High-dose rapamycin excerts a temporary impact on T. reesei through gene trFKBP12

doi:10.21203/rs.3.rs-110351/v1

Download PDF

Research

High-dose rapamycin excerts a temporary impact on T. reesei through gene trFKBP12

https://doi.org/10.21203/rs.3.rs-110351/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 26 Mar, 2021

Read the published version in Biotechnology for Biofuels and Bioproducts →

You are reading this latest preprint version

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.

Biotechnology and Bioengineering

rapamycin

T. reesei

trFKBP12

Cellulolytic fungi like Neurospora crassa and Trichoderma reesei, are involved in the degradation of plant biomass and play important roles in ecosystems (1, 2). These fungi have evolved an excellent capability to secret (hemi)cellulase to convert insoluble polysaccharides into fermentable sugars for surviving under lignocellulolytic conditions. The (hemi)cellulase should be produced in a strictly controlled way. Otherwise, too much cellular resources are directed towards protein synthesis, which is detrimental for cell survival (1). Therefore, cellulolytic fungi have developed a complex regulatory machinery to coordinate nutrients for growth and hydrolytic enzyme production. For instance, the cellulase production of T. reesei is regulated in response to various environmental stresses such as light (3, 4), organic solvents (5), and metal ions (6). Several signal pathways like mitogen-activated protein kinase (MAPK) pathways (7), Ca²⁺-responsive signaling pathway (8) and light regulation pathway (3) have been reported to be involved in the regulation of cellulase production. A better understanding of regulatory systems for cellulase production is prerequisite for exploitation of such regulatory networks to increase enzyme secretion, improve fermentation efficiency and reduce the enzyme production cost.

The TOR (Target of Rapamycin) signaling pathway is regarded as a central signaling hub integrating cell growth and metabolism with environmental inputs, including nutrients and growth factors in eukaryotes (9). The TOR signaling network responds to signals such as nutritional state, cellular energy state, and growth factors to orchestrate cellular growth, proliferation, and stress responses. In response to nutrients, it activates anabolic processes like protein, lipid and nucleotide synthesis, and suppresses catabolic processes such as autophagy, thus promoting cell growth. Several studies have reported that the TOR signaling pathway plays a part in regulating cellulase production. Gene Sch9 is a critical component of the TOR signaling pathway in Saccharomyces cerevisiae. Deletion of its homolog stk-10 in N. crassa severely decreased cellulase production (10). Similarly, loss of schA (the homolog of stk-10) in Aspergillus nidulans caused defects in protein production and cellulolytic enzyme activities (11). Also, knockdown of sch9 in T. reesei slightly compromised the cellulase production (12). Despite these preliminary studies, how and to what extent the TOR signaling pathway is involved in cellulase production remains elusive. Even worse, the relevant study of the TOR signaling pathway in filamentous fungi is lack, though it has been extensively studied in yeast S. cerevisiae and Schizosaccharomyces pombe (13, 14), mammalian cells (15), and plant cells (16, 17).

The TOR kinases are first identified in S. cerevisiae as the targets of rapamycin. Rapamycin, a lipophilic macrocytic lactone isolated from Streptomyces hygroscopicus in the early 1970s (18), displays antifungal, anticancer and immunosuppressive activities (9). Rapamycin and its derivatives, which are called “rapalogues”, have been applied in clinic to inhibit tumor growth and prevent organ rejection (9). When diffusing into the cell, rapamycin forms a complex with the peptidyl-prolyl cis/trans isomerase FKBP12, which subsequently binds to the TOR kinases and inhibits their functions (19). Both FKBP12 and TOR kinases are conserved in eukaryotic organisms from fungi to human (20). Rapamycin is an indispensable tool for studying the role of the TOR signaling pathway in organisms. However, the impact of rapamycin on T. reesei and cellulase production in filamentous fungi has not been reported, as far as we know.

As a first step toward understanding the role of the TOR pathway in cellulase production, the impact of rapamycin on T. reesei RUT-C30 grown on different carbon sources was investigated in terms of cellulase production, morphology, cell growth, sporulation and lipid content. T. reesei was not insensitive to rapamycin and only a temporary effect was observed with high concentration of rapamycin at the early cultivation stage, including decreased cellulase production, cell growth and sporulation, and altered cell morphology. The molecular mechanism behind this phenomenon was explored by comparative transcriptional profiling and gene knockout of trFKBP12.

Rapamycin at high concentration temporarily inhibits cellulase production

The effect of rapamycin on (hemi)cellulase production of T. reesei RUT-C30 was investigated (Fig. 1). In the presence of 0.01 µM rapamycin, all the tested cellulase activities were not changed at 24 h, but was increased at 72 h and higher than those of RUT-C30 without rapamycin during the rest fermentation time. The pNPGase, pNPCase, CMCase, and FPase activities were improved by 28.7%, 24.0%, 19.2%, and 26.2% separately at 72 h. Nevertheless, when the concentration of rapamycin was increased to 1 µM, the pNPGase, pNPCase, and CMCase activities were reduced at 24 h, but increased at 120 h and beyond. The FPase activity did not reach the same level as that without rapamycin until 168 h. A higher concentration of rapamycin than 1 µM caused a more serious reduction on cellulase activities at the early fermentation process. Especially, the addition of 100 µM rapamycin led to a decline by 67.2%, 60.2%, 56.6%, and 70.3% for pNPGase, pNPCase, CMCase, and FPase activities respectively at 24 h. Interestingly, T. reesei had the ability to recover and produce the same or even a larger amount of cellulase at 168 h, regardless of the concentrations of rapamycin. Obviously, rapamycin improved the cellulase production at low concentration, but at high concentration inhibited severely the celluase production at the early stage which was restored at the late stage. The secreted protein level followed a similar trend as celluase activity. The concentration of secreted protein was halved at 100 µM rapamycin at 24 h, as compared to the untreated sample.

The pNPXase activity did not have obvious change at 0.01 µM rapamycin. At 1 µM rapamycin, the pNPXase activity was decreased at 48 h and 72 h, but was restored to the level of that without rapamycin at 120 h. At 10 µM and 100 µM rapamycin, the pNPXase activity was sharply reduced by 68.0% and 71.9% at 24 h, and remained lower than that without rapamycin at 168 h, suggesting that high concentration of rapamycin decreased hemicellulase activity which was unable to be recovered.

A similar effect of 100 µM rapamycin on the (hemi)cellulase production was found in T. reesei grown on glucose or lactose, except that the pNPCase activity was not changed in the presence of rapamycin when using glucose as the carbon source (Figure S1). Nevertheless, the temporary inhibition effect of rapamycin on the total extracellular protein concentration was not found at the early fermentation stage of T. reesei cultivated on glucose or lactose. In addition, the inhibition effect of rapamycin on the pNPXase activity can be relieved at the late stage, different from that of T. reesei cultured on cellulose. Obviously, the effect of rapamycin on the (hemi)cellulase production in T. reesei was not dependent on the carbon sources, though some subtle differences were observed among different carbon sources.

Rapamycin induces transit morphology defect of T. reesei on cellulose, but not on lactose or glucose.

The effect of rapamycin on the morphology of T. reesei RUT-C30 cultivated in TMM + 2% cellulose, 2% lactose or 2% glucose was investigated by confocal laser scanning microscopy (CLSM) (Fig. 2 and Figure S2). The morphology of T. reesei was not affected by 0.01 µM rapamycin throughout the whole cellulase production on cellulose. When the concentration of rapamycin was increased to 1 µM and beyond, the abnormal hyphal morphology was observed at 24 h. The filamentation of T. reesei was strongly inhibited by rapamycin, showing spherical form which was reminiscent of hypha-yeast transition. However, the morphological defects by 1 µM or 10 µM rapamycin were not found at 72 h and later. Even at 100 µM rapamycin, only a few mycelia exhibited aberrant morphology at 72 h and then disappeared after 72 h. All these findings implicated that rapamycin suppresses the hyphal formation in the early stage of T. reesei grown on cellulose temporarily even at high concentration, which was restored at the late stage. However, when the carbon source was lactose or glucose, there was no notable effect of rapamycin on the mycelium morphology (Figure S2).

Rapamycin hinders cell growth and sporulation of T. reesei tentatively, but not the lipid content

The effect of rapamycin on the growth of T. reesei RUT-C30 on cellulose, lactose or glucose was studied (Fig. 3). As compared to the untreated samples, the cell growth of T. reesei on cellulose, lactose or glucose at 24 h was retarded notably with the treatment of rapamycin at concentrations no less than 1 µM. This cell growth impairment was rescued completely at later stage under cellulose or glucose condition, but not under lactose condition. Meanwhile, the spore amount of T. reesei grown on cellulose, lactose or glucose was also reduced at 24 h, which was recovered at 120 h with no significant change even with 100 µM rapamycin (Fig. 3).

It has been reported that rapamycin treatment led to an increase in the number and size of lipid droplets in the fungus S. cerevisiae (21), Podospora anserine (22, 23), and Ustilago maydis (24). Therefore, to see whether the lipid content of T. reesei was altered by rapamycin, the lipid content of T. reesei grown on cellulose, lactose or glucose was stained by Nile Red and checked under CLSM (Fig. 3 and Figure S3). There was no significant difference in the fluorescent intensity and lipid form between rapamycin-treated RUT-C30 and the untreated one, which was independent on the carbon source, implying that the lipid synthesis in T. reesei RUT-C30 was not affected by rapamycin. Overall, the cell growth and sporulation of T. reesei were reduced by high-dose rapamycin at the early fermentation stage, but not at the late stage. On the contrary, its lipid content was not influenced by rapamycin. T. reesei is highly resistant to rapamycin regardless of carbon source.

Transcription pattern of T. reesei treated with high-dose rapamycin

To gain insight into how rapamycin influences T. reesei RUT-C30 at the transcriptional level, RNA-seq analysis was performed using RUT-C30 cultured in TMM medium with or without 100 µM rapamycin for 24 h. The sequences of the total reads were mapped to the reference genome of T. reesei RUT-C30 (https://www.ncbi.nlm.nih.gov/genome/323%3fgenomeassembly_id%3d49799) with coverage of 97.68–97.76%. A total of 10048 unique transcripts were detected. Genes were considered to be differentially expressed between the two conditions when the average reads of the corresponding transcripts differed with |log₂Ratio| ≥ 1 and p value ≤ 0.05. By comparing rapamycin-treated RUT-C30 to the untreated one, 484 differentially expressed genes (DEGs) were obtained, of which 201 were upregulated and 283 were downregulated (Table S1).

The enriched molecular function was mainly related to “catalytic activity” (Fig. 4A), which comprised 69 DEGs. Among them, 50 DEGs show hydrolase activity, of which 30 act on glycosyl bonds. DEGs in “cellulose binding”, “cellulase activity”, “beta-glucosidase activity” and “xylanase activity” categories were all downregulated, which are related to cellulose and hemicellulose degradation. In addition, the category “ATPase activity, coupled to transmembrane movement of substances” included 8 DEGs. Among them, 6 were predicted to be ABC transporters regarding multidrug resistance that were all upregulated to different degrees (Table S2). The increased expressions of these ABC transporters in T. reesei might be a defense mechanism against rapamycin by exporting it out of the cells, which might be worth exploring in future study.

For the enriched cellular components, 37 DEGs were under “extracellular region” category, demonstrating that rapamycin greatly impacts the extracellular enzymes (Fig. 4B). Among them, two cellobiohydrolase (CBH) (CEL7A and CEL6A), seven endoglucanase (EG) (CEL7B, CEL5A, CEL12A, CEL61A, CEL45A, CLE74A, and CEL61B), three β-glucosidase (BGL) (CEL3A, CEL3C and CEL3D), and one β-xylosidase (BXL1) were downregulated. The enriched biological processes were mainly cellulose and hemicellulose catabolic processes, of which all DEGs were downregulated (Fig. 4C). These results were consistent with the remarkably decreased pNPCase, CMCase, pNPGase and pNPXase activities in RUT-C30 treated with 100 µM rapamycin for 24 h, respectively. In addition, the most enriched pathways affected by rapamycin included “Biosynthesis of secondary metabolites”, “Biosynthesis of antibiotic”, “Starch and sucrose metabolism”, “Valine, leucine and isoleucine degradation”, and “Fructose and mannose metabolism” (Fig. 4D).

DEGs involved in the cellulase production were downregulated by rapamycin

Thirty nine DEGs were related to cellulose degradation, of which thirty seven were downregulated and only two were upregulated (Fig. 5 and Table S3). Fifteen cellulases including two cellobiohydrolases (CEL7A and CEL6A), seven endoglucanases except CEL5B, and six β-glucosidases (CEL3A, CEL1A, CEL1B, CEL3C, CEL3H and CEL3D) were notably down-expressed, which agreed with the reduced pNPCase, CMCase, pNPGase and total filter paper FPase activities as found above. The downregulated mRNA levels of BXL1 and xylanases (XYN2, XYN3, XYN4, and XYN6) were in line with decreased pNPXase activity. Moreover, nonenzymatic cellulose attacking enzymes swollenin (25), Cip1 and Cip2 (26), which acted in synergy with cellulases and hemicellulases to enhance the hydrolytic efficiency of cellulose were also downregulated significantly. Unexpectedly, the hemicellulase α-galactosidase AGL1 and β-mannosidase (M419DRAFT_93487), which hydrolyses α-D-galactosides and mannans, were upregulated, though another α-galactosidase (M419DRAFT_71638) and two other β-mannosidases (M419DRAFT_67432 and M419DRAFT_122377) were downregulated noticeably.

In addition, 7 transcriptional factors involved in cellulase production were identified to be DEGs with marked downregulation (Fig. 5D). They were cellulase transcription activators Xyr1 (27), Ace3 (28) and Clr2 (29), MFS sugar transporters Crt1 and Stp1 (30, 31), xylanase promoter binding protein Xpp1 (32), and the carbon catabolite repressor Cre4 (33). Xpp1 was firstly described as a repressor of xylanases (34) and later as a repressor of secondary metabolism (32). The downregulation of Xpp1 did not lead to the increase of xylanase production (Fig. 1), but the expression of 11 DEGs involved in KEGG category “Biosynthesis of secondary metabolites” was found to be increased significantly (Fig. 4D and Table S4), in agreement with the role of Xpp1 as a repressor of secondary metabolism.

FKBP12 is required for the temporary inhibition of rapamycin on cellulase production

Rapamycin forms a gain-of-function complex with FKBP12 first (19), which then inhibits the TOR kinases. By searching T. reesei genome for FKBP12 homologs of S. cerevisiae, we identified three FKBP orthologues including trFKBP12-1 (M419DRAFT_72966), trFKBP12-2 (M419DRAFT_140396), and trFKBP12-3 (M419DRAFT_61673). Among these three FKBP orthologues, only the transcription level of trFKBP12-1 was significantly upregulated by 1.7 fold in T. reesei treated with rapamycin (Table 1), indicating that trFKBP12-1 is probably the cellular receptor of rapamycin. Protein trFKBP12-1 was referred to as trFKBP12 in this study.

Table 1

Transcriptional level of FKBP12 and TOR complexes in *T. reesei*
Protein ID^a	log₂FC	p value	Name	Protein ID^b	Identity
M419DRAFT_72966	1.71	0.0035	FKBP12-1	CAA86890	47.32%
M419DRAFT_140396	-0.20	0.0077	FKBP12-2		46.85%
M419DRAFT_61673	0.02	0.7787	FKBP12-3		44.44%
M419DRAFT_24714	-0.33	5.00E − 05	TOR1	NP_012600.1	49.27%
M419DRAFT_24714	-0.33	5.00E − 05	TOR2	NP_012719.2	49.77%
M419DRAFT_137337	0.11	0.2403	Lst8	NP_014392.3	67.00%
M419DRAFT_82078	-0.24	0.0072	Kog1	NP_012056.1	45.92%
M419DRAFT_26158	0.10	0.2521	Avo1	NP_014563.1	24.23%
M419DRAFT_128246	-0.19	0.0194	Avo3	NP_011018.1	26.88%
^a Protein ID was assigned based on the genome database (https://fungi.ensembl.org/Trichoderma_reesei_rut_c_30_gca_000513815/Info/Index)
^b Protein ID was assigned based on the genome database (https://www.ncbi.nlm.nih.gov/)

To determine whether trFKBP12 is the cellular receptor of rapamycin in T. reesei, gene trFKBP12 was knockout using T. reesei KU70 as the parent strain (35), obtaining mutant strain ΔtrFKBP12. KU70 was chosen as a host strain for its high efficiency of gene targeting, where ku70 was deleted in RUT-C30 (36). Similar to strain RUT-C30, the inhibition effect of 100 µM rapamycin on cellulase production was observed in strain KU70 at the early fermentation stage (Fig. 6A, 6B, and 6C), which was completely relieved at the late stage (Fig. 6D). This inhibition effect was not found in strain ΔtrFKBP12 during the whole fermentation process (Fig. 6), demonstrating that trFKBP12 was indispensable for the temporary inhibition effect of rapamycin on cellulase production in T. reesei at the early cultivation stage. It is worth noting that knockout of trFKBP12 alone led to a delay in cellulose production, similar to that caused by high-dose rapamycin. No morphology change was found in strain KU70 or ΔtrFKBP12 with the treatment of 100 µM rapamycin (Figure S4).

DEGs involved in the TOR pathway

The TOR kinase is the target of FKBP-rapamycin complex and can interact with multiple proteins to form two complexes TORC1 and TORC2. Both TORC1 and TORC2 play important roles in cell growth and metabolism, but only TORC1 was rapamycin-sensitive (13, 37). In silico analysis revealed there is only one TOR (M419DRAFT_24714) in T. reesei, with 49.27% and 49.77% sequence identity to TOR1 and TOR2 from S. cerevisiae respectively, which was coined trTOR here. The expression of gene trTOR was slightly downregulated by rapamycin (Table 1). The essential components of TORC1 (Lst8 and Kog1), and TORC2 (Avo1, Avo3, and Lst8) were identified in T. reesei, with little change at transcription levels (Table 1). The other components of the TOR complexes (Tco89, Avo2, and Bit61) were not identified in T. reesei. It seems that the effect of rapamycin on the TOR complexes was not very significant in T. reesei. The effort to delete gene trTOR was failed, indicating that trTOR is an essential gene.

Moreover, based on sequence comparisons with the homologues of genes in the TOR signal pathways (13, 38, 39), we identified a series of genes involved in TOR signal pathways in T. reesei RUT-C30, including 15 genes in ribosome biogenesis, 8 genes in cell cycle/growth, 25 genes in nutrient uptake, 1 gene in stress, 5 genes in lipid metabolism, 6 genes in cell wall integrity, and 5 genes in autophagy (Table S5). All these genes were not differentially expressed. These findings matched well with the phenotype profiling results that T. reesei displayed high resistance to rapamycin regardless of carbon sources (Fig. 1, Fig. 2, and Fig. 3).

DEGs related to gene expression

Thirty five out of the total 484 DEGs are related to the gene expression in T. reesei, showing that rapamycin has prominent effects on both the transcription and translation level of T. reesei grown on cellulose (Table S6). DEGs related to DNA metabolism were found, like DNA topoisomerase IV alpha subunit, and DNase I protein. In particular, 14 DEGs are specifically involved in regulation of transcription by RNA polymerase II that is responsible for the transcription of cellulase production (40). On the other hand, three tRNA were downregulated, including tRNA-Arg, tRNA-Glu and tRNA-Lys, while threonyl/alanyl tRNA synthetase and eukaryotic translation initiation factor 2c were upregulated. In addition, two DEGs function as ribonuclease for RNA degradation. Therefore, 100 µM rapamycin exerted an effect on both the translation and transcription of T. reesei at the early fermentation stage on cellulose.

DEGs associated with transporters

Forty seven out of the total 484 DEGs are transporters (Table S1), including 1 putative efflux pump antibiotic resistance protein, 1 xenobiotic transmembrane transporter, 3 amino acid transporters, 3 aquaporins, 8 ATPase-coupled transmembrane transporters, 10 carboxylate transporters, 11 major facilitator superfamily transporters, 3 transporters for transporting inorganic compounds (1 phosphate permease, 1 acetate transporter, and 1 ammonium permease), 4 transporters for transporting metal ions (1 metal ion transmembrane transporter, 1 siderophore transporter, 1 calcium transmembrane transporter, and 1 zinc ion transmembrane transporter), and 3 other transporters. All the four metal ion transporters were downregulated, while the three amino acid transporters were upregulated.

The cellulase production, cell growth, sporulation ability and lipid content of T. reesei grown on cellulose, lactose or glucose, were not affected by rapamycin even at the high concentration of 100 µM which was much higher than the concentrations inhibiting other organisms. For example, 0.11 µM rapamycin were enough to activate TORC1-controlled transcriptional activators Gln3p, Gat1p, Rtg1p, and Rtg3p and resulted in a fast lipid droplet replenishment in S. cerevisiae (41). Rapamycin at 1.09 µM completely abolished the growth of P. anserine (23) and at 0.27 µM induced autophagy in hyphae of F. graminearum (20). Obviously, T. reesei is highly resistant to rapamycin, which is independent on carbon sources. The resistance to rapamycin were also observed in S. pombe (42), and land plants like A. thaliana, the bryophyte Physcomitrella patens, the monocot O. sativa, and the dicots Nicotiana tabacum and Brassica napus (16), though rapamycin susceptibility is widespread among eukaryotes, inhibiting the growth of most fungi and animal cells (21, 41, 43). The reason why T. reesei is insensitive to rapamycin is still unknown.

It has been reported that mutations of FKBP12 prevent the formation of FKBP-rapamycin complex, and mutations in the FRB (FKPB12-rapamycin-binding) domain of TOR1 block the binding of FKBP-rapamycin to TOR1, both conferring rapamycin resistance. Therefore, we performed sequence alignments of rapamycin binding domains of trTOR and trFKBP12 with those of the corresponding homologs from organisms that are sensitive (S. cerevisiae and H. sapiens) or insensitive (A. thaliana, S. pombe, and O. sativa) to rapamycin (Figure S5). Most of the amino acids required for the formation of hydrophobic rapamycin-binding pocket (44) are well reserved in trFKBP12, including Tyr³¹, Phe⁴⁸, Ile⁶⁸, Trp⁷¹, Tyr⁹⁴, Ile¹⁰³ and Phe¹¹¹ (numbered according to trFKBP12) (Figure S5A). Similarly, the amino acid residue Trp²¹⁰¹, Phe²¹⁰⁸, or Ser²⁰³⁵ of mTOR, mutation of which confers rapamycin resistance (45), is also present in trTOR (Figure S5B). These findings demonstrate that the high resistance of T. reesei to rapamycin was not due to mutations of trFKBP12 and trTOR with comparison to counterparts from organisms that are susceptible to rapamycin.

Rapamycin can bind to ABC transporters with high affinity, acting as a substrate for transport by ABC transporters out of cells and causing rapamycin resistance (46). In the presence of rapamycin, 8 ABC transporters were significantly changed at mRNA level (Table S2), of which 6 related to multidrug resistance were upregulated including M419DRAFT_75459, M419DRAFT_90404, M419DRAFT_109180, M419DRAFT_116127, M419DRAFT_97054 and M419DRAFT_114328. The noticeable increase of these ABC transporters might contribute to the rapamycin resistance in T. reesei RUT-C30.

T. reesei might represent an excellent platform to study the resistance of cells to rapamycin which is commonly countered when rapamycin is explored as antifungal or anticancer agents. mTOR (mammalian Target of Rapamycin) has become a focus for cancer drug development (47). Rapamycin is a highly specific inhibitor of mTOR and potently suppresses tumor cell growth by retarding cells in G1 phase or potentially inducing apoptosis. Currently, rapamycin and its analogues are being evaluated as anticancer agents in clinical trials. However, many human cancers have displayed intrinsic resistance or acquired resistance to rapamycin (48). Though several predicted mechanisms behind the resistance of cancer cells to rapamycin have been proposed, the detailed mechanisms remain to be explored. The high rapamycin resistance of T. reesei might endow it as a model for the eukaryotic cell to unravel mechanism of rapamycin resistance.

The expression level of gene trFKBP12 was remarkably increased in the presence of high concentration rapamycin. In Fusarium fujikuroi and Botrytis cinerea, rapamycin treatment also led to an increased transcription level of FKBP12-encoding gene fpr1 (49, 50). Knockout of gene trFKBP12 caused a transient inhibition effect on cellulase production at the early fermentation stage (Fig. 5), as compared to the parent strain KU70. Furthermore, trFKBP12 knockout completely abolished the inhibition effect of high dose rapamycin on cellulase production in T. reesei, as indicated by the unchanged cellulase production in strain ΔtrFKBP12 treated with high-dose rapamycin. These findings imply that the temporary inhibition of rapamycin on cellulase requires trFKBP12, and free trFKBP12 might play a role in the initial production of cellulase. Both the deletion of trFKBP12 and the addition of rapamycin can result in a reduced level of free trFKBP12. Rapamycin can bind trFKBP12 to form the rapamycin-trFKBP12 complex, leading to the reduction of free trFKBP12. In contrast to the profound upregulation of trFKBP12, the expression of trTOR was only slightly downregulated and genes involved in the TOR signaling pathways were not changed markedly, which might be associated with the high rapamycin resistance in T. reesei. FKBP12 mediated rapamycin sensitivity via TOR in most eukaryote cells (51–53).

Despite that T. reesei exhibited high resistance to rapamycin, its cellulase production, cell growth and sporulation were inhibited by high concentration of rapamycin at the early fermentation stage, which was independent on carbon sources and was relieved at the late stage. In addition, the presence of high concentration rapamycin induced morphology defect in strain RUT-C30 at the early cultivation on cellulose. However, this morphology defect was not found in RUT-C30 grown on glucose or lactose, and strain KU70 or ΔtrFKBP12 propagated on cellulose, indicating that it is a very specific phenotype for RUT-C30 cultured on cellulose. Similar hyphal morphology alterations with the treatment of rapamycin were previously reported in other filamentous fungi like P. anserine (23) and U. maydis (54).

In this study, we have investigated the effect of rapamycin on cellulase production, filamentous morphology, cell growth, sporulation, and lipid content of T. reesei RUT-C30 grown on different carbon sources (cellulose, lactose and glucose). The high dose rapamycin (100 µM) induced a delay in cellulase production, cell growth and sporulation regardless of the carbon sources and specifically caused morphology defect in RUT-C30 grown on cellulose, both of which were transient and restored at the late fermentation stage. In contrast, the lipid content of T. reesei RUT-C30 cultivated on cellulose, lactose or glucose were not changed by rapamycin. These findings implicate that T. reesei RUT-C30 is highly resistant to rapamycin. In line with the phenotype profiling results, transcriptomic analysis found that the mRNA levels of genes associated with the cellulase production were decreased severely by rapamycin. Furthermore, the transcriptional level of the putative rapamycin receptor trFKBP12 was increased significantly, while those of gene trTOR (the downstream effector of the rapamycin-FKBP12 complex) and genes associated with the TOR signaling pathways were not changed markedly, matching well with the super rapamycin resistance of T. reesei we observed. Upon the deletion of gene trFKBP12, there is no influence of rapamycin on cellulase production, indicating the transient inhibition of high-dose rapamycin on cellulase production is assisted by trFKBP12. Overall, we first discovered that T. reesei is highly resistant to rapamycin, probably owing to that trTOR and its relevant signaling pathways were not very sensitive to rapamycin. These results deepen our understanding of the impact of rapamycin and the role of the TOR signaling pathways in T. reesei, a cellulose-producing workhouse in industry.

Microbial strains, plasmids and cultivation conditions

Escherichia coli DH5α was explored for propagation and construction of plasmids. Agrobacterium tumefaciens AGL-1 was used as a T-DNA donor for fungal transformation. T. reesei KU70, derived from T. reesei RUT-C30 by deleting gene ku70, was provided friendly by Professor Wei Wang from East China University of Science and Technology (55). Plasmid pXBthg was a gift from Professor Zhihua Zhou from Key Laboratory of Synthetic Biology, Shanghai (56). E. coli DH5α and A. tumefaciens AGL-1 were cultivated in Luria-Bertani (LB) with 220 rpm at 37 ℃ and 28 ℃, respectively. T. reesei were grown on potato dextrose agar (PDA) plates at 28 ℃ for conidia production and in Trichoderma minimal media (TMM) (57) with 2% (w/t) cellulose, lactose or glucose at 28 ℃ with 220 rpm. All chemicals used in this research were ordered from Sigma-Aldrich, USA.

Shake flask cultivation

Five percent (v/v) 10⁷ /mL conidia of T. reesei were inoculated into 10 mL (sabouraud dextrose broth) SDB and incubated at 28 ℃ with 220 rpm for 2 days. Ten percent (v/v) pre-grown mycelia were inoculated into 50 mL TMM media (pH 6) with different concentrations of rapamycin as indicated in the text using 2% cellulose, lactose, or glucose as the carbon source, and then incubated at 28 ℃ with 200 rpm for 7 days. Samples were taken at varied time points as indicated in the context for (hemi)cellulase activity assay, confocal observation of fungal mycelia, biomass measurement, Nile Red staining and RNAseq analysis. If necessary, samples were centrifuged at 8000 rpm for 30 min to separate the mycelia and the supernatant.

Analysis method

(Hemi)cellulase activities, confocal observation of fungal mycelia, DNA content measurement, sporulation assay and RNAseq analysis were performed as described in our previous research (58–60). The growth of strain T. reesei grown in TMM + 2% cellulose with and without rapamycin was assayed by DNA content measurement, while by measuring the optical density of the culture suspension at 600 nm (OD 600 nm) with a UV-vis spectrophotometer (UV-2600, Shimadzu, Japan) when cultured in TMM + 2% lactose or TMM + 2% glucose.

Lipid content analysis by Nile Red staining

For Nile Red staining, fresh mycelia collected as above were washed with PBS for two times, and incubated with Nile Red solution (25 ng/mL) at 28 ℃ for 10 min. Then, the samples were washed with PBS for two times, and checked under a confocal microscope (TCS SP8, Leica, Germany) with the excitation wavelength of 510–560 nm and the emission wavelength of 600–680 nm.

Deletion of gene trFKBP12 in T. reesei

The upstream and downstream sequences (~ 1500 bp) of gene trFKBP-12 were amplified separately by PCR using genomic DNA of T. reesei as a template, and cloned into plasmid pXBthg at XhoI and at BamHI respectively using ClonExpress™ II One Step Cloning Kit (Vazyme, China), leading to plasmid pXBthg-FKBP. The resulting plasmid pXBthg-FKBP was transformed into T. reesei KU70 by the Agrobacterium tumefaciens-mediated transformation (AMT) method (61). Transformants were selected on PDA medium with 50 µg/mL hygromycin for three rounds and were verified by PCR and sequencing at Sangon Biotech. The primers used were listed in Table S7.

TOR: Target of Rapamycin; BGL: β-glucosidase; EG: endoglucanase; CBH: cellobiohydrolase; GH: glycoside hydrolase; pNPGase: the β-glucosidase activity; pNPCase: the CBH activity; CMCase: the CMC activity; FPase: the filter paper activity; pNPXase: the β-xylosidase activity; AMT: Agrobacterium-mediated fungal transformation; SDB: sabouraud dextrose broth; TMM: Trichoderma minimal medium; PDA: potato dextrose agar.

Ethical Approval and Consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Acknowledgements

Not applicable.

Funding

This work was supported by grants from the National Natural Science Foundation of China (31700040), the National Key R&D Program of China (No. 2016YFA0501604), and the Fundamental Research Funds for the Central Universities.

Authors’ contributions

APP, ZL, and FL conceived and designed the study. APP carried out the majority of the experiments. HW, FZ and XH carried out some experiments on T. reesei cultivation, enzyme activity, and confocal imaging respectively. WW and ZZ provided strain T. reesei KU70 and plasmid pXBthg, respectively. FGW helped analyze the data. APP and FL analyzed the data and drafted the manuscript. All authors read and approved the final manuscript.

Authors’ Information

¹State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China

² Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

³ State Key Lab of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, China.

Tian CG, Beeson WT, Iavarone AT, Sun JP, Marletta MA, Cate JHD, et al. Systems analysis of plant cell wall degradation by the model filamentous fungus Neurospora crassa. Proc Natl Acad Sci USA. 2009;106(52):22157-62.
Martinez D, Berka RM, Henrissat B, Saloheimo M, Arvas M, Baker SE, et al. Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina). Nat Biotechnol. 2008;26(5):553-60.
Schmoll M. Regulation of plant cell wall degradation by light in Trichoderma. Fungal Biol Biotechnol. 2018;5(1):10.
Beier S, Hinterdobler W, Bazafkan H, Schillinger L, Schmoll M. CLR1 and CLR2 are light dependent regulators of xylanase and pectinase genes in Trichoderma reesei. Fungal Genet Biol. 2020;136:10.
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.
Chen Y, Shen Y, Wang W, Wei D. Mn²⁺ modulates the expression of cellulase genes in Trichoderma reesei Rut-C30 via calcium signaling. Biotechnol Biofuels. 2018;11(1):54.
Wang MY, Zhao QS, Yang JH, Jiang BJ, Wang FZ, Liu KM, 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(8):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. Mol microbiol. 2016;100(3):560-75.
Kim J, Guan KL. mTOR as a central hub of nutrient signalling and cell growth. Nat Cell Biol. 2019;21(1):63-71.
Lin L, Wang S, Li X, He Q, Benz JP, Tian C. STK-12 acts as a transcriptional brake to control the expression of cellulase-encoding genes in Neurospora crassa. PLoS Genet. 2019;15(11):e1008510.
Brown NA, de Gouvea PF, Krohn NG, Savoldi M, Goldman GH. Functional characterisation of the non-essential protein kinases and phosphatases regulating Aspergillus nidulans hydrolytic enzyme production. Biotechnol Biofuels. 2013;6(1):91.
Lv XX, Zhang WX, Chen GJ, Liu WF. Trichoderma reesei Sch9 and Yak1 regulate vegetative growth, conidiation, and stress response and induced cellulose production. J Microbiol. 2015;53(4):236-42.
Loewith R, Hall M. Target of Rapamycin (TOR) in nutrient signaling and growth control. Genetics. 2011;189:1177-201.
Reidman S, Cohen A, Kupiec M, Weisman R. The cytosolic form of aspartate aminotransferase is required for full activation of TOR complex 1 in fission yeast. J Biol Chem. 2019;294(48):18244-55.
Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell. 2012;149(2):274-93.
Menand B, Desnos T, Nussaume L, Berger F, Bouchez D, Meyer C, et al. Expression and disruption of the Arabidopsis TOR (target of rapamycin) gene. Proc Natl Acad Sci USA. 2002;99(9):6422-7.
Dobrenel T, Caldana C, Hanson J, Robaglia C, Vincentz M, Veit B, et al. TOR signaling and nutrient sensing. Annu Rev Plant Physiol. 2016;67:261-85.
Sehgal SN, Baker H, Vezina C. Rapamycin (AY-22,989), a new antifungal antibiotic .Ⅱ. fermentation, isolation and characterization. The Journal of Antibiotics. 1975;28(10):727-32.
Heitman J, Movva NR, Hall MN. Targets for cell-cycle arrest by the immunosuppressant rapamycin in yeast. Science. 1991;253(5022):905-9.
Yu F, Gu Q, Yun Y, Yin Y, Xu JR, Shim WB, et al. The TOR signaling pathway regulates vegetative development and virulence in Fusarium graminearum. New Phytol. 2014;203(1):219-32.
Madeira JB, Masuda CA, Maya-Monteiro CM, Matos GS, Montero-Lomeli M, Bozaquel-Morais BL. TORC1 inhibition induces lipid droplet replenishment in yeast. Mol Cell Biol. 2015;35(4):737-46.
Pinan-Lucarré B, Iraqui I, Clavé C. Podospora anserina target of rapamycin. Curr Genet. 2006;50(1):23-31.
Dementhon K, Paoletti M, Pinan-Lucarré B, Loubradou-Bourges N, Sabourin M, Saupe SJ, et al. Rapamycin mimics the incompatibility reaction in the fungus Podospora anserina. Eukaryot Cell. 2003;2(2):238-46.
Romero-Aguilar L, Guerra-Sánchez G, Tenorio EP, Tapia-Rodriguez M, Matus-Ortega G, Flores-Herrera O, et al. Rapamycin induces morphological and physiological changes without increase in lipid content in Ustilago maydis. Arch Microbiol. 2020;202(3).
Saloheimo M, Paloheimo M, Hakola S, Pere J, Swanson B, Nyyssonen E, et al. Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials. Eur J Biochem. 2002;269(17):4202-11.
Kuhad RC, Deswal D, Sharma S, Bhattacharya A, Jain KK, Kaur A, et al. Revisiting cellulase production and redefining current strategies based on major challenges. Renewable Sustainable Energy Rev. 2016;55:249-72.
Mello-de-Sousa TM, Rassinger A, Derntl C, Pocas-Fonseca MJ, Mach RL, Mach-Aigner AR. The relation between promoter chromatin status, Xyr1 and cellulase expression in Trichoderma reesei. Curr Genomics. 2016;17(2):145-52.
Häkkinen M, Valkonen MJ, Westerholm-Parvinen A, Aro N, Arvas M, Vitikainen M, et al. Screening of candidate regulators for cellulase and hemicellulase production in Trichoderma reesei and identification of a factor essential for cellulase production. Biotechnol Biofuels. 2014;7(1):14.
Coradetti ST, Craig JP, Xiong Y, Shock T, Tian C, Glass NL. Conserved and essential transcription factors for cellulase gene expression in ascomycete fungi. Proc Natl Acad Sci USA. 2012;109(19):7397-402.
Zhang W, Kou Y, Xu J, Cao Y, Zhao G, Shao J, et al. Two major facilitator superfamily sugar transporters from Trichoderma reesei and their roles in induction of cellulase biosynthesis. J Biol Chem. 2013;288(46):32861-72.
Novy V, Nielsen F, Seiboth B, Nidetzky B. The influence of feedstock characteristics on enzyme production in Trichoderma reesei: a review on productivity, gene regulation and secretion profiles. Biotechnol Biofuels. 2019;12(1).
Derntl C, Kluger B, Bueschl C, Schuhmacher R, Mach RL, Mach-Aigner AR. Transcription factor Xpp1 is a switch between primary and secondary fungal metabolism. Proc Natl Acad Sci USA. 2017;114(4):E560-E9.
Kubicek CP, Mikus M, Schuster A, Schmoll M, Seiboth B. Metabolic engineering strategies for the improvement of cellulase production by Hypocrea jecorina. Biotechnol Biofuels. 2009;2:19.
Derntl C, Rassinger A, Srebotnik E, Mach RL, Mach-Aigner AR. Xpp1 regulates the expression of xylanases, but not of cellulases in Trichoderma reesei. Biotechnol Biofuels. 2015;8(1):112.
Zhang L, Zhao X, Zhang G, Zhang J, Wang X, Zhang S, et al. Light-inducible genetic engineering and control of non-homologous end-joining in industrial eukaryotic microorganisms: LML 3.0 and OFN 1.0. Sci Rep. 2016;6:20761.
Guangtao Z, Hartl L, Schuster A, Polak S, Schmoll M, Wang T, et al. Gene targeting in a nonhomologous end joining deficient Hypocrea jecorina. J Biotechnol. 2009;139(2):146-51.
Zheng XF, Florentino D, Chen J, Crabtree GR, Schreiber SL. TOR kinase domains are required for two distinct functions, only one of which is inhibited by rapamycin. Cell. 1995;82(1):121-30.
Conrad M, Schothorst J, Kankipati HN, Van Zeebroeck G, Rubio-Texeira M, Thevelein JM. Nutrient sensing and signaling in the yeast Saccharomyces cerevisiae. FEMS Microbiol Rev. 2014;38(2):254-99.
Bracharz F, Redai V, Bach K, Qoura F, Brück T. The effects of TORC signal interference on lipogenesis in the oleaginous yeast Trichosporon oleaginosus. BMC Biotechnol. 2017;17(1):27.
Zheng F, Cao Y, Yang R, Wang L, Lv X, Zhang W, et al. Trichoderma reesei XYR1 activates cellulase gene expression via interaction with the Mediator subunit TrGAL11 to recruit RNA polymerase II. PLoS Genet. 2020;16(9).
Madeira JB, Masuda CA, Maya-Monteiro CM, Matos GS, Montero-Lomeli M, Bozaquel-Morais BL. TORC1 inhibition induces lipid droplet replenishment in yeast. Mol Cell Biol. 2015;35(4):737-46.
Weisman R, Choder M, Koltin Y. Rapamycin specifically interferes with the developmental response of fission yeast to starvation. J Bacteriol. 1997;179(20):6325-34.
Paghdal KV, Schwartz RA. Sirolimus (rapamycin): From the soil of Easter Island to a bright future. J Am Acad Dermatol. 2007;57(6):1046-50.
Cruz MC, Cavallo LM, Gorlach JM, Cox G, Perfect JR, Cardenas ME, et al. Rapamycin antifungal action is mediated via conserved complexes with FKBP12 and TOR kinase homologs in Cryptococcus neoformans. Mol Cell Biol. 1999;19(6):4101-12.
Weisman R, Choder M. The fission yeast TOR homolog, tor1⁺, is required for the response to starvation and other stresses via a conserved serine. J Biol Chem. 2001;276(10):7027-32.
Arceci RJ, Stieglitz K, Bierer BE. Immunosuppressants FK506 and rapamycin function as reversal agents of the multidrug resistance phenotype. Blood. 1992;80(6):1528-36.
Manning BD. Game of TOR - The Target of Rapamycin rules four kingdoms. New Engl J Med. 2017;377(13):1297-9.
Kurmasheva RT, Huang S, Houghton PJ. Predicted mechanisms of resistance to mTOR inhibitors. Br J Cancer. 2006;95(8):955-60.
Teichert S, Wottawa M, Schoenig B, Tudzynski B. Role of the Fusarium fujikuroi TOR kinase in nitrogen regulation and secondary metabolism. Eukaryot Cell. 2006;5(10):1807-19.
Melendez HG, Billon-Grand G, Fevre M, Mey G. Role of the Botrytis cinerea FKBP12 ortholog in pathogenic development and in sulfur regulation. Fungal Genet Biol. 2009;46(4):308-20.
Liu GY, Sabatini DM. mTOR at the nexus of nutrition, growth, ageing and disease. Nat Rev Mol Cell Biol. 2020;21(4):183-203.
Dumas SN, Lamming DW. Next generation strategies for geroprotection via mTORC1 inhibition. J Gerontol A Biol Sci Med Sci. 2020;75(1):14-23.
Brown EJ, Albers MW, Shin TB, Ichikawa K, Keith CT, Lane WS, et al. A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature. 1994;369(6483):756-8.
Romero-Aguilar L, Guerra-Sánchez G, Tenorio EP, Tapia-Rodriguez M, Matus-Ortega G, Flores-Herrera O, et al. Rapamycin induces morphological and physiological changes without increase in lipid content in Ustilago maydis. Arch Microbiol. 2020;202(5):1211-21.
Wu C, Chen Y, Huang X, Sun S, Luo J, Lu Z, et al. An efficient shortened genetic transformation strategy for filamentous fungus Trichoderma reesei. J Gen Appl Microbiol. 2020;65(6):301-7.
Ma L, Zhang J, Zou G, Wang C, Zhou Z. Improvement of cellulase activity in Trichoderma reesei by heterologous expression of a beta-glucosidase gene from Penicillium decumbens. Enzyme Microb Technol. 2011;49(4):366-71.
Minty JJ, Singer ME, Scholz SA, Bae CH, Ahn JH, Foster CE, et al. Design and characterization of synthetic fungal-bacterial consortia for direct production of isobutanol from cellulosic biomass. Proc Natl Acad Sci USA. 2013;110(36):14592-7.
Li C, Lin F, Zhou L, Qin L, Li B, Zhou Z, et al. Cellulase hyper-production by Trichoderma reesei mutant SEU-7 on lactose. Biotechnol Biofuels. 2017;10:228.
Li C, Lin F, Li Y, Wei W, Wang H, Qin L, et al. A beta-glucosidase hyper-production Trichoderma reesei mutant reveals a potential role of cel3D in cellulase production. Microb Cell Fact. 2016;15(1):151.
Li C, Lin F, Sun W, Yuan S, Zhou Z, Wu FG, et al. Constitutive hyperproduction of sorbicillinoids in Trichoderma reesei ZC121. Biotechnol Biofuels. 2018;11:291.
Zhong YH, Wang XL, Wang TH, Jiang Q. Agrobacterium-mediated transformation (AMT) of Trichoderma reesei as an efficient tool for random insertional mutagenesis. Appl Microbiol Biotechnol. 2007;73(6):1348-54.

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.

Download PDF

Journal Publication

published 26 Mar, 2021

Read the published version in Biotechnology for Biofuels and Bioproducts →

Editorial decision: Major revision
07 Feb, 2021
Review #3 received at journal
06 Feb, 2021
Reviewer #3 agreed at journal
02 Jan, 2021
Review #2 received at journal
21 Dec, 2020
Review #1 received at journal
21 Dec, 2020
Reviewer #2 agreed at journal
08 Dec, 2020
Reviewers invited by journal
04 Dec, 2020
Reviewer #1 agreed at journal
04 Dec, 2020
First submitted to journal
16 Nov, 2020
Editor assigned by journal
16 Nov, 2020
Submission checks completed at journal
16 Nov, 2020
Editor invited by journal
16 Nov, 2020

You are reading this latest preprint version

High-dose rapamycin excerts a temporary impact on T. reesei through gene trFKBP12

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Results

Discussion

Conclusion

Methods

Abbreviations

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1