residues of TOR (B) in red confers rapamycin resistance.
- TableS1TotalDEGsinT.reeseiRUTC30.xlsx
Table S1 Transcriptional level of total DEGs in T. reesei RUT-C30 with or without 100 μM rapamycin.
- TableS2DEGsrelatedtotransportersinT.reeseiRUTC30.xlsx
Table S2 Transcriptional level of DEGs related to transporters in T. reesei RUT-C30.
- TableS3Genesinvolvedinhemicellulaseproduction.xlsx
Table S3 Transcriptional level of genes involved in (hemi)cellulase production in T. reesei RUT-C30.
- TableS4DEGsinBiosynthesisofsecondarymetabolites.xlsx
Table S4 Transcriptional level of DEGs related to “Biosynthesis of secondary metabolites” in T. reesei RUT-C30.
- TableS5GenesinvolvedinTORsignalpathways.xlsx
Table S5 Transcriptional level of genes involved in TOR signal pathways in T. reesei RUT-C30.
- TableS6DEGsrelatedtogeneexpression.xlsx
Table S6 Transcription level of genes associated with gene expression in T. reesei RUT-C30.
- TableS7PrimersfortrFKBPdeletionandconfirmation.docx
Table S7 Primers for trFKBP deletion and confirmation.
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