Induction of cellulase production by Sr2+ in Trichoderma reesei via calcium signaling transduction
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SUBMITTER: Li N
PROVIDER: S-EPMC10992071 | biostudies-literature | 2022 Sep
REPOSITORIES: biostudies-literature
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Project description:BackgroundTrichoderma reesei is renowned for its cellulase-producing ability and is used for biofuel production from lignocellulose. In plants and fungi, cellulase production is induced by cellulose and suppressed by glucose; however, whether metformin can enhance cellulase production and mitochondrial function in T. reesei remains unclear. Metformin reduces blood glucose levels by inhibiting hepatic gluconeogenesis; therefore, it is worth investigating whether metformin transmission modulates cellulase biosynthesis in T. reesei.ResultsMetformin increased cellulase production and the transcription of cellulase-related genes. It also enhanced the concentrations of Ca2+ in the cytosol and mitochondria and regulated the transcription levels of cellulase-related genes by modulating calcium homeostasis in T. reesei QM6a. In addition, metformin was identified as an antioxidant that can enhance cellulase activity by reducing reactive oxygen species (ROS). Our results demonstrated that metformin influences the state of mitochondria by enhancing mitochondrial activity and membrane potential to promote cellulase production.ConclusionCollectively, these results indicate that metformin enhances cellulase production, scavenges ROS, and protects mitochondrial activity in T. reesei.
| S-EPMC11580550 | biostudies-literature
Project description:In this study, the effect of polyethylene glycol 8000 (PEG8000) stress on cellulase biosynthesis in Trichoderma reesei CICC2626 via calcium signaling was investigated, and a plausible mechanism by which intracellular Ca2+ regulates the transcription of cellulase genes was proposed. The results indicated that the total cellulase (filter paper-hydrolyzing activity [FPase]), endoglucanase (carboxymethyl cellulase activity [CMCase]), and β-glucosidase activities of the strain were 1.3-, 1.2-, and 1.3-fold higher than those of the control (no PEG8000 addition) at a final concentration of 1.5% (w/v) PEG8000. Moreover, the transcriptional levels of cellulase genes, protein concentrations, and biomass increased. With the synergistic use of commercial cellulase and T. reesei CICC2626 cellulase to hydrolyze alkali-pretreated rice straw, the released reducing sugar concentration reached 372.7 mg/g, and the cellulose content (22.7%, 0.32 g) was significantly lower than the initial content (62.5%, 1.88 g). Transcriptome data showed that 12 lignocellulose degradation-related genes were significantly upregulated in the presence of 1.5% PEG8000. Furthermore, the addition of Ca2+ inhibitors and deletion of crz1 (calcineurin-responsive zinc finger 1-encoding gene, which is related to the calcium signaling pathway) demonstrated that calcium signaling plays a dominant role in PEG8000-induced cellulase genes overexpression. These results revealed a link between PEG8000 induction and calcium signaling transduction in T. reesei CICC2626. Moreover, this study also provides a novel inducer for enhanced cellulase production. KEY POINTS: • Cellulase biosynthesis in Trichoderma reesei could be enhanced by PEG8000 • PEG8000 could induce a cytosolic Ca2+ burst in Trichoderma reesei • The activated calcium signaling was involved in cellulase biosynthesis.
| S-EPMC10817842 | biostudies-literature
Project description:BackgroundThe filamentous fungus Trichoderma reesei is one of the best producers of cellulase and has been widely studied for the production of cellulosic ethanol and bio-based products. We previously reported that Mn2+ and N,N-dimethylformamide (DMF) can stimulate cellulase overexpression via Ca2+ bursts and calcium signalling in T. reesei under cellulase-inducing conditions. To further understand the regulatory networks involved in cellulase overexpression in T. reesei, we characterised the Mn2+/DMF-induced calcium signalling pathway involved in the stimulation of cellulase overexpression.ResultsWe found that Mn2+/DMF stimulation significantly increased the intracellular levels of cAMP in an adenylate cyclase (ACY1)-dependent manner. Deletion of acy1 confirmed that cAMP is crucial for the Mn2+/DMF-stimulated cellulase overexpression in T. reesei. We further revealed that cAMP elevation induces a cytosolic Ca2+ burst, thereby initiating the Ca2+ signal transduction pathway in T. reesei, and that cAMP signalling causes the Ca2+ signalling pathway to regulate cellulase production in T. reesei. Furthermore, using a phospholipase C encoding gene plc-e deletion strain, we showed that the plc-e gene is vital for cellulase overexpression in response to stimulation by both Mn2+ and DMF, and that cAMP induces a Ca2+ burst through PLC-E.ConclusionsThe findings of this study reveal the presence of a signal transduction pathway in which Mn2+/DMF stimulation produces cAMP. Increase in the levels of cAMP activates the calcium signalling pathway via phospholipase C to regulate cellulase overexpression under cellulase-inducing conditions. These findings provide insights into the molecular mechanism of the cAMP-PLC-calcium signalling pathway underlying cellulase expression in T. reesei and highlight the potential applications of signal transduction in the regulation of gene expression in fungi.
| S-EPMC7941909 | biostudies-literature
Project description:BackgroundCellulase can convert lignocellulosic feedstocks into fermentable sugars, which can be used for the industrial production of biofuels and chemicals. The high cost of cellulase production remains a challenge for lignocellulose breakdown. Trichoderma reesei RUT C30 serves as a well-known industrial workhorse for cellulase production. Therefore, the enhancement of cellulase production by T. reesei RUT C30 is of great importance.ResultsTwo sets of novel minimal transcriptional activators (DBDace2-VP16 and DBDcre1-VP16) were designed and expressed in T. reesei RUT C30. Expression of DBDace2-VP16 and DBDcre1-VP16 improved cellulase production under induction (avicel or lactose) and repression (glucose) conditions, respectively. The strain TMTA66 under avicel and TMTA139 under glucose with the highest cellulase activities outperformed other transformants and the parental strain under the corresponding conditions. For TMTA66 strains, the highest FPase activity was approximately 1.3-fold greater than that of the parental strain RUT C30 at 120 h of cultivation in a shake flask using avicel as the sole carbon source. The FPase activity (U/mg biomass) in TMTA139 strains was approximately 26.5-fold higher than that of the parental strain RUT C30 at 72 h of cultivation in a shake flask using glucose as the sole carbon source. Furthermore, the crude enzymes produced in the 7-L fermenter from TMTA66 and TMTA139 supplemented with commercial β-glucosidase hydrolyzed pretreated corn stover effectively.ConclusionsThese results show that replacing natural transcription factors with minimal transcriptional activators is a powerful strategy to enhance cellulase production in T. reesei. Our current study also offers an alternative genetic engineering strategy for the enhanced production of industrial products by other fungi.
| S-EPMC5956553 | biostudies-literature
Project description:Recent research has revealed the calcium signaling significance in the production of cellulases in Trichoderma reesei. While vacuoles serve as the primary calcium storage within cells, the function of vacuolar calcium transporter proteins in this process remains unclear. In this study, we conducted a functional characterization of four vacuolar calcium transport proteins in T. reesei. This was accomplished by the construction of the four mutant strains ∆trpmc1, ∆tryvc1, ∆tryvc3, and ∆tryvc4. These mutants displayed enhanced growth when subjected to arabinose, xylitol, and xylose. Furthermore, the mutants ∆trpmc1, ∆tryvc1, and ∆tryvc4 showed a reduction in growth under conditions of 100 mM MnCl2, implying their role in manganese resistance. Our enzymatic activity assays revealed a lack of the expected augmentation in cellulolytic activity that is typically seen in the parental strain following the introduction of calcium. This was mirrored in the expression patterns of the cellulase genes. The vacuolar calcium transport genes were also found to play a role in the expression of genes involved with the biosynthesis of secondary metabolites. In summary, our research highlights the crucial role of the vacuolar calcium transporters and, therefore, of the calcium signaling in orchestrating cellulase and hemicellulase expression, sugar utilization, and stress resistance in T. reesei.
| S-EPMC11677643 | biostudies-literature
Project description:BackgroundThe filamentous fungus Trichoderma reesei Rut-C30 is one of the most vital fungi for the production of cellulases, which can be used for biofuel production from lignocellulose. Nevertheless, the mechanism of transmission of external stimuli and signals in modulating cellulase production in T. reesei Rut-C30 remains unclear. Calcium is a known second messenger regulating cellulase gene expression in T. reesei.ResultsIn this study, we found that a biologically relevant extracellular Mn2+ concentration markedly stimulates cellulase production, total protein secretion, and the intracellular Mn2+ concentration of Rut-C30, a cellulase hyper-producing strain of T. reesei. Furthermore, we identified two Mn2+ transport proteins, designated as TPHO84-1 and TPHO84-2, indicating that they are upstream in the signaling pathway that leads to cellulase upregulation. We also found that Mn2+ induced a significant increase in cytosolic Ca2+ concentration, and that this increased cytosolic Ca2+ might be a key step in the Mn2+-mediated regulation of cellulase gene transcription and production. The utilization of LaCl3 to block plasma membrane Ca2+ channels, and deletion of crz1 (calcineurin-responsive zinc finger transcription factor 1) to interrupt calcium signaling, showed that Mn2+ exerts the induction of cellulase genes via calcium channels and calcium signaling. To substantiate this, we identified a Ca2+/Mn2+ P-type ATPase, TPMR1, which could play a pivotal role in Ca2+/Mn2+ homeostasis and Mn2+ induction of cellulase genes in T. reesei Rut-C30.ConclusionsTaken together, our results revealed for the first time that Mn2+ stimulates cellulase production, and demonstrates that Mn2+ upregulates cellulase genes via calcium channels and calcium signaling. Our research also provides a direction to facilitate enhanced cellulase production by T. reesei.
| S-EPMC5831609 | biostudies-literature
Project description:BackgroundLignocellulose is the most abundant renewable resource in the world and has attracted widespread attention. It can be hydrolyzed into sugars with the help of cellulases and hemicellulases that are secreted by filamentous fungi. Several studies have revealed that the Ras small GTPase superfamily regulates important cellular physiological processes, including synthesis of metabolites, sporulation, and cell growth and differentiation. However, it remains unknown how and to what extent Ras small GTPases participate in cellulase production.ResultsIn this study, we found that the putative Ras small GTPase RSR1 negatively regulated the expression of cellulases and xylanases. Deletion of rsr1 (∆rsr1) significantly increased cellulase production and decreased the expression levels of ACY1-cAMP-protein kinase A (PKA) signaling pathway genes and the concentration of intracellular cyclic adenosine monophosphate (cAMP). Loss of acy1 based on ∆rsr1 (∆rsr1∆acy1) could further increase cellulase production and the expression levels of cellulase genes, while overexpression of acy1 based on ∆rsr1 (∆rsr1-OEacy1) significantly reduced cellulase production and transcriptional levels of cellulase genes. In addition, our results revealed that RSR1 negatively controlled cellulase production via the ACY1-cAMP-PKA pathway. Transcriptome analysis revealed significantly increased expression of three G-protein coupled receptors (GPCRs; tre62462, tre58767, and tre53238) and approximately two-fold higher expression of ACE3 and XYR1, which transcriptionally activated cellulases with the loss of rsr1. ∆rsr1∆ tre62462 exhibited a decrease in cellulase activity compared to ∆rsr1, while that of ∆rsr1∆tre58767 and ∆rsr1∆tre53238 showed a remarkable improvement compared to ∆rsr1. These findings revealed that GPCRs on the membrane may sense extracellular signals and transmit them to rsr1 and then to ACY1-cAMP-PKA, thereby negatively controlling the expression of the cellulase activators ACE3 and XYR1. These data indicate the crucial role of Ras small GTPases in regulating cellulase gene expression.ConclusionsHere, we demonstrate that some GPCRs and Ras small GTPases play key roles in the regulation of cellulase genes in Trichoderma reesei. Understanding the roles of these components in the regulation of cellulase gene transcription and the signaling processes in T. reesei can lay the groundwork for understanding and transforming other filamentous fungi.
| S-EPMC10204303 | biostudies-literature
Project description:BACKGROUND:The induction of cellulase production by insoluble carbon source cellulose was a common and efficient strategy, but has some drawbacks, such as difficult fermentation operation, substantial cellulase loss, long fermentation time, and high energy-consumption, resulting in high cost of cellulase production in industry. These drawbacks can be overcome if soluble carbon sources are utilized as the inducers for cellulase production. However, until now the induction efficiency of most soluble carbon sources, especially lactose and glucose, is still inferior to cellulose despite extensive efforts have been made by either optimizing the fermentation process or constructing the recombinant strains. Therefore, strain improvement by metabolic engineering for high induction efficiency of soluble carbon sources is of great interest. RESULTS:Trichoderma reesei mutant SEU-7 was constructed from T. reesei RUT-C30 with the overexpression of endogenous gene β-glucosidase (BGL1) by insertional mutagenesis via Agrobacterium tumefaciens-mediated transformation (AMT). Compared to RUT-C30, SEU-7 displays substantially enhanced activities of both cellulase and hemicellulase when grown on either lactose or cellulose. The induction efficiency with lactose was found to be higher than cellulose in strain SEU-7. To the best of our knowledge, we achieved the highest FPase activity in SEU-7 in both batch culture (13.0 IU/mL) and fed-batch culture (47.0 IU/mL) on lactose. Moreover, SEU-7 displayed unrivaled pNPGase activity on lactose in both batch culture (81.0 IU/mL) and fed-batch culture (144.0 IU/mL) as compared to the other reported T. reesei strains in the literature grown in batch or fed-batch experiments on cellulose or lactose. This superiority of SEU-7 over RUT-C30 improves markedly the saccharification ability of SEU-7 on pretreated corn stover. The overexpression of gene BGL1 was found either at the mRNA or at the protein level in the mutant strains with increased cellulase production in comparison with RUT-C30, but only SEU-7 displayed much higher expression of gene BGL1 on lactose than on cellulose. Two copies of gene BGL1 were inserted into the chromosome of T. reesei SEU-7 between KI911141.1:347357 and KI911141.1:347979, replacing the original 623-bp fragment that is not within any genes' coding region. The qRT-PCR analysis revealed that the mRNA levels of both cellulase and hemicellulase were upregulated significantly in SEU-7, together with the MFS transporter CRT1 and the XYR1 nuclear importer KAP8. CONCLUSIONS:Recombinant T. reesei SEU-7 displays hyper-production of both cellulase and hemicellulase on lactose with the highest FPase activity and pNPGase activity for T. reesei, enabling highly efficient saccharification of pretreated biomass. For the first time, the induction efficiency for cellulase production by lactose in T. reesei was reported to be higher than that by cellulose. This outperformance of T. reesei SEU-7, which is strain-specific, is attributed to both the overexpression of gene BGL and the collateral mutation. Moreover, the increased transcription levels of cellulase genes, the related transcription factors, and the MFS transporter CRT1 contribute to the outstanding cellulase production of SEU-7. Our research advances strain improvement to enhance the induction efficiency of soluble carbon sources to produce cost-effective cellulase and hemicellulase in industry.
| S-EPMC5628480 | biostudies-literature
Project description:BackgroundThe filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) displays increased cellulase expression while growing on inducers such as lactose or cellulose. However, the mechanism of cellulase induction in T. reesei is not yet completely characterized. Here, a protein annotated as β-glucosidase (BGL3I) was found to be involved in cellulase induction in T. reesei. The effects of BGL3I on cellulase production have not yet been fully understood.ResultsDeletion of the bgl3i gene had no influence on the growth of T. reesei, but significantly increased its cellulase activities. Deletion of bgl3i also resulted in decreased extracellular galactosidase activity, but significantly increased transcription of lactose permeases, which might be involved in lactose transport. Furthermore, deletion of bgl3i enhanced the transcription levels of intracellular β-glucosidases cel1a, cel1b and the regulator xyr1, which are all essential for lactose induction in T. reesei. BGL3I was found to have a relatively high ability to hydrolyze sophorose, which is proposed to be the strongest natural inducer of cellulase synthesis in T. reesei.ConclusionsBGL3I may take part in the complex regulating system of cellulase induction. The deletion of bgl3i offers a new strategy to improve T. reesei strain performance.
| S-EPMC6240962 | biostudies-literature
Project description:BackgroundInduction of cellulase in cellulolytic fungi Trichoderma reesei is strongly activated by cellulosic carbon sources. The transport of cellulosic inducer and the perception of inducing signal is generally considered as the critical process for cellulase induction, that the inducing signal would be perceived by a sugar transporter/transceptor in T. reesei. Several sugar transporters are coexpressed during the induction stage, but which function they serve and how they work collaboratively are still difficult to elucidate.ResultsIn this study, we found that the constitutive expression of the cellulose response transporter-like protein CRT2 (previously identified as putative lactose permease TRE77517) improves cellulase induction on a cellulose, cellobiose or lactose medium. Functional studies indicate that the membrane-bound CRT2 is not a transporter of cellobiose, lactose or glucose in a yeast system, and it also does not affect cellobiose and lactose utilization in T. reesei. Further study reveals that CRT2 has a slightly similar function to the cellobiose transporter CRT1 in cellulase induction. Overexpression of CRT2 led to upregulation of CRT1 and the key transcription factor XYR1. Moreover, overexpression of CRT2 could partially compensate for the function loss of CRT1 on cellulase induction.ConclusionsOur study uncovers the novel function of CRT2 in cellulase induction collaborated with CRT1 and XYR1, possibly as a signal transductor. These results deepen the understanding of the influence of sugar transporters in cellulase production.
| S-EPMC10364367 | biostudies-literature