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Carbon nutrient sensing and responses in lignocellulose degradation
The lignocellulose degrading process is mainly mediated by extracellular enzymes involved in the cleavage of polysaccharides. Nutrient sensing pathways, especially pathways enabling the use of preferred carbon sources are very important for the survival strategy used of the fungi. In filamentous fungi, those pathways function as the upstream of the direct activation of genes encoding CAZymes (Fig. 5). This activation results in the inhibition of the energy-consuming production of lignocellulose degrading enzymes (Huberman et al., 2016). Up to date, the best-studied pathway is carbon catabolite repression (CCR).
The utilization of a diverse array of carbon sources derived from lignocellulose degradation requires the coordination of the cellular metabolism and the preferential consumption of glucose prior to other carbon sources, a phenomenon known as carbon catabolite repression (CCR). CCR mechanism represses transcription of secreted and intracellular metabolic enzymes and has been described in both yeasts and filamentous fungi (Brown et al., 2014). The transcription of these genes is tightly regulated by a Cys2-His2 type DNA-binding zinc finger factor, named Mig1 in S. cerevisiae, and CreA/Cre1 in filamentous fungi (Fig. 5, 6).
In S. cerevisiae, the repressor protein ScMig1 is imported into the nucleus when glucose is added to derepressed cells (Fig. 6A). In the nucleus, ScMig1 binds to upstream regulatory elements (URE) of genes coding for transcription factors. This leads to inhibit the expression of those transcription factors which transcriptionally regulate alternative carbon usage. In addition to ScMig1, CCR is also regulated by nuclear chromatin structure which can be linked to intrinsic cellular program and environmental factors (Brosch et al., 2008). In yeast, ScMig1 reacts with the corepressors ScTup1 and ScSsn6 (Fig. 6A). The obtained complex binds to promoters of alternative carbon usage genes leading to their repression through the modulation of nucleosome positioning (Li et al., 2007a; Treitel and Carlson, 1995).
In filamentous fungi, the carbon nutrient-dependent nuclear localization of CreA/Cre1 (Fig. 6B) is similar to ScMig1 in S. cerevisiae. However, this repressor is not only regulated by glucose but also by other sugars resulting from carbon nutrient sources (Brown et al., 2013; Sun and Glass, 2011). The role of CreA nuclear localization in regulation of CCR has been confirmed in A. nidulans, and N. crassa by use of GFP fusions (Brown et al., 2013; Sun and Glass, 2011).
Orthologues of the S. cerevisiae corepressors ScTup1 and ScSsn6 have also been widely identified in filamentous fungi (García et al., 2008). In A. nidulans, the homolog of ScTup1 is AnRcoA (Fig. 6B). This corepressor is dispensable for CCR and affects the repressed chromatin structures in the alcR promoter during repression (García et al., 2008). alcR is a trans-acting gene containing a specific site allowing the binding of the repressor CreA (Kulmburg et al., 1993). In comparison with S. cerevisiae, the regulatory role of the CreA/Cre1 complex to CCR is less well-known in detail in filamentous fungi. The role of CreA in nucleosome positioning has been only shown in T. reesei. In particular, TrCreA is essential for correct nucleosome positioning in the cellulase promoter cbh2 under repressing and inducing conditions (Zeilinger et al., 2003).
Figure 6. The molecular mechanism of carbon catabolite repression in S. cerevisiae (A), and filamentous fungi (B).
In presence of glucose, S. cerevisiae and filamentous fungi repress the expression of alternative carbon utilization genes via binding of a repressor protein to upstream regulatory elements (URE), which inhibit the expression of the transcription factors (TF) and metabolic enzymes necessary for alternative carbon utilization. Besides, chromatin structure hampers transcription. In the condition of limited carbon nutrient, Snf1/SnfA mediates phosphorylation and relocalisation of the repressor protein leading to depression, while local modifications to chromatin structure enable transcription. (Grey Snf1/SnfA complex = unactive; yellow star = activate Snf1/SnfA complex; P = phosphorylation; orange circle = nucleosome). From Brown et al (Brown et al., 2014).
In addition to CreA/Cre1 considered as a crucial regulator in CCR, the protein kinase Sucrose non-fermenting complex (Snf1/SnfA) (Fig. 6A, B) and the protein kinase A (PKA) play also a regulatory role for CCR and for the expression of lignocellulolytic enzymes (Fig. 5). In S. cerevisiae, ScSnf1 phosphorylates the repressor ScMig1 under carbon limitation and promotes the expression of glucose-repressed genes (Fig. 6A) (García‐Salcedo et al., 2014). ScSnf1, therefore, releases the CCR in glucose depleted conditions. When present at high levels, glucose is imported into the cell and the intracellular production of phosphorylated glucose lead to the inactivation of ScSnf1, which in turn inhibits ScMig1 phosphorylation. As a result, ScMig1 is localized in the nucleus (Fig. 6A). In filamentous fungi as A. nidulans, a similar function for SnfA (Fig. 6B) to phosphorylate CreA was confirmed (Brown et al., 2013; Ries et al., 2016).
Besides the cAMP/PKA pathway, the TOR (Target of Rapamycin) pathway has also been proposed possibly having an important role in regulating the expression of cell wall degrading enzymes in response to lignocellulosic material (Brown et al., 2013; Xiong et al., 2014). The TOR signaling pathway is a highly conserved nutrient sensing pathway in eukaryotes, and is found to work in parallel with the cAMP/PKA pathway to regulate common targets relating to growth in S. cerevisiae (Zurita-Martinez and Cardenas, 2005). The roles of TOR signaling in filamentous fungi will be separately discussed in detail in one following part.
In summary, there are several carbon sensing pathways having a role in regulation of growth of filamentous fungi in response to the lignocellulosic material. Among those, CCR and cAMP/PKA appear to be the major pathways. However, the regulatory role of other nutrient sensing pathways, for instance, the TOR pathway, remains to be unraveled and may provide novel solutions to enhance the extracellular enzyme productivity.
Fungi are able to use many nitrogen sources such as amino acids, ammonium ions, and other sources such as nucleotidic bases (Bahn et al., 2007; Dijck et al., 2017). Sensing and uptake of these nutrients are essential for the synthesis of many cellular components. However, the number of described nitrogen sensing receptors is smaller than carbon sensing receptors. To date, there are no non-transporting receptors for ammonium sensing described in yeasts. That observation is also true for amino acid sensing with the absence of described transceptors that mediate amino acid sensing in filamentous fungi. For ammonium sensing, transceptors have been found only in the ectomycorrhizal fungus Hebeloma cylindrosporum (Javelle et al., 2003) and in the phytopathogenic fungi Fusarium fujikuroi and Colletotrichum gloeosporioides (Shnaiderman et al., 2013; Teichert et al., 2008).
Non-transporting receptor Ssy1 for amino acid sensing in S. cerevisiae
In S. cerevisiae, extracellular amino acids are detected by a multiprotein complex composed of ScSsy1-ScPtr3-ScSsy5 (SPS). This detection induces the transcriptional expression of amino acid transporters genes and other genes encoding nitrogen-metabolizing enzymes (Kodama et al., 2002). ScSsy1 is an amino acid permease (AAP) and functions as a receptor in the SPS complex (Klasson et al., 1999). ScPtr3 is a linker between ScSsy1 and ScSsy5. ScSsy5 contains an inhibitory domain that dissociates in response to extracellular amino acids, freeing a catalytic domain to activate the transcription factor ScStp1p (Pfirrmann et al., 2010).
The receptor induction by binding of amino acids to ScSsy1 results in activating the endoproteolytic cleavage of transcription factors Stp1/2 in a process called receptor-activated proteolysis (RAP) in which Stp1/2 functions as latent cytoplasmic precursors (Pfirrmann et al., 2010). Stp1/2, in turn, induces the expression of genes encoding amino acid–metabolizing enzymes and amino acid permeases. In S. cerevisiae, Stp1 is regulated by the TOR signaling pathway (Fig. 4). Indeed, in presence of amino acids, activated Stp1 localizes in nucleus and induces expression of its target genes. In contrast, in absence of amino acids or in presence of rapamycin at a concentration at which TOR kinase activity is inhibited (TOR pathway will be discussed later), Stp1 is degraded in a manner dependent on the activity of a protein phosphatase 2A (PP2A)-like phosphatase, and Sit4, which have been known as effectors of TOR. This degradation results in the disappearance of Sit4 from the nucleus (Shin et al., 2009).
Transceptors in S. cerevisiae
In the model yeast S. cerevisiae, all identified transceptors belong to the AAP family. The best-studied receptor for amino acid sensing is Gap1, which transports all L-amino acids, several D-amino acids, and polyamines (Uemura et al., 2005). The role as an amino acid receptor of Gap1 was found when studying the amino acid induced rapid activation of the PKA pathway, which has been known to be activated in a cAMP dependent reaction (Fig. 4). In this pathway, Gap1 is involved in the downstream relay of the signal according to an unknown mechanism (Donaton et al., 2003).
In S. cerevisiae, three genes encoding for ammonium transporters have been identified, but only one is a transceptor named ScMep2. ScMep2 acts as a regulator of pseudohyphal growth and PKA activation when ammonium is added to nitrogen-starved cells (Boeckstaens et al., 2007). ScMep2 in controlled by the ScNpr1, a kinase that is the target of ScTORC1 (Fig. 4). Under nitrogen starvation or ScTOR inactivation, ScNpr1 is activated by dephosphorylation. In presence of sufficient ammonium, ScNpr1 is phosphorylated and inactive (Boeckstaens et al., 2007).
Table of contents :
I. Fungal adaptation to environment
I.1 Fungi have to adapt to their environment
I.2 Fungal sensing of the environment
I.2.1 Nutrient sensing
Carbon nutrient sensing and responses in lignocellulose degradation
Target Of Rapamycin (TOR) signaling as a key regulator of growth in the adaptation of filamentous fungi
I.2.2 Stress sensing and responses for adaptation to environment
II. Wood and wood degrading fungi
II.1 Wood components
II.1.1 Structural compositions
II.I.2 Wood extractives
II.I.2.1 Phenolic compounds
II.1. 2.1.5 Quinones
II.2 Wood decaying fungi
II.2.1 Brown rot fungi
II.2.2 White rot fungi
II.2. 3 General biological characteristics of Phanerochaete chrysosporium
II. 3 Antifungal mechanisms of wood extractives and fungal adaptation
Discussion and conclusions (Article I & II)
Mutagenesis and screening mutant resistant to CTWE
Phenotypes of chy mutants
Identification of the causal mutation(s) leading to CTWE resistance
Involvement of AGR57_10098 in resistance against CTWE
DISCUSSION AND PERSPECTIVES
Wood extractives as tools to characterize wood decaying fungi
The possible relationships between identified proteins PcTOR and PcDUF1630 protein and secretion
The possible relationships between PcTOR and PcNACHT protein
Understanding the functions of TOR signaling in P. chrysosporium
Proteomic analysis could be used to characterize deeply the role of TOR in regulating the secretome response of P. chrysosporium
The possible relationship between TOR and the intracellular detoxification system