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In Trichoderma reesei, carbon catabolite repression (CCR) significantly downregulates the transcription of cellulolytic enzymes, which is usually mediated by the zinc finger protein Cre1. It was found that there is a conserved region at the C-terminus of Cre1/CreA in several cellulase-producing fungi that contains up to three continuous S/T phosphorylation sites. Here, S387, S388, T389, and T390 at the C-terminus of Cre1 in T. reesei were mutated to valine for mimicking an unphosphorylated state, thereby generating the transformants Tr_Cre1S387V, Tr_Cre1S388V, Tr_Cre1T389V, and Tr_Cre1T390V, respectively. Transcription of cel7a in Tr_ Cre1S388V was markedly higher than that of the parent strain when grown in glucose-containing media. Under these conditions, both filter paperase (FPase) and p-nitrophenyl-β-D-cellobioside (pNPCase) activities, as well as soluble proteins from Tr_Cre1S388V were significantly increased by up to 2- to 3-fold compared with that of other transformants and the parent strain. The results suggested that S388 is critical site of phosphorylation for triggering CCR at the terminus of Cre1. To our knowledge, this is the first report demonstrating an improvement of cellulase production in T. reesei under CCR by mimicking dephosphorylation at the C-terminus of Cre1. Taken together, we developed a precision engineering strategy based on the modification of phosphorylation sites of Cre1 transcription factor to enhance the production of cellulase in T. reesei under CCR., Graphical Abstract Schematic diagram on transcriptional regulation of cellulase expression in mutant and parent strains. We developed a precision engineering strategy by mimicking dephosphorylation at a key residue S388 of Cre1 to improve the production of cellulase in fungal species under CCR, highlighting that this technology can accelerate the industrial process of lignocellulosic biorefinery.

Carbon catabolite repression (CCR), which is mainly mediated by Cre1 and triggered by glucose, leads to a decrease in cellulase production in Trichoderma reesei. Many studies have focused on modifying Cre1 for alleviating CCR. Based on the homologous alignment of CreA from wild-type Penicillium oxalicum 114–2 (Po-0) and cellulase hyperproducer JUA10-1(Po-1), we constructed a C-terminus substitution strain—Po-2—with decreased transcriptional levels of cellulase and enhanced CCR. Results revealed that the C-terminal domain of CreAPo−1 plays an important role in alleviating CCR. Furthermore, we replaced the C-terminus of Cre1 with that of CreAPo−1 in T. reesei (Tr-0) and generated Tr-1. As a control, the C-terminus of Cre1 was truncated and Tr-2 was generated. The transcriptional profiles of these transformants revealed that the C-terminal chimera greatly improves cellulase transcription in the presence of glucose and thus upregulates cellulase in the presence of glucose and weakens CCR, consistent with truncating the C-terminus of Cre1 in Tr-0. Therefore, we propose constructing a C-terminal chimera as a new strategy to improve cellulase production and alleviate CCR in the presence of glucose., Graphical abstract Image 1

Background Biofuels derived from lignocellulosic biomass are a viable alternative to fossil fuels required for transportation. Following plant biomass pretreatment, the furan derivative furfural is present at concentrations which are inhibitory to yeasts. Detoxification of furfural is thus important for efficient fermentation. Here, we searched for new genetic attributes in the fungus Neurospora crassa that may be linked to furfural tolerance. The fact that furfural is involved in the natural process of sexual spore germination of N. crassa and that this fungus is highly amenable to genetic manipulations makes it a rational candidate for this study. Results Both hypothesis-based and unbiased (random promotor mutagenesis) approaches were performed to identify N. crassa genes associated with the response to furfural. Changes in the transcriptional profile following exposure to furfural revealed that the affected processes were, overall, similar to those observed in Saccharomyces cerevisiae. N. crassa was more tolerant (by ~ 30%) to furfural when carboxymethyl cellulose was the main carbon source as opposed to sucrose, indicative of a link between carbohydrate metabolism and furfural tolerance. We also observed increased tolerance in a Δcre-1 mutant (CRE-1 is a key transcription factor that regulates the ability of fungi to utilize non-preferred carbon sources). In addition, analysis of aldehyde dehydrogenase mutants showed that ahd-2 (NCU00378) was involved in tolerance to furfural as well as the predicted membrane transporter NCU05580 (flr-1), a homolog of FLR1 in S. cerevisiae. Further to the rational screening, an unbiased approach revealed additional genes whose inactivation conferred increased tolerance to furfural: (i) NCU02488, which affected the abundance of the non-anchored cell wall protein NCW-1 (NCU05137), and (ii) the zinc finger protein NCU01407. Conclusions We identified attributes in N. crassa associated with tolerance or degradation of furfural, using complementary research approaches. The manipulation of the genes involved in furan sensitivity can provide a means for improving the production of biofuel producing strains. Similar research approaches can be utilized in N. crassa and other filamentous fungi to identify additional attributes relevant to other furans or toxic chemicals.

Background Biofuels derived from lignocellulosic biomass are a viable alternative to fossil fuels required for transportation. Following plant biomass pretreatment, the furan derivative furfural is present at concentrations which are inhibitory to yeasts. Detoxification of furfural is thus important for efficient fermentation. Here, we searched for new genetic attributes in the fungus Neurospora crassa that may be linked to furfural tolerance. The fact that furfural is involved in the natural process of sexual spore germination of N. crassa and that this fungus is highly amenable to genetic manipulations makes it a rational candidate for this study. Results Both hypothesis-based and unbiased (random promotor mutagenesis) approaches were performed to identify N. crassa genes associated with the response to furfural. Changes in the transcriptional profile following exposure to furfural revealed that the affected processes were, overall, similar to those observed in Saccharomyces cerevisiae. N. crassa was more tolerant (by ~ 30%) to furfural when carboxymethyl cellulose was the main carbon source as opposed to sucrose, indicative of a link between carbohydrate metabolism and furfural tolerance. We also observed increased tolerance in a Δcre-1 mutant (CRE-1 is a key transcription factor that regulates the ability of fungi to utilize non-preferred carbon sources). In addition, analysis of aldehyde dehydrogenase mutants showed that ahd-2 (NCU00378) was involved in tolerance to furfural as well as the predicted membrane transporter NCU05580 (flr-1), a homolog of FLR1 in S. cerevisiae. Further to the rational screening, an unbiased approach revealed additional genes whose inactivation conferred increased tolerance to furfural: (i) NCU02488, which affected the abundance of the non-anchored cell wall protein NCW-1 (NCU05137), and (ii) the zinc finger protein NCU01407. Conclusions We identified attributes in N. crassa associated with tolerance or degradation of furfural, using complementary research approaches. The manipulation of the genes involved in furan sensitivity can provide a means for improving the production of biofuel producing strains. Similar research approaches can be utilized in N. crassa and other filamentous fungi to identify additional attributes relevant to other furans or toxic chemicals.

Background Cellulolytic enzymes produced by the filamentous fungus Trichoderma reesei are commonly used in biomass conversion. The high cost of cellulase is still a significant challenge to commercial biofuel production. Improving cellulase production in T. reesei for application in the cellulosic biorefinery setting is an urgent priority. Results Trichoderma reesei hyper-cellulolytic mutant SS-II derived from the T. reesei NG14 strain exhibited faster growth rate and more efficient lignocellulosic biomass degradation than those of RUT-C30, another hyper-cellulolytic strain derived from NG14. To identify any genetic changes that occurred in SS-II, we sequenced its genome using Illumina MiSeq. In total, 184 single nucleotide polymorphisms and 40 insertions and deletions were identified. SS-II sequencing revealed 107 novel mutations and a full-length wild-type carbon catabolite repressor 1 gene (cre1). To combine the mutations of RUT-C30 and SS-II, the sequence of one confirmed beneficial mutation in RUT-C30, cre196, was introduced in SS-II to replace full-length cre1, forming the mutant SS-II-cre196. The total cellulase production of SS-II-cre196 was decreased owing to the limited growth of SS-II-cre196. In contrast, 57 genes mutated only in SS-II were selected and knocked out in RUT-C30. Of these, 31 were involved in T. reesei growth or cellulase production. Cellulase activity was significantly increased in five deletion strains compared with that in two starter strains, RUT-C30 and SS-II. Cellulase production of T. reesei Δ108642 and Δ56839 was significantly increased by 83.7% and 70.1%, respectively, compared with that of RUT-C30. The amount of glucose released from pretreated corn stover hydrolyzed by the crude enzyme from Δ108642 increased by 11.9%. Conclusions The positive attribute confirmed in one cellulase hyper-producing strain does not always work efficiently in another cellulase hyper-producing strain, owing to the differences in genetic background. Genome re-sequencing revealed novel mutations that might affect cellulase production and other pathways indirectly related to cellulase formation. Our strategy of combining the mutations of two strains successfully identified a number of interesting phenotypes associated with cellulase production. These findings will contribute to the creation of a gene library that can be used to investigate the involvement of various genes in the regulation of cellulase production. Electronic supplementary material The online version of this article (10.1186/s12934-019-1131-z) contains supplementary material, which is available to authorized users.

Carbon catabolite repression (CCR) mediated by CRE1 in Trichoderma reesei emerged as a mechanism by which the fungus could adapt to new environments. In the presence of readily available carbon sources such as glucose, the fungus activates this mechanism and inhibits the production of cellulolytic complex enzymes to avoid unnecessary energy expenditure. CCR has been well described for the growth of T. reesei in cellulose and glucose, however, little is known about this process when the carbon source is sophorose, one of the most potent inducers of cellulase production. Thus, we performed high-throughput RNA sequencing to better understand CCR during cellulase formation in the presence of sophorose, by comparing the mutant ∆cre1 with its parental strain, QM9414. Of the 9129 genes present in the genome of T. reesei, 184 were upregulated and 344 downregulated in the mutant strain ∆cre1 compared to QM9414. Genes belonging to the CAZy database, and those encoding transcription factors and transporters are among the gene classes that were repressed by CRE1 in the presence of sophorose; most were possible indirectly regulated by CRE1. We also observed that CRE1 activity is carbon-dependent. A recent study from our group showed that in cellulose, CRE1 repress different groups of genes when compared to sophorose. CCR differences between these carbon sources may be due to the release of cellodextrins in the cellulose polymer, resulting in different targets of CRE1 in both carbon sources. These results contribute to a better understanding of CRE1-mediated CCR in T. reesei when glucose comes from a potent inducer of cellulase production such as sophorose, which could prove useful in improving cellulase production by the biotechnology sector.

Background The enzymes for efficient hydrolysis of lignocellulosic biomass are a major factor in the development of an economically feasible cellulose bioconversion process. Up to now, low hydrolysis efficiency and high production cost of cellulases remain the significant hurdles in this process. The aim of the present study was to develop a versatile cellulase system with the enhanced hydrolytic efficiency and the ability to synthesize powerful inducers by genetically engineering Trichoderma reesei. Results In our study, we employed a systematic genetic strategy to construct the carbon catabolite-derepressed strain T. reesei SCB18 to produce the cellulase complex that exhibited a strong cellulolytic capacity for biomass saccharification and an extraordinary high β-glucosidase (BGL) activity for cellulase-inducing disaccharides synthesis. We first identified the hypercellulolytic and uracil auxotrophic strain T. reesei SP4 as carbon catabolite repressed, and then deleted the carbon catabolite repressor gene cre1 in the genome. We found that the deletion of cre1 with the selectable marker pyrG led to a 72.6% increase in total cellulase activity, but a slight reduction in saccharification efficiency. To facilitate the following genetic modification, the marker pyrG was successfully removed by homologous recombination based on resistance to 5-FOA. Furthermore, the Aspergillus niger BGLA-encoding gene bglA was overexpressed, and the generated strain T. reesei SCB18 exhibited a 29.8% increase in total cellulase activity and a 51.3-fold enhancement in BGL activity (up to 103.9 IU/mL). We observed that the cellulase system of SCB18 showed significantly higher saccharification efficiency toward differently pretreated corncob residues than the control strains SDC11 and SP4. Moreover, the crude enzyme preparation from SCB18 with high BGL activity possessed strong transglycosylation ability to synthesize β-disaccharides from glucose. The transglycosylation product was finally utilized as the inducer for cellulase production, which provided a 63.0% increase in total cellulase activity compared to the frequently used soluble inducer, lactose. Conclusions In summary, we constructed a versatile cellulase system in T. reesei for efficient biomass saccharification and powerful cellulase inducer synthesis by combinational genetic manipulation of three distinct types of genes to achieve the customized cellulase production, thus providing a viable strategy for further strain improvement to reduce the cost of biomass-based biofuel production. Electronic supplementary material The online version of this article (10.1186/s13068-017-0963-1) contains supplementary material, which is available to authorized users.

Research on regulation of cellulases and hemicellulases gene expression may be very useful for increasing the production of these enzymes in their native producers. Mechanisms of gene regulation of cellulase and hemicellulase expression in filamentous fungi have been studied, mainly in Aspergillus and Trichoderma. The production of these extracellular enzymes is an energy-consuming process, so the enzymes are produced only under conditions in which the fungus needs to use plant polymers as an energy and carbon source. Moreover, production of many of these enzymes is coordinately regulated, and induced in the presence of the substrate polymers. In addition to induction by mono- and oligo-saccharides, genes encoding hydrolytic enzymes involved in plant cell wall deconstruction in filamentous fungi can be repressed during growth in the presence of easily metabolizable carbon sources, such as glucose. Carbon catabolite repression is an important mechanism to repress the production of plant cell wall degrading enzymes during growth on preferred carbon sources. This manuscript reviews the recent advancements in elucidation of molecular mechanisms responsible for regulation of expression of cellulase and hemicellulase genes in fungi.

Legume rhizobium symbiosis is initiated upon perception of bacterial secreted lipo-chitooligosaccharides (LCOs). Perception of these signals by the plant initiates a signaling cascade that leads to nodule formation. Several studies have implicated a function for cytokinin in this process. However, whether cytokinin accumulation and subsequent signaling are an integral part of rhizobium LCO signaling remains elusive. Here, we show that cytokinin signaling is required for the majority of transcriptional changes induced by rhizobium LCOs. In addition, we demonstrate that several cytokinins accumulate in the root susceptible zone 3 h after rhizobium LCO application, including the biologically most active cytokinins, trans-zeatin and isopentenyl adenine. These responses are dependent on calcium- and calmodulin-dependent protein kinase (CCaMK), a key protein in rhizobial LCO-induced signaling. Analysis of the ethylene-insensitive Mtein2/Mtsickle mutant showed that LCO-induced cytokinin accumulation is negatively regulated by ethylene. Together with transcriptional induction of ethylene biosynthesis genes, it suggests a feedback loop negatively regulating LCO signaling and subsequent cytokinin accumulation. We argue that cytokinin accumulation is a key step in the pathway leading to nodule organogenesis and that this is tightly controlled by feedback loops.

Nucleosome positioning within the promoter and coding regions of the cellobiohydrolase-encoding cbh1 gene of Trichoderma reesei was investigated. T. reesei is a filamentous fungus that is able to degrade dead plant biomass by secreting enzymes such as cellulases, a feature which is exploited in industrial applications. In the presence of different carbon sources, regulation of one of these cellulase-encoding genes, cbh1, is mediated by various transcription factors including CRE1. Deletion or mutation of cre1 caused an increase in cbh1 transcript levels under repressing conditions. CRE1 was shown to bind to several consensus recognition sequences in the cbh1 promoter region in vitro. Under repressing conditions (glucose), the cbh1 promoter and coding regions are occupied by several positioned nucleosomes. Transcription of cbh1 in the presence of the inducer sophorose resulted in a loss of nucleosomes from the coding region and in the re-positioning of the promoter nucleosomes which prevents CRE1 from binding to its recognition sites within the promoter region. Strains expressing a non-functional CRE1 (in strains with mutated CRE1 or cre1-deletion) exhibited a loss of positioned nucleosomes within the cbh1 coding region under repressing conditions only. This indicates that CRE1 is important for correct nucleosome positioning within the cbh1 coding region under repressing conditions.

We isolated the putative glucose repressor gene cre1 from the phytopathogenic fungus Sclerotinia sclerotiorum. cre1 encodes a 429 amino acid protein 59% similar to the carbon catabolite repressor CREA from Aspergillus nidulans. In addition to the overall amino acid sequence relatedness between CRE1 and CREA proteins, cre1 can functionally complement the A. nidulans creAd30 mutation as assessed by repression of the alcohol dehydrogenase I gene expression. The CRE1 region carrying the two zinc fingers is also very similar to the DNA binding domains of the Saccharomyces cerevisiae glucose repressors Mig1p and Mig2p. Despite the presence in the CRE1 protein of several motifs involved in the regulation of Mig1p activity, cre1 cannot complement mig deficiencies in S. cerevisiae. These data suggest that glucose repression pathways may have evolved differently in yeasts and filamentous fungi.