How nutritional status signalling coordinates metabolism and lignocellulolytic enzyme secretion.

Fungal Genetics and Biology xxx (2014) xxx–xxx

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Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi

How nutritional status signalling coordinates metabolism and lignocellulolytic enzyme secretion Neil Andrew Brown a,⇑, Laure Nicolas Annick Ries a, Gustavo Henrique Goldman a,b,⇑ a b

Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Campinas, Brazil

a r t i c l e

i n f o

Article history: Received 14 February 2014 Revised 26 June 2014 Accepted 28 June 2014 Available online xxxx Keywords: Carbon catabolite repression Lignocellulolytic enzymes Carbon starvation Protein secretion Saccharomyces cerevisiae Ascomycete fungi

a b s t r a c t The utilisation of lignocellulosic plant biomass as an abundant, renewable feedstock for green chemistries and biofuel production is inhibited by its recalcitrant nature. In the environment, lignocellulolytic fungi are naturally capable of breaking down plant biomass into utilisable saccharides. Nonetheless, within the industrial context, inefficiencies in the production of lignocellulolytic enzymes impede the implementation of green technologies. One of the primary causes of such inefficiencies is the tight transcriptional control of lignocellulolytic enzymes via carbon catabolite repression. Fungi coordinate metabolism, protein biosynthesis and secretion with cellular energetic status through the detection of intra- and extra-cellular nutritional signals. An enhanced understanding of the signals and signalling pathways involved in regulating the transcription, translation and secretion of lignocellulolytic enzymes is therefore of great biotechnological interest. This comparative review describes how nutrient sensing pathways regulate carbon catabolite repression, metabolism and the utilisation of alternative carbon sources in Saccharomyces cerevisiae and ascomycete fungi. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Lignocellulose is the most abundant natural material on Earth and is seen as an attractive source of renewable energy. However, its recalcitrant nature impedes the efficient utilisation of this feedstock in green technologies. Lignocellulolytic fungi that colonise live and/or dead plant matter secrete an array of lignocellulolytic enzymes that efficiently degrade plant biomass, facilitating colonisation and providing a source of carbon to sustain growth. Therefore, such fungi must perceive this complex mixture of saccharides and adopt the most efficient strategy for coordinating polysaccharide degradation, carbon uptake and metabolism. Subsequently, several ascomycete fungi, including Aspergillus species and Trichoderma reesei have become the main commercial source of lignocellulolytic enzymes utilised in industry. The ability to coordinate proliferation with cellular energetic status is a fundamental requisite for life and is conserved across all kingdoms. The catabolism of carbohydrates, through glycolysis,

⇑ Corresponding authors. Address: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café S/N, CEP 14040-903, Ribeirão Preto, São Paulo, Brazil. Fax: +55 16 36024178. E-mail addresses: [email protected] (N.A. Brown), [email protected] (G.H. Goldman).

respiration or fermentation is the predominant route for energy production. Glucose catabolism represents the greatest energetic gain and is therefore the preferred carbon source for the majority of microbes. Subsequently, an ancestral capability to sense intraor extra-cellular energy sources enables the coordination of cellular metabolism and the preferential consumption of glucose prior to other carbon sources, referred to as carbon catabolite repression (CCR), which is a common strategy utilised by microbes including budding yeasts and filamentous fungi. The efficient production of biofuels and green chemistries from low cost plant residues, composed of varying saccharides, are all impaired via CCR. In the natural context the sole use of a single carbon source until exhaustion is beneficial. However, in the industrial situation, the autoinhibition of protein overproduction is undesirable. For example, the inhibition of lignocellulolytic enzyme secretion via the presence of readily consumable sugars, such as those released from lignocellulose deconstruction, impedes enzyme production and represents a major factor preventing the implementation of next-generation biofuel production (Himmel and Bayer, 2009). The efficient depolymerisation of lignocellulose into simple sugars represents the initial step for the majority of green chemistries. Therefore, how microbes sense the presence of differing carbon sources thus regulating CCR and lignocellulolytic enzyme production is the focus of this review.

http://dx.doi.org/10.1016/j.fgb.2014.06.012 1087-1845/Ó 2014 Elsevier Inc. All rights reserved.

Please cite this article in press as: Brown, N.A., et al. How nutritional status signalling coordinates metabolism and lignocellulolytic enzyme secretion. Fungal Genet. Biol. (2014), http://dx.doi.org/10.1016/j.fgb.2014.06.012

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N.A. Brown et al. / Fungal Genetics and Biology xxx (2014) xxx–xxx

The fundamental knowledge of nutrient sensing in microbes is predominantly founded upon studies of Saccharomyces cerevisiae, where the components and function of the relevant nutrient sensing signalling cascades that modulate growth and metabolism are known (for reviews see Rolland et al., 2001; Santangelo, 2006; Zaman et al., 2008). The equivalent genes in several model filamentous fungal systems have been identified but their regulation, mechanism and function are less well understood. The genes encoding for proteins involved in such nutrient sensing pathways have demonstrated a high level of conservation in S. cerevisiae and filamentous fungi, yet deviations in pathway composition and organisation do exist. The array and activity of the lignocellulolytic enzymes secreted by a filamentous fungal species depends upon the niche naturally occupied by a particular species. Saprophytic ascomycete fungal species including Aspergillus nidulans, Neurospora crassa and T. reesei have been widely adopted as the model organisms for the study of lignocellulolytic enzyme production (for reviews see Culleton et al., 2013; Glass et al., 2013; Kubicek et al., 2009). However, due to their efficient secretion systems, commercial enzyme cocktails are predominantly based on A. niger and T. reesei (Berka et al., 1991; Durand et al., 1988). Comparatively, the genome of T. reesei encodes fewer lignocellulolytic enzymes than other lignocellulolytic fungi (Martinez et al., 2008), while A. niger possesses a more versatile range of cellulases, hemicellulases and esterases that will become more important once undesirable lignocellulose pretreatment steps are abolished (Pel et al., 2007). Subsequently, S. cerevisiae has been used as a framework for the basis of this comparative review with the three model ascomycetes, A. nidulans, N. crassa and T. reesei, which focuses upon the three phases of lignocellulolytic enzyme regulation; (1) repression in the presence of preferred carbon sources, (2) derepression under carbon limitation and (3) induction of lignocellulolytic enzyme transcription and secretion. Finally, the remaining questions and application of such knowledge in S. cerevisiae and filamentous fungi within the industrial situation are discussed.

2. Signalling for the repression of lignocellulolytic enzymes in the presence of readily metabolised carbon sources 2.1. The mechanism and regulation of repression Through the action of functionally conserved repressor proteins, CCR in fungi prevents the utilisation of alternative carbon sources via inhibiting the transcription of secreted and intracellular metabolic enzymes. The transcription of alternative carbon usage genes in S. cerevisiae and filamentous fungi is tightly controlled by a Cys2-His2 type DNA-binding zinc finger repressor protein, named Mig1p in S. cerevisiae and Cre1, CRE1 or CreA in filamentous fungi (Fig. 1) (Zaman et al., 2008; Shroff et al., 1997; Strauss et al., 1995). The S. cerevisiae Mig1p and A. nidulans CreA proteins show 70% identity (e-value 4e-29) within the double zinc finger domain. These repressor proteins in S. cerevisiae and filamentous fungi bind to GC boxes within the promoter of the repressed gene. The Mig1p binding motif (50 -ATAAAATGCGGGGAA-30 ) is flanked by AT-rich regions that are proposed to assist in protein-induced target site bending (Lundin et al., 1994). In A. nidulans and T. reesei, two closely spaced CreA/CRE1 consensus motifs 50 -SYGGRG-30 represent double binding sites that are key to repression (Cubero and Scazzocchio, 1994; Dowzer and Kelly, 1991; Takashima et al., 1996). In filamentous fungi additional repressor proteins have been identified, such as ACE1 of T. reesei, which has been shown to bind to the promoter of cellulase genes. The ACE1 homologue in A. nidulans StzA is described as a stress response factor (e-value 3e-86, 40.8% identity, 56.0% similarity; www.aspgd.org).

The industrial T. reesei RutC30 strain is a well-documented cellulase hyperproducer that is known to have a defective CCR system caused by a cre1 truncation, in addition to enhanced protein secretion and altered protein glycosylation (IImen et al., 1996; Peterson and Nevalainen, 2012; Seidl et al., 2008). Similarly to the cre1 deficient T. reesei strain, the ace1 gene deletion strain demonstrates higher cellulase induction (Aro et al., 2003). In N. crassa CRE-1 has been also characterized and its role in the regulation of cellulase/hemicellulose gene expression has been analysed (Sun and Glass, 2011; Ziv et al., 2008) In A. nidulans, a complete creA gene deletion results in severe morphological defects, while partial gene disruption can enhance enzyme production (Shroff et al., 1997). However, the simple removal of the repressor protein is not sufficient to create an efficient system for enzyme hyperproduction. Therefore, a thorough understanding of the mechanism of CCR is required to enable the further enhancement of enzyme production. 2.1.1. Repression in S. cerevisiae The nuclear localisation of Mig1p in S. cerevisiae cells regulates repressor function, with Mig1p being imported into the nucleus within minutes of the addition of glucose to derepressed cells (de Vit et al., 1997) (Fig. 1A). An additional level of gene regulation, which also governs CCR, is mediated through changes in nuclear chromatin structure, accompanied by short- or long-term alterations in transcriptional activity, which can be triggered by intrinsic cellular programs and/or environmental factors (Brosch et al., 2008). Chromatin organisation is influenced by nucleosome positioning and histone acetylation or methylation which in turn regulates gene expression via obstructing transcription factor binding (Li et al., 2007). The Tup1p/Ssn6p corepressor complex in S. cerevisiae, can be recruited by a number of DNA-binding proteins, including Mig1p, promoting histone deacetylation and nucleosome positioning (Smith and Johnson, 2000). The Mig1p repressor recruits the Tup1p/Ssn6p complex to the promoters of alternative carbon usage genes, enhancing repression via the modulation of nucleosomes positioning (Treitel and Carlson, 1995) (Fig. 1A). Chromatin distribution in the nucleus is non-random and the nuclear periphery is a key site in the transcriptional regulation of glucose-repressed genes. The reverse recruitment model describes how genes are activated by coming into contact with distinctly localised transcription factories that are tethered to nuclear pores (Menon et al., 2005). The Sucrose non-fermenting 1 (Snf1) complex is perinuclear during Mig1p derepression in S. cerevisiae, while a target gene SUC2 associates with the nuclear pore when derepressed, compared to being highly mobile and randomly distributed throughout the nucleus when repressed (Sarma et al., 2007). Consequently, recent studies have shown that Mig1p physically interacts with the nuclear pore complex, which mediates its repressing function through a mechanism that is independent of Mig1p nucleocytoplasmic shuttling (Sarma et al., 2011). 2.1.2. Repression in filamentous fungi In filamentous fungi, as with S. cerevisiae, the nuclear localisation of CreA/CRE1/Cre1 is also dependent on carbon availability (Fig. 1B). However, due to differences in lifestyle and the ability to catabolise various carbon sources, repressor localisation is not solely regulated by glucose in filamentous fungi. In A. nidulans, Fusarium oxysporum, N. crassa and Sclerotinia sclerotiorum, the regulation of CreA/1-GFP nuclear localisation plays a role in CCR (Brown et al., 2013; Sun and Glass, 2011; Jonkers and Rep, 2009a; Vautard-Mey et al., 1999). However, this carbon source response is only visible if the fusion protein is under the control of the native promoter. Similar studies of CreA/1-GFP fusion proteins in A. nidulans and N. crassa, under the control of a constitutive promoter (Roy et al., 2008; Sun and Glass, 2011), demonstrated constitutive nuclear localisation, irrespective of the presence of glucose,



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Fig. 1. The competitive inhibition/induction mechanism of carbon catabolite repression in S. cerevisiae (A) and filamentous fungi (B). In the presence of glucose S. cerevisiae and filamentous fungi repress the expression of alternative carbon usage genes via a double lock mechanism. A repressor protein binds to upstream regulatory elements (URE) inhibiting the expression of the transcription factors (TF) and metabolic enzymes required for alternative carbon usage. In addition, chromatin structure impedes transcription. During carbon-limitation in S. cerevisiae and the majority of filamentous fungi, Snf1-mediated phosphorylation and relocalisation of the repressor protein results in derepression, while local modifications to chromatin structure permit transcription. Note: the protein identifiers in (B) are based upon Aspergillus nidulans. In addition, the role of importins/exportins or the SnfA regulatory subunits is yet to be studied in filamentous fungi. Legend: grey Snf1/SnfA complex = unactive; yellow star = activate Snf1/SnfA complex; P = phosphorylation; orange cirucle = nucleosome. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

suggesting that the regulation mechanism can be oversaturated. Interestingly, in A. nidulans, creA transcription has been shown to be higher under derepressing conditions, and is negatively autoregulated under repressing conditions, while CreA–DNA complexes formed more efficiently under repressing conditions (Strauss et al., 1999). The exact mechanisms orchestrating the localisation and affinity of repressor binding remain to be clearly defined in S. cerevisiae and filamentous fungi. In filamentous fungi, fewer details are known about the complex regulation of CreA/Cre1/CRE1 function. However, correct nucleosomal positioning is involved in the repression of the

alcohol utilisation regulon in A. nidulans, and is dependent upon CreA (Mathieu et al., 2005). Homologues of the S. cerevisiae Tup1p are widely found in filamentous fungi. Deletion of the Tup1p and Ssn6p homologues in A. nidulans revealed the Tup1p homologue was dispensable for CCR, yet essential for repressed chromatin structure in the alcR promoter during repression, while the Ssn6p homologue was shown to be an essential gene (Gracia et al., 2008) (Fig. 1B). In T. reesei, the CRE1-dependent repositioning of nucleosomes from the cellobiohydrolase-encoding cbh1 coding region to the promoter post transfer from glucose-repressed to cellulase-inducing conditions, shows the importance of CRE1 in


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correct nucleosome positioning and cellulase regulation (Ries et al., 2014). Furthermore, Zeilinger et al. (2003) demonstrated that CRE1 is required for correct nucleosome positioning in the cbh2 promoter of T. reesei under inducing and repressing conditions. Therefore, the recruitment of corepressors and nucleosome organisation appears to play a supplementary role in CCR in both S. cerevisiae and filamentous fungi. Future evidence for the role of genome and chromatin organisation in the regulation of lignocellulolytic enzyme production exists in T. reesei. In contrast to other lignocellulolytic fungi, within the T. reesei genome, genes encoding lignocellulolytic enzymes are organised in small gene clusters (Martinez et al., 2008). In Aspergilli the methyltransferase LaeA is a global regulator of the expression of secondary metabolite gene clusters in response to environmental cues via the cAMP/PKA, Velvet and mitogen-activated protein kinase pathways (Bok and Keller, 2004; Palmer and Keller, 2010). Subsequently, the T. reesei methyltransferase, LAE1, influences lignocellulolytic enzyme production. The deletion of lae1 results in the complete loss of induction in multiple cellulase, b-glucosidases and xylanases, while the overexpression of lae1 results in increased expression. Furthermore, expression of xyr1, encoding the main inducer of lignocellulolytic enzyme-encoding genes, was shown to be LAE1 dependent (Seiboth et al., 2012). Comparative studies of the influence of LaeA on lignocellulolytic enzyme production in Aspergilli and N. crassa have not been undertaken due to the absence of CAZyme gene clusters in the respective genomes. Beyond the functions of repressors and chromatin organisation in the regulation of lignocellulolytic enzyme encoding genes, additional mechanisms influence enzyme translation. The regulation of gene expression by natural antisense transcripts (NATs) is known in fungi. These complementary RNAs to protein encoding mRNA, promote mRNA destruction and subsequently have a regulatory role. In A. niger and T. reesei a dramatic switch in antisense/sense ratio has been documented between nutrient-rich growth and growth on lignocellulose (Delmas et al., 2012; Ries et al., 2013). The mechanism and influence of this level of regulation, influencing protein translation, remains to be determined. Consequently, it is clear that a multifaceted, complex, system of CCR regulation and repressor binding occurs in both yeast and filamentous fungal systems. 2.2. Sugar sensing and the activation of repression In order to efficiently block the transcription and synthesis of unrequired proteins, a microbe needs to rapidly detect the presence of a preferred carbon source and induce CCR. The addition of glucose to derepressed S. cerevisiae or filamentous fungal cells induces CCR within minutes. The following section will focus upon how glucose is sensed and how these signalling cascades influence CCR. 2.2.1. Sugar sensing in S. cerevisiae Glucose sensing and the subsequent modulation of the cellular response are predominantly coordinated via the cAMP/protein kinase A (PKA) pathway as depicted in Fig. 2. In S. cerevisiae, the addition of glucose to deprived cells, causes a short burst in the production of cAMP, increasing 5–50-fold within 2 min (Santangelo, 2006) and the transcriptional modulation of approximately 40% of the genome (Wang et al., 2004). Extracellular glucose is sensed by the G-protein coupled receptor Gpr1p and results in the activation of the associated intracellular heterotrimeric protein homologue Gpa2, which activates the adenylate cyclase Cyr1p (Xue et al., 1998). Alternatively, the internalisation and subsequent phosphorylation of glucose via the gluco- or hexo-kinases, Glk1p and Hxk1p/2p, activates Ras1p/Ras2p signalling which stimulates

Cyr1p (Colombo et al., 2004; Matsumoto et al., 1982). The absence of the ability to phosphorylate glucose in the glk1D hxk1D hxk2D mutant results in a loss of glucose induced Ras2p activation (Colombo et al., 2004) demonstrating that glucose phosphorylation is required for Ras/cAMP induction. Inactivation of Gpr1p has a less dramatic influence on cAMP production than inactivation of Ras2p, the latter of which resulted in dramatically reduced cAMP levels (Tanaka et al., 1991). Therefore, the Ras/cAMP pathway could represent the major route for glucose sensing as 92% of the glucose induced transcriptional responses are also induced in the activated Ras2p strain during growth in the absence of glucose (Wang et al., 2004). However, these experiments are not conclusive, since there is no evidence that the wild-type strain ever displays such high Ras activity which may be restricted to the experimental conditions used. All glucose induced transcriptional changes mediated via the S. cerevisiae Gpr1p or Ras2p pathway are entirely dependent upon PKA activity (Wang et al., 2004). The sensing of intra- or extra-cellular glucose by either of the aforementioned routes subsequently results in the activation of PKA, as cAMP blocks the binding of the PKA regulatory subunits, enabling the activation and migration of the PKA catalytic subunit to the nucleus (Portela and Moreno, 2006). Within the nucleus, activated PKA regulates transcription factors, thus influencing stress responses, ribosomal biogenesis and carbohydrate metabolism (Griffioen and Thevelein, 2002). The activation and localisation of PKA is also partially influenced by TOR signalling, representing the cross-talk with other nutrient signalling pathways (Schmelzle et al., 2004). Inactivation of the PKA pathway in the presence of abundant nutrition results in cell cycle arrest in G1, while the response is too rapid to be a consequence of the influence of PKA on ribosome biogenesis and cell size (Zaman et al., 2008). Alternatively, hyper-activation of PKA, via the deletion of the Bcy1p regulatory subunit encoding gene, results in the failure to arrest in G1 during carbon starvation (Toda et al., 1987). The regulation of the cAMP/ PKA pathway subsequently links nutrition and metabolism to cell cycle and proliferation. Moreover, in S. cerevisiae, the import of glucose into the cell and the production of phosphorylated glucose results in the inactivation of the protein kinase Snf1p, inhibiting Mig1p phosphorylation and promoting nuclear localisation and repression. The addition of glucose to glucose-deprived cells results in the rapid post-translational modification of the serine/threonine protein phosphatases, PP2A and PP1, via the PKA pathway (Castermans et al., 2012). The PP1 phosphatase Glc7p and its regulatory subunit Reg1p dephosphorylate the activation loop of Snf1p (Tu and Carlson, 1994, 1995) inhibiting the use of alternative carbon sources. In addition, the PP2A phosphatase Sit4p and the PP2C phosphatase Ptc1p also regulate Snf1p phosphorylation state in a glucosedependent manner (Ruiz et al., 2011, 2013). Phosphorylated glucose, but not glucose metabolism, also inhibits the nuclear localisation of Snf1p, by promoting nuclear export of the Gal83p subunit (Vincent et al., 2001). The hexokinase Hxk2p has been detected in the nucleus and has been shown to interact with Mig1p suggesting that it has an additional role apart from phosphorylating glucose (Ahuatzi et al., 2007). Therefore, in S. cerevisiae glucose sensing and PKA activation inhibit phosphorylation of Hxk2p, which forms a dimer, blocking Reg1p-Glc7p dephosphorylation and maintaining Snf1p inactive (Santangelo, 2006). Furthermore, S. cerevisiae also senses the concentration of extracellular glucose via the high and low affinity glucose transporter homologues, Snf3p and Rgt2p, which have lost the capacity to transport glucose. Both Snf3p and Rgt2p show strong sequence similarity to hexose transporters but possess an extended cytoplasmic tail (Özcan et al., 1996, 1998). Activation of Snf3p and Rgt2p induces the transcription of distinct sets of hexose transporters with differing saccharide affinities appropriate to carbon



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Fig. 2. The cAMP/PKA glucose sensing pathway in S. cerevisiae in the presence (A) or absence of glucose (B). The two major routes of cAMP-dependent PKA activation, (1) extracellular glucose sensing via the Gpr1, G-coupled protein receptor and (2) the intracellular sensing of phosphorylated glucose via the Ras1/Ras2 pathway. Of the two routes, the sensing of phosphorylated glucose has the major impact on cAMP production. The regulatory subunit of PKA, Bcy1 dissociates from the catalytic subunit, Tpk, resulting in its activation, nuclear translocation and the induction of a glucose response. Concomitantly, activated PKA promotes the Reg1-Glc7 phosphatase to deactivate the Snf1 complex, impeding alternative carbon usage. In the absence of glucose, Bcy1 does not dissociate from Tpk and the PKA complex is retained in the cytosol. The drop in Reg1-Glc7 activity plus the induction of upstream kinases (predominately Sak1 during carbon stress) results in Snf1 phosphorylation and nuclear migration. Snf1-mediated carbon catabolite derepression promotes alternative carbon usage. Legend: grey = unactive complex; yellow star = activate complex; G = glucose; P = phosphorylation; arrow = induction; blunt ended arrow = repression; red X = absence of glucose. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

availability (Özcan et al., 1996, 1998). The binding of glucose to either sensor results in the recruitment and casein kinasedependent phosphorylation of the Mth1p and Std1p corepressors, targeting them for proteosomal degradation (Moriya and Johnston, 2004). In the absence of the corepressors, Rgt1p-mediated repression of HXT gene expression is alleviated by PKA phosphorylation, thus regulating the transcription of the appropriate hexose transporters (Lakshmanan et al., 2003; Polish et al., 2005). The Snf3p/Rgt2p pathway is not essential for the glucose response, in that CCR can still take place even in the absence of both sensors; and is proposed to fine-tune or commensurate the glucose signal (Santangelo, 2006).

2.2.2. Sugar sensing in filamentous fungi In filamentous fungi, the cAMP-dependent PKA pathway has been extensively studied for its role in germination, morphological development and secondary metabolism (Shimizu and Keller, 2001; Shimizu et al., 2003; Fillinger et al., 2002). In the lignocellulolytic filamentous fungi, T. reesei and Aspergilli, the cAMP-dependent PKA pathway has also been shown to detect glucose, inhibiting alternative carbon usage and lignocellulolytic enzyme production (Dong et al., 1995; Farkas et al., 1989). However, orthologues of the S. cerevisiae Gpr1p glucose sensor at the head of respective filamentous fungal pathway are yet to be identified. Nonetheless, in T. reesei, two G-protein a subunits, a single RAS GTPase, the adenylate cyclase


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and PKA have been shown to influence cellulase production in response to light in the presence of the saccharide inducer (Schmoll et al., 2009; Schuster et al., 2012; Seibel et al., 2009; Zhang et al., 2012). Interestingly, in N. crassa, the constitutive activation of PKA, through the removal of the regulatory subunit Mcb, increases cellulase secretion (Lee et al., 1998). These results suggest that the PKA performs additional functions, beyond a glucoseinduced response, that influence lignocellulolytic enzyme production in filamentous fungi. Similar to the S. cerevisiae Mig1p repressor, CreA/CRE1 proteins contain PKA phosphorylation sites, yet their functionality and influence on repressor protein function is unknown. In the filamentous fungus A. nidulans the single inactivation of the hexose kinase orthologues, glkA and hxkA, had little effect on growth on glucose, while the double DglkA DhxkA knockout resulted in the abolishment of glucose phosphorylation and a complete loss of growth on glucose (Flipphi et al., 2003). The impact of these kinases on cAMP synthesis and PKA activity is not yet confirmed. Nonetheless, glucose-mediated repression of genes involved in acetate catabolism, ethanol utilisation and xylan degradation was severely impaired in the double mutant, but only partially derepressed in the single mutants, suggesting a compensatory role (Flipphi et al., 2003). This observation is supported by the fact that the addition of 6-deoxyglucose, a compound which cannot be phosphorylated during glycolysis, to derepressed A. nidulans hyphae, resulted in the absence of CreA from the nucleus (Brown et al., 2013). Similarly, in T. reesei, only the construction of a double Dglk1 Dhxk1 mutant strain resulted in derepression (Kubicek et al., 2009). These results demonstrate that phosphorylation of glucose is essential for CCR in filamentous fungi. Whether phosphorylated glucose itself or a downstream or derived metabolite acts as a signal, remains unclear. The DglkA DhxkA strain also demonstrates enhanced endocellulase and endoxylanase activity when grown on lignocellulose as a carbon source (Brown and Goldman, unpublished result). These observations suggest that the removal of the inhibitory effect of glucose phosphorylation via genetic intervention or during growth on lignocellulose may enhance SnfA activity and CreA derepression. In addition, two non-essential protein phosphatases with homology to Sit4p and Ptc1p in S. cerevisiae, were identified as being required for wildtype cellulase production during growth on cellulose in A. nidulans (Brown et al., 2013), yet it remains to be shown if these phosphatases influence SnfA activity. The only putative glucose sensor in a filamentous fungus to be identified to date is the RCO3 sensor from N. crassa (Madi et al., 1997), although no targets controlled by this sensor have been described. Similar to the Snf3p/Rgt2p pathway in S. cerevisiae, RCO3 of N. crassa demonstrated significant sequence similarity to S. cerevisiae glucose transporters. In contrast to Snf3p and Rgt2p in S. cerevisiae, RCO3 lacks an extended cytoplasmic tail and instead possesses an extended cytoplasmic intergenic region which is proposed to mediate protein–protein interactions (Madi et al., 1997). No studies of the influence of an orthologous Snf3p/ Rgt2p pathway, in filamentous fungi, on lignocellulolytic enzyme production exist. Interestingly, in contrast to S. cerevisiae, filamentous fungi do not only induce CCR in response to glucose but also to high concentrations of xylose or cellobiose. How these different saccharides are sensed is unknown. Nonetheless, as saccharide concentration is important, the uptake system and/or production of an intracellular metabolic signal may play a key role. For example, in the case of cellobiose-induced repression, the production of intracellular phosphorylated glucose is required in some way to mediate repression. This was demonstrated by the genetic disruption of the three main b-glucosidases and two cellobiose transporters in N. crassa (Znameroski et al., 2014). Therefore, the transport of

simple saccharide into the cell appears to be central to the induction of CCR. 2.3. Parallel and conflicting nutrient sensing pathways In S. cerevisiae additional nutrient sensing mechanisms that overlap or counteract the PKA pathway include the Sch9p and Yak1p pathways, respectively. The pro-survival Sch9p kinase (S6K-like kinase) regulates a similar gene set to PKA, inducing ribosome biogenesis and repressing carboxylic acid metabolism, while SCH9 disruption suppresses mutational defects in the Ras/PKA pathway (Toda et al., 1988). Sch9p has been cited to be involved in stress and nutrient sensing pathways. Glucose both increases Sch9p protein and phosphorylation levels, yet it appears to play a minor role in glucose-mediated changes (Urban et al., 2007). However, Sch9p plays a more significant role in TOR-dependent nutrient sensing and is required for TOR-mediated ribosome biogenesis, entry into G0 phase, and has been shown to influence cell lifespan (Urban et al., 2007). In the filamentous fungus A. nidulans, the Sch9p homologue, SchA, has also been shown to have overlapping functions with PKA, influencing germination, trehalose metabolism and growth kinetics (Fillinger et al., 2002). In addition, SchA has recently been shown to influence lignocellulolytic enzyme production via the modulation of CreA nuclear localisation (Brown et al., 2013). Similar to PKA, S6K kinase phosphorylation sites with undetermined functionality exist within the A. nidulans CreA repressor protein, suggesting that SchA may perform a role in post-translational CreA regulation. In S. cerevisiae, the Yak1p kinase functions to counteract the PKA pathway, inhibiting growth and stimulating stress responses (Zaman et al., 2008). Yak1p-dependent phosphorylation of the Pop2p deacetylation complex, which regulates the translation and stability of mRNAs involved in alternative carbon usage, is necessary for glucose derepression (Moryia et al., 2001). Upon glucose starvation, the PKA regulatory subunit Bcy1p is confined to the cytoplasm in a Yak1p-dependent manner (Griffioen et al., 2001). In the absence of Yak1p phosphorylation of Pop2p, cells fail to enter stationary phase. Recently, in the filamentous fungus A. nidulans, YakA and PKA were both identified as being required for wild-type growth on cellulose and endocellulase secretion (Brown et al., 2013). Therefore, in A. nidulans the PKA, SchA and YakA pathways, which have overlapping and opposing functions, all appear to be involved in modulating the response to the change in nutritional status post transfer to growth on cellulose. 3. Signalling for carbon catabolite derepression during carbon limitation Carbon deprivation dramatically alters cellular growth and metabolism. During carbon starvation in S. cerevisiae, the concomitant down-regulation of genes encoding the transcriptional/translation machinery plus components of the TCA cycle and oxidative phosphorylation (Wu et al., 2004) reflects the modulation of the cell cycle and the provision of the metabolic intermediates and ATP required for maintaining cellular growth. This carbon starvation response is accompanied by the transcriptional induction (greater than fivefold) of 268 genes, including those that encode proteins involved in shifting metabolism from the catabolism of saccharides to the utilisation of alternative carbon sources such as lipids and amino acids (Wu et al., 2004). In lignocellulolytic fungi, such as A. nidulans and N. crassa, carbon starvation also causes a similar transcriptional response, while also inducing the transcription of secreted lignocellulolytic enzymes (Krohn et al., 2014; Xie et al., 2004). Subsequently, during carbon limitation, lignocellulolytic fungi shift primary metabolism to the utilisation of pre-existing car-



bon sources from within the cell and promote the transcription of secreted hydrolytic enzymes. The short term use of existing energy sources may enable the synthesis of such scavenging enzymes, which can potentially liberate potent hydrolytic enzyme inducers from recalcitrant polysaccharides. Such carbon starvation-induced alterations to metabolism are regulated by CCR and inhibited in the presence of glucose, while the regulation of Snf1p/SnfA/SNF1 activity plays a central role in the alleviation of CCR in S. cerevisiae and the majority of filamentous fungi (Brown et al., 2013; Lee et al., 2009; Ospina-Giraldo et al., 2003; Tonukari et al., 2000; Treitel et al., 1998), yet subtle differences in mechanism do exist. The alteration of energy levels, metabolism and the increased demand for protein secretion ultimately imposes additional stresses upon the cell. The role of several central mechanisms influencing carbon catabolite derepression will be focused upon in this section. 3.1. The mechanism and regulation of Snf1-mediated derepression Similar to the mammalian AMP-activated kinase (AMPK) homologue, Snf1p in S. cerevisiae maintains cellular energy homeostasis when energy is low via stimulating glucose uptake, lipid oxidation and inhibiting ATP consuming processes (Hedbacker and Carlson, 2008). The Snf1p pathway is required for growth on alternative non-fermentable carbon sources such as glycerol and ethanol, influencing a range of metabolic processes, pseudohyphal growth and the nitrogen limitation response (Hedbacker and Carlson, 2008; Kuchin et al., 2003; Orlova et al., 2006). Furthermore, lipids represent an energy source that can be mobilised upon carbohydrate starvation and this coordination of lipid metabolism with glucose availability is regulated by Snf1p (Woods et al., 1994). In S. cerevisiae Snf1p-mediated Mig1p phosphorylation promotes Mig1p DNA dissociation and nuclear export (Treitel et al., 1998). The relocalisation of Mig1p results in a loss of corepressor recruitment, assisting in the alteration of nucleosome positioning (Treitel and Carlson, 1995). Snf1p forms a heterotrimeric protein complex, consisting of the Snf1p catalytic, Snf4p regulatory and one of three b subunits, Gal83p, Sip1p or Sip3p. Binding of Snf4p to Snf1p relieves autoinhibition, while phosphorylation of the activation loop by one of three upstream kinases Sak1p, Elm1p or Tos3p, promotes activation (Hong et al., 2003). In response to carbon stress, Sak1p is the key upstream kinase and controls the nuclear localisation of the Snf1p-Gal83p complex, where Snf1p phosphorylates Mig1p promoting derepression (Hedbacker et al., 2004). Similarly to S. cerevisiae, phosphorylation of CreA/CRE1 in lignocellulolytic fungi is proposed to perform the key role in the relocalisation of the repressor protein. In the lignocellulolytic fungi A. nidulans and S. sclerotiorum, the orthologous Snf1p kinases are essential for CreA/CRE1 derepression. The absence of SnfA in A. nidulans results in an inability to remove CreA from the nucleus when grown under derepressing conditions, including starvation and growth on cellulose, therefore inhibiting cellulase production in the presence of cellulose (Brown et al., 2013). In S. sclerotiorum Snf1-dependent phosphorylation has also been shown to be essential for derepression but not Cre1 relocalisation (Vautard-Mey and Fèvre, 2000). The subcellular localisation of these Snf1p orthologues from lignocellulolytic fungi, under repressing and derepressing conditions, is unclear. In contrast to other lignocellulolytic fungi, CRE1 DNA-binding in T. reesei during repression is positively regulated by phosphorylation (Cziferszky et al., 2002). In addition, the T. reesei Snf1p orthologue is able to phosphorylate Mig1p when heterologously expressed in S. cerevisiae, but cannot phosphorylate CRE1 in vivo (Cziferszky et al., 2003), suggesting that CCR in T. reesei may be distinct. Therefore, the general mechanisms leading to CCR appear to be largely conserved in S. cerevisiae and filamentous fungi during growth in the presence of glucose (Fig. 1). However, growth of S.

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cerevisiae on non-glucose carbon sources results in Snf1p-mediated Mig1p derepression. In contrast, in filamentous fungi, including A. nidulans, N. crassa and T. reesei, CreA/Cre1/CRE1 derepression only occurs in the presence of complex carbohydrates such as lignocellulose whereas high concentrations of alternative, simple carbon sources, such as cellobiose and xylose in A. nidulans and ethanol in F. oxysporum, predominantly results in CreA/Cre1 nuclear localisation (Brown et al., 2013; Jonkers and Rep, 2009a). Therefore, it appears that carbon limitation or metabolic stress is required for complete carbon catabolite derepression and increased lignocellulolytic enzyme production in filamentous fungi.

3.2. The role of repressor protein degradation in derepression Protein turnover plays an essential role in the regulation of protein function and in filamentous fungi this has been linked to the regulation of CCR. Proteins destined for degradation are typically polyubiquitinated by E3 ligases, targeting them to the proteasome (Hershko and Ciechanover, 1998). The Skp1, Cullin and F-box protein (SCF) complex represents the largest family of ubiquitin ligases. The F-box proteins, which form part of the SCF complex, assist in binding the complex to the target substrate (Kipreos and Pagano, 2000). Filamentous fungal F-box proteins are responsible for multiple biological functions including cell cycle, circadian clocks, transcription, development, signal transduction and nutrient sensing (Jonkers and Rep, 2009b). In multiple plant pathogenic fungi, which secrete hydrolytic enzymes that attack the plant cell wall, F-box proteins have been shown to be essential for full virulence including Botrytis cinerea, F. graminearum and F. oxysporum (Duyvesteijn et al., 2005; Han et al., 2007; Jonkers et al., 2011). Interestingly, the inability to grow on a range of alternative carbon sources plus the reduction in hydrolytic enzyme expression and virulence of the F. oxysporum F-box deletion strain, fpr1, was rescued via a cre1 mutation (Jonkers and Rep, 2009a), suggesting that F-box proteins perform a role in Cre1 derepression. This also appears to be the case in A. nidulans where a screen of a F-box deletion collection, consisting of 42 F-box domain encoding genes, identified FbxA as having deregulated xylanase production and increased sensitivity to non-metabolisable, toxic, 2-deoxyglucose (Colabardini et al., 2012). Two additional Cre proteins exist in Aspergilli, the deubiquinating enzyme CreB and the WD40 repeat containing, proline-rich CreC, which have been shown to form a complex along with CreA, influencing CreA stability and proteosomal degradation (Lockington and Kelly, 2001, 2002). In A. nidulans, A. oryzae and T. reesei deletion of CreB orthologues resulted in increased cellulase and xylanase secretion in the absence of glucose (Denton and Kelly, 2011; Hunter et al., 2013). Acetate can also lead to strong CCR in A. nidulans, where acetate repression involved CreA but not CreB or CreC (Georgakopoulos et al., 2012). Interestingly, a screen for mutations impaired in acetate, but not glucose repression revealed orthologues of S. cerevisiae SAGA components SPT3 and SPT8 (Georgakopoulos et al., 2012). However, the difference between glucose and acetate CreA-mediated repression appear to be related to metabolic differences rather than an independent repression pathway. Collectively the aforementioned studies stress the importance of F-box proteins and ubiquitination in the regulation of CreA/Cre1 protein turnover, introducing a new layer of complexity to the coordination of CCR in filamentous fungi. In comparison, the S. cerevisiae genome encodes approximately 20 F-box domain containing proteins, yet no direct evidence that demonstrates a role for protein turnover in Mig1p regulation has been established. However, the role of protein turnover in the regulation of Rgt1p and HXT expression via the antagonistic action of the SCFGrr1 complex has been demonstrated (Flick et al., 2003),


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suggesting that unforeseen mechanisms in Mig1p regulation involving protein turnover may exist. 3.3. The role of cellular energy stress in derepression and the use of intra- or extra-cellular non-carbohydrate carbon sources The mitochondria supply cellular energy and perform a role in the adaptation of the cell to metabolic stress. In mammalian cells, the ATM kinase acts as a redox sensor controlling mitochondrial function and operates upstream of the AMPK (Snf1p orthologue), mTOR and S6K kinases (Sch9p orthologue), regulating growth, metabolism and lifespan (Ditch and Paull, 2012). In S. cerevisiae, mitochondria, epigenetics and telomere function have been implicated in influencing cell lifespan. Mitochondrial ROS signalling in S. cerevisiae is sensed by Tel1p (ATM orthologues) which communicates mitochondrial functional status to the nucleus (Schroeder et al., 2013). The subsequent loss of Rph1p histone demethylase within the sub-telomeric region results in specific sub-telomeric silencing of transcription (Schroeder et al., 2013). This signals telomere dysfunction that alters mitochondrial biogenesis and function in both mammalian and S. cerevisiae cells. Therefore, telomere dysfunction and ATM/Tel1p control mitochondrial function while activating p53-mediated growth arrest and apoptosis (Nautiyal et al., 2002; Sahin et al., 2011). Traditionally, studies of ATM in filamentous fungi, such as A. nidulans and N. crassa, have focused on its role in the DNA damage response and in the regulation of the cell cycle (Malavazi et al., 2006, 2007; Wakabayashi et al., 2010). However recently, in A. nidulans, AtmA has also been shown to control mitochondrial function, oxidative phosphorylation and glucose uptake (Krohn et al., 2014). During carbon starvation, responses including autophagy, the shifting metabolism to the glyoxylate cycle, and the secretion of

hydrolytic enzymes were shown to be AtmA-dependent (Krohn et al., 2014) (Fig. 3). The XprG, p53-like transcription factor, was influenced by AtmA activity, where the absence of atmA inhibited starvation-induced protease secretion and cell death in an XprG-dependent manner (Katz et al., 2013; Krohn et al., 2014). Therefore, ATM may represent an ancestral pathway that links mitochondrial function to growth, possibly through influencing TOR and XprG functions. Carbon limitation and stress SnfAmediated responses, including the induction of the Hsf1 heat shock protein and lipid beta-oxidation, were absent in the DatmA strain, suggesting that AtmA may also perform a role in the activation of SnfA (Krohn et al., 2014), as is the case in mammalian cells. The reduced growth of the DatmA strain on cellulose correlated with the reduced transcription and secretion of cellulases (Brown et al., 2013). Despite the necessity of AtmA for the wild-type induction of lignocellulolytic enzymes during carbon starvation or growth on cellulose, CreA::GFP relocalisation under such derepressing conditions was not impaired (Brown et al., 2013). Therefore, the AtmA and XprG pathway appears to influence a CreA-independent mechanism that impacts upon lignocellulolytic enzyme transcription during growth on cellulose. During carbon-starvation of A. nidulans, a number of other proteins involved in the utilisation of intra- and extra-cellular alternative carbon sources were up-regulated (Kim et al., 2011). For example, the expression of aspartate transaminase, which metabolizes aspartate to form the citric acid cycle intermediate a-ketoglutarate (Fahien and MacDonald, 2002), an entry point for amino acid utilisation, and of the aldehyde dehydrogenase (AldA), required for ethanol utilisation, was increased (Flipphi et al., 2003). The CpcA transcription factor of the cross-pathway control, which responds to an imbalance in the amino acid pool by derepressing amino acid biosynthesis, and the AmrD transcription factor that

Fig. 3. The signalling pathways involved in the carbon limitation stress response which is activated in A. nidulans during growth on lignocellulose as a sole carbon source. A reduction in cellular carbon, glycolysis and respiration causes a drop in intracellular energy levels and mitochondrial dysfunction during growth on lignocellulose. These conditions result in the cAMP-independent route of PKA activation, for which the exact function remains unclear. The lack of saccharides for primary metabolism results in the need to switch carbon metabolism to fatty acid/amino acid catabolism, where the pyruvate dehydrogenase kinase (PDK) acts as a metabolic switch for the pyruvate dehydrogenase complex (PDC). In the absence of PDK there is no growth on lignocellulose. The ATM kinase senses mitochondrial dysfunction and activates alternative energy pathway including the TOR/SchA and SakA/SnfA pathways. Induction of these alternative pathways results in CreA derepression and scavenging hydrolytic enzyme secretion. The increased demand for secretion is accompanied by the reduction in available carbon and nitrogen, which jointly impact on endoplasmic reticulum (ER) functionality and protein folding. This results in hacA splicing (hacu = unspliced; hacAi = induced) and translation which induces the unfolded protein response (UPR). The repression under secretion stress (RESS) and UPR responses reduce protein loading into the ER and enhance secretion capacity. Legend: grey = unactive; blue = activate; G = glucose; arrow = induction; bluntended arrow = repression; red X = absence of glucose; UUR = unidentified upstream regulator. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)



regulates amino acid catabolism, were up-regulated in A. nidulans during growth on cellulose (Brown et al., 2013). In addition, amino acid transporters in A. nidulans, A. niger and T. reesei were also upregulated during growth on cellulose or lignocellulose (Brown et al., 2013; Delmas et al., 2012; Ries et al., 2013). Subsequently, this period of carbon limitation, when fungi are first transferred to lignocellulose, may require the utilisation of pre-existing internal and/or external non-carbohydrate carbon sources, such as amino acids. In mammalian cells the mitochondrial pyruvate dehydrogenase complex (PDC) represents an important metabolic switch for fuel selection, which during carbon starvation needs to be inactivated to conserve three-carbon compounds for gluconeogenesis (Randle, 1986). The PDK (pyruvate dehydrogenase kinase) negatively regulates the pyruvate dehydrogenase complex responsible for the uptake of pyruvate into the mitochondria and its conversion into acetyl CoA (Popov et al., 1993). The alteration in ADP/ATP, acetyl-CoA/CoA and NADH/NAD ratios within the mitochondria of carbon-starved mammalian cells results in PDK activation, shifting metabolism towards the catabolism of amino acids and lipids (Randle, 1986; Wu et al., 1999). Germination in Aspergilli is known to require the acquisition of extracellular energy sources (MacCabe et al., 2003). The A. nidulans strain lacking pkpA, which encodes a PDK, is unable to germinate on cellulose or CMC (Brown et al., 2013). When pre-grown in fructose and then transferred to cellulose for 5 days, the DpkpA strain demonstrated reduced cellulase production (Brown et al., 2013). In accordance, pkpA was transiently up-regulated during the initial phase of growth on cellulose and then subsequently repressed, suggesting that metabolism has subsequently shifted back to the utilisation of carbohydrates. Therefore, PkpA performs an essential role in shifting metabolism to the use of alternative non-carbohydrate carbon sources during carbon limitation, which permits growth on cellulose (Fig. 3). Autophagy performs a major role in withstanding nutrient limitation stress in S. cerevisiae and filamentous fungi via recycling cellular components. Autophagy is tightly controlled by the highly conserved autophagy-related genes (ATGs) (Nakatogawa et al., 2009). The Atg1p kinase, responsible for the induction of autophagy, is regulated by the essential nutrient sensing kinase TORC1, Sch9p and the PKA pathway (Kamada et al., 2010; Stephan et al., 2009; Yorimitsu et al., 2007). Multiple nutrient sensing kinases with described roles in the regulation of autophagy, including AtmA, PkaA and SchA, were identified as being required for wildtype cellulase production in A. nidulans during growth on cellulose (Brown et al., 2013). In addition, processes involved in autophagy are up-regulated during the growth of filamentous fungi on cellulose (Brown et al., 2013; Delmas et al., 2012; Ries et al., 2013) (Fig. 3). This induction of autophagy is another example of a cellular response that reflects the recalcitrant, nutrient poor, nature of lignocellulose. Traditionally, PKA has been studied for its role in glucose sensing and is known to inhibit autophagy independently of the TOR pathway (Stephan et al., 2009). However, recently the existence of a cAMP-independent PKA pathway has begun to emerge in multiple organisms (Blackstone and Chang, 2011; Brown et al., 2013; Graef and Nunnari, 2011; McInnis et al., 2010). In S. cerevisiae the existence of a glucose-sensing cAMP-independent signaling pathway for activation of PKA has been described previously (Budhwar et al., 2010; Lu and Hirsch, 2005). Starvation in mammalian cells triggers autophagy, yet mitochondria enlarge in size, which has been shown to sustain ATP production and prevent mitophagy (Blackstone and Chang, 2011). This process is the result of the inhibition of mitochondrial fission through PKA-mediated phosphorylation of the GTPase DRP1. However, such a mechanism has not been demonstrated in fungi to date. Nonetheless, during amino acid starvation in S. cerevisiae, defects in mitochondrial

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respiration induce PKA activity, suppressing autophagy (Graef and Nunnari, 2011). In Schizosaccharomyces pombe deletion of the adenylate cyclase caused a marked increase in the levels of the PKA catalytic subunit, which was hyper-phosphorylated (McInnis et al., 2010). Interestingly, in wild-type S. pombe cells, glucose starvation and stationary phase stresses typically associated with reduced cAMP-dependent PKA activity, induced PKA hyperphosphorylation, while this hyperphosphorylation did not occur under any test conditions in S. pombe strains lacking the PKA regulatory subunit (McInnis et al., 2010). These results demonstrate the existence of a cAMP-independent mechanism of PKA catalytic subunit phosphorylation, which may serve as a mechanism for inducing PKA functions specific to conditions where its cAMP-dependent functions are down-regulated. The maintenance of PKA activity in the absence of cAMP has previously been noted in A. nidulans (Fillinger et al., 2002). In addition, PKA activity in A. nidulans has been shown to double during growth on cellulose or during carbon starvation, when compared to growth on simple carbon sources such as glucose, xylose or cellobiose (Brown et al., 2013). However, in filamentous fungi the function of the cAMP-independent route of PKA activation is unknown. Nonetheless, PKA would appear to perform additional functions during carbon limitation, which are regulated in a cAMP-independent manner, that contribute to lignocellulolytic enzyme production. Therefore, the shifting and maintenance of mitochondrial function in lignocellulolytic fungi during the initial period of carbon limitation, post transfer to lignocellulose, appears to play an essential role in providing the required energy to sustain life during the scavenge for an alternative carbon source. 3.4. The role of secretion stress in repression under derepressing conditions In eukaryotes protein secretion involves the transport of proteins through the endoplasmatic reticulum (ER), Golgi apparatus and vesicles to the cell membrane. The industrially adopted microbe, A. niger, is a more efficient secretor of proteins than S. cerevisiae and A. nidulans, and subsequently scientific advancements in this area have focused on this organism. Correct protein glycosylation is essential for protein folding, ER functionality and secretion (Geysens et al., 2009). Protein glycosylation is a high energy consuming process, requiring ATP and also glucose as a substrate. Inhibition of glycosylation or the induction of secretion stress, via the addition of dithiothreitol (DTT), tunicamycin or brefeldin A, results in the accumulation of misfolded proteins in the ER and the induction of the unfolded protein response (UPR) (Traver et al., 2000; Patil and Walter, 2001). Triggering the UPR results in the up-regulation of the secretory system including chaperones, foldases, glycosylation enzymes, vesicle transport proteins and ER-associated degradation proteins, while genes that encode secreted enzymes, are down-regulated (Guillemette et al., 2007). The application of DTT or brefeldin A to T. reesei, decreases the mRNA level of secreted protein-encoding genes, including cellobiohydrolases and hydrophobins, but not the intracellular b-glucosidases (Saloheimo et al., 2002). In A. nidulans and T. reesei secretion stress also induces the cross-pathway control regulator CpcA, which influences amino acid and protein biosynthesis and ER functionality (Arvas et al., 2006). The repression under secretion stress (RESS) response, which specifically down-regulates genes encoding for secreted proteins, appears to occur under conditions inductive of the UPR and is CRE1-independent, as transcripts where still down-regulated in the cre1 mutant strain (Pakula et al., 2003; Saloheimo et al., 2002). Therefore, transfer from growth on a monosaccharide to lignocellulose results in a reduction in available energy and/or glucose, which will impact glycosylation and cause the


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accumulation of misfolded proteins. Simultaneously, an increased demand for enzyme secretion further stresses the secretion system (Fig. 3). In S. cerevisiae and filamentous fungi, the HAC1 transcription factor, that activates the UPR response, is modulated by the splicing of an unconventional intron (Mori et al., 2000; Saloheimo et al., 2003). The transfer of S. cerevisiae from fermentable to non-fermentable carbon sources resulted in increased HAC1 splicing, previously associated with the UPR (Kuhn et al., 2001). The demand of producing heterologous proteins in fungi, for industrial or scientific purposes, induces secretion stress. Genetic modifications of the UPR have been shown to enhance heterologous protein secretion, demonstrating the efficiency of the UPR to assist in secretion (Valkonen et al., 2003). The construction of an A. niger strain where HacA was constitutively activated enabled the transcriptional assessment of HacA function. In addition to known HacA targets such as ER foldases and chaperones, gene ontology analyses revealed the up-regulation of genes involved in protein glycosylation, intracellular protein transport and exocytosis (Carvalho et al., 2012). The two A. nidulans transcripton factors involved in the UPR, hacA and cpcA, were upregulated during growth on lignocellulose (Brown et al., 2013), while the state of hacA splicing was unknown, indicating an increased requirement for protein secretion under such conditions. In N. crassa, an increase in hac1 splicing is observed when N. crassa is grown on cellulose, consistent with up-regulation of genes involved in secretion stress (Benz et al., 2014). Therefore, the UPR and hacA splicing mechanism, which has received significant industrial attention for its properties in enhancing protein secretion efficiencies, may represent a key mechanism to enable fungi to secrete sufficient lignocellulolytic enzymes under carbon limiting conditions. 4. Signalling for the elevated transcriptional induction of polysaccharide-specific hydrolytic enzymes Despite carbon limitation inducing the transcription of hydrolytic enzymes, as demonstrated in Aspergilli and N. crassa (Krohn

et al., 2014; Xie et al., 2004), lignocellulolytic enzyme transcription and secretion is further dramatically up-regulated by the detection of the saccharides released during polysaccharide breakdown. The regulation and production of this enhanced hydrolytic arsenal, fitting to the detected substrate, is coordinated through the action of pathway-specific transcription factors (Fig. 4). Significant progress has been made on this subject, such as the identification of the pathway-specific transcription factors and their inducers. However, the hierarchical organisation of transcriptional control when multiple, overlapping, DNA-binding proteins are involved remains to be clarified. In addition, how the saccharide inducers of lignocellulolytic enzymes are sensed remains unknown. 4.1. Polysaccharide-responsive transcriptional inducers Recent transcriptomic and genetic studies of N. crassa during growth on cellulose identified two cellulase-inducing transcription factors, Clr1 and Clr2, which are highly conserved in Aspergilli and have been shown to be required for cellulase production (Coradetti et al., 2012). In A. oryzae the homologue of Clr2, ManR, is a known positive regulator of mannolytic enzymes that also acts as a regulator of cellulolytic enzymes including b-glucosidase, cellobiohydrolases and endoglucanases (Ogawa et al., 2013). In A. nidulans a cellulose responsive cis-element in the promoter of the major endoglucanases, eglA and eglB, is essential for gene induction in the presence of cellulose (Endo et al., 2008). The SRF-MADS box protein, McmA, has subsequently been shown to bind to the consensus CC(A/T)6GG sequence in the eglA/B promoter and represents a novel regulator of cellulolytic gene expression (Yamakawa et al., 2013). In T. reesei deletion of the transcriptional regulator of b-glucosidase genes, BGLR, increases cellulase production during growth on cellobiose, a response which is proposed to be caused by an inability to produce a glucose signal for CCR (Nitta et al., 2012). BGLR shows limited homology to the A. oryzae amylase regulator, AmyR, which is involved in starch degradation (Tani et al., 2001). The detection of starch and maltose induces AmyR binding

Fig. 4. The signals and transcriptional activators responsible for the induction of cellulases and xylanases during growth on lignocellulose. Scavenging hydrolytic enzymes result in the release of saccharide (cellobiose, xylobiose) that act as potent pathway specific inducers. In the case of T. reesei, b-glucosidases convert cellobiose into sophorose, which act as the inducer. Lignocellulose detection results in the induction of numerous transcription factors that overlap in their regulation of the lignocellulolytic regulon. These transcription factors interact with accessory proteins, such as the Hap complex, which modifies chromatin structure. Lignocellulolytic gene transcription is fine tuned via additional stimuli, including pH and nitrogen availability, through the action of the PacC and AreA transcription factors. Note: the protein identifiers are based upon A. nidulans, where possible, and appear in blue. T. reesei specific transcription factors and signals appear in grey. Legend: G = glucose, X = xylose, PPP = pentose phosphate pathway; UUR = unidentified upstream regulator. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)



to the CGGN8CGG or CGGAAATTTAA sequence in the promoter of genes involved in starch degradation, which in A. niger also includes a b-glucosidase. Hemicellulase utilisation is controlled by two widely studied transcriptional regulators. The xylanase regulator, XlnR, which binds to the GGCTAR motif, is constitutively expressed and post-translationally activated via proteolytic cleavage of the C-terminus, resulting in nuclear import, when in the presence of xylose (Hasper et al., 2004; Mach-Aigner et al., 2008; Stricker et al., 2006). The arabinolytic regulator AraR is induced in the presence of arabinose and also regulates pectinases and pentose catabolism. Interestingly, the partially overlapping XlnR and AraR pathways appear to function antagonistically. In the wild-type A. nidulans background, XlnR-dependent genes are expressed on xylose, while AraR-dependent genes are expressed on arabinose. However, in the DxlnR or DaraR strains, xylanolytic genes were expressed on arabinose and arabinolytic genes on xylose (Battaglia et al., 2011). Analyses of the transcriptional profile of multiple lignocellulolytic fungi on cellulose have demonstrated the dual activation of both cellulases and xylanases (Brown et al., 2013; Znameroski et al., 2012). Multiple positive transcriptional regulators of cellulase expression also influence the cellulose-induced production of xylanases. In T. reesei ACE2 binds to the 50 -GGCTAATAA site in the promoter of major cellulase and xylanase genes modulating their expression in response to cellulose (Aro et al., 2001). In response to cellobiose- and cellulose, the A. aculeatus ClbR regulator of cellulase and xylanase induction functions in both XlnR-dependent and -independent pathways, while there was no impact on xlyanase production during growth on xylose (Kunitake et al., 2013). Conversely, the major xylanase regulator, XlnR/XYR1, binds to the promoters of xylanolytic and pentose catabolic genes in addition to cellulase-encoding genes (Battaglia et al., 2011; Hasper et al., 2004; Mach-Aigner et al., 2008; Stricker et al., 2006). Less prominent polysaccharides in lignocellulose include galactomannan, pectin and inulin. In A. nidulans a two-step galactose regulatory system, consisting of GalX and GalR, exists, where GalX regulates the expression of galR and some galactose metabolic genes, while the majority are under the control of GalR (Christensen et al., 2011). Disruption of the mannanolytic regulator-encoding gene, manR in A. oryzae, results in reduced growth on galactomannan (Ogawa et al., 2012). The pectinolytic regulator RhaR responds to galactouronic acid, rhamnose and arabinose and controls most pectinases and the rhamnose catabolic pathway (Watanabe et al., 2008). The gene encoding the regulator of inulin degradation, inuR, is induced by the detection of sucrose. InuR demonstrates similarity to AmyR in A. oryzae and is proposed to have arisen from a common ancestor (Yuan et al., 2008). In the 50 region upstream of many eukaryotic genes a CCAAT sequence is found, which is involved in protein binding. The first CCAAT binding complex to be described was the Hap2/3/4/5 complex in S. cerevisiae, which regulates the expression of oxidative phosphorylation in response to growth on non-fermentable carbon source (Gancedo, 1998). CCAAT sequences exist in the promoter of many lignocellulolytic genes of filamentous fungi. The homologous Hap complex in A. nidulans, termed AnCF, performs a role in creating an open chromatin structure, facilitating gene transcription (Steidl et al., 1999). Mutation of the CCAAT sequence or the ACE2-binding motif in the promoter of cbh2 within T. reesei results in a reduction in gene transcription, while simultaneous deletion causes a complete loss of transcription (Zeilinger et al., 2001). Hence, transcription factors interact with accessory proteins to facilitate/fine-tune lignocellulolytic gene transcription.

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4.2. Lignocellulolytic pathway inducers The identification of soluble molecules released from lignocellulose that elicit the activation of lignocellulolytic pathways are of keen biotechnological interest. As described above, multiple soluble saccharides induce different, or multiple, polysaccharide catabolic processes. However, which elicitors are released during, and important to growth on lignocellulose is not clear. Whether these soluble saccharides act as ligands for external receptors or they are detected via their internalisation represents another important unanswered question. Progress in this area has focused specifically on the induction of cellulases. Most of the studies of the elicitors for cellulase induction have utilised the exogenous supply of such compounds in excess, which again is abstract from the real conditions encountered during growth on cellulose. Fungi possess an uptake system for cellobiose, the major soluble end product of cellulose degradation (Kubicek et al., 1993). Cellobiose, in low concentrations, induces cellulase production in a range of lignocellulolytic fungi (Chikamatsu et al., 1999; Eberhart et al., 1977; Vaheri et al., 1979). In contrast, at high concentrations cellobiose does not induce cellulases in N. crassa (Znameroski et al., 2012), while in A. nidulans high cellobiose concentrations have been shown to promote CreA nuclear localisation and CCR (Brown et al., 2013). The enzymatic action of b-glucosidases on cellobiose can either form sophorose via a transglycosylation reaction or glucose through hydrolysis. In T. reesei sophorose is produced by an extracellular b-glucosidase, which potently induces cellulase production (Sternberg and Mandels, 1979). However, sophorose does not demonstrate the same inducing efficiency in Aspergilli or N. crassa, suggesting that T. reesei adopted a unique mechanism, possibly due to its evolutionary origin as a mycoparasite. In N. crassa the inhibition of b-glucosidase activity increases cellulase activity, while the simultaneous deletion of three b-glucosidases (D3bG), that were highly induced during growth on cellulose (including an extracellular, a cell wall anchored and an intracellular b-glucosidase), also increased cellulase activity during growth on cellobiose (Znameroski et al., 2012). The introduction of the DcreA into the D3bG strain (DcreA D3bG) further increased cellulase activity, while the simultaneous deletion of two cellodextrin transporters (CDT-1 and -2) in the D3bG background generated a strain that was unable to respond to cellobiose. Cellulase induction during growth on cellobiose in the Dcdt1 Dcdt2 D3bG strain resulted in a cellulase induction profile similar to a carbon starvation response, while induction in the DcreA D3bG resembled wildtype cellulase production on cellulose (Znameroski et al., 2013). Subsequently, the transport of cellobiose appears to be essential for full level cellulase induction, while the produced glucose has a repressing effect. These studies demonstrate that even during growth on lignocellulose, glucose-induced CCR is still having an inhibitory effect on enzyme production. 4.3. Additional pathways contributing to lignocellulolytic activity In addition to sensing the saccharide content of the respective environment, other non-saccharide related factors influence lignocellulolytic enzyme secretion, including pH, light, nitrogen availability and the developmental stage of the fungus (Fig. 4). Ambient pH influences multiple processes in fungi including extracellular enzymatic activity, sugar uptake and internal metabolism. Consequently, the secretion of enzymes applicable to the respective pH is controlled and regulated in lignocellulolytic fungi by the alkaline-responsive transcription factor, PacC (Penalva et al., 2008). Deletion of pacC in N. crassa and the plant pathogenic fungus S. sclerotiorum resulted in a growth defect on cellulose and reduced hydrolytic enzyme secretion, suggesting involvement of PacC in


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lignocellulolytic enzyme production (Coradetti et al., 2012; Rollins, 2003). Transfer to a lignocellulosic substrate resulted in the modulation of multiple amino acid-related processes in several fungi, indicating an alteration in C:N ratio as well as an additional strain exerted upon nitrogen metabolism. Therefore, nitrogen availability also influences cellulase secretion, as demonstrated by the deletion of the global nitrogen regulator-encoding gene, areA, which resulted in reduced cellulase production in A. nidulans (Lockington et al., 2002). The T. reesei ACE1 repressor homologue in A. nidulans, StzA, has been shown to compete or interact with AreA in the promoter of genes involved in amino acid metabolism (Chilton et al., 2008). Interestingly, binding sites of the cross-pathway control regulator, CpcA (50 -TGAC/GTCA) exist in the promoters of both ace1 and stzA, while the addition of methionine can enhance cellulase production (Gremel et al., 2008). In A. nidulans the cross-pathway control regulator CpcA influences amino acid biosynthesis and is involved in the UPR; as well as being induced during growth on cellulose (Brown et al., 2013). This further demonstrates the impact that growth on cellulose has on the intracellular C:N ratio. The cross-talk between carbon and nitrogen pathways has been suggested to involve the TOR signalling pathway, which is yet to be directly linked to lignocellulolytic enzyme regulation. Collectively, this data suggests the existence of a link between lignocellulolytic enzyme production and nitrogen availability, through the actions of CpcA, AreA and ACE1/StzA. Light influences circadian rhythms and the transcription of numerous genes in fungi (Lewis et al., 2002). In N. crassa the two white collar, blue-light, photoreceptors, WC-1 and WC-2, interact to form the white collar complex (WCC), which functions in collaboration with the photoadaptor VVD (Idnurm and Heitman, 2005). In N. crassa, the WCC binds to the promoter of the cellulase inducer clr1 (Smith et al., 2010). This positive effect on cellulase transcription is counteracted by an unknown posttranslational mechanism which results in reduced cellulase activity in the presence of light. Deletion of WC-1, WC-2 and VVD influence cellulase production (Schmoll et al., 2012). Deletion of WC-1 and WC-2 (Dwc-1 and Dwc-2 strains) results in a downregulation of amino acid and glycogen metabolism, energy supply and protein folding. The ability to respond to energy deprivation and amino acid starvation, via the up-regulation of the crosspathway control regulator, cpc1, was subsequently shown to be beneficial to cellulase gene expression (Schmoll et al., 2012). Transcriptome analyses of T. reesei have also demonstrated that light levels influence cellulase production. Functional genomics has identified WC-1, WC-2 and VVD homologues in T. reesei, which have been correlated with the positive regulation of cellulase transcription in the presence of light. In addition, in T. reesei, the PKA pathway has been shown to influence cellulase production in response to light (Schmoll et al., 2009; Schuster et al., 2012; Seibel et al., 2009; Zhang et al., 2012), linking this nutrient sensing pathway to light-mediated gene regulation. In T. reesei and several phytopathogens the up-regulation of lignocellulolytic enzymes is also coordinated with sexual and asexual development (Chen et al., 2012). This possibly represents an ancestral evolutionary link to the requirement for sex in order to sporulate and in turn be disseminated from the current location, where nutrients have become limited poor. In accordance, a N. crassa strain lacking the pheromone gene ppg1 showed lower cellulase activity (Schmoll et al., 2012). Therefore, in addition to the saccharide and energetic status signals, generated during carbon limitation and growth on lignocellulose, multiple other environmental stimuli result in the fine-tuning of lignocellulolytic pathways.

5. Conclusions The mechanisms responsible for the coordination of cellular metabolism and the secretion of lignocelluloytic enzymes with carbon source availability remain to be clarified. The multifaceted regulation of lignocellulolytic enzyme production represents a complex and dynamic process involving numerous signals and feedback loops. The simple removal of repressor proteins does increase enzyme transcription in the presence of the elicitor, but also has an undesirable impact upon the microbe’s health. Therefore, a greater understanding of lignocellulolytic gene induction/repression is required. Despite substantial advancement in the identification of saccharidespecific transcription factors, the current understanding of the elicitor molecules and pathways involved in transcription factor regulation remains poor. S. cerevisiae has and will continue to serve as a useful comparative tool from which signalling pathways in filamentous fungi can be reconstructed. The positive signals and pathways inducing CCR demonstrate a high degree of conservation in S. cerevisiae and filamentous fungi. However, the true elicitors of lignocellulolytic enzyme induction and the pathways which regulate them are poorly defined. What is clear is that not only soluble saccharides influence gene induction, but multiple additional environmental and intracellular cues coordinate efficient enzyme secretion with the ecological niche. A better understanding of induction/repression and the signal transduction pathways modulating these processes will assist in the development of new strategies for enhanced lignocellulolytic enzyme secretion. Genetic manipulations that circumvent regulatory feedback loops or metabolic/biosynthesis bottlenecks will improve enzyme production. For example, metabolic engineering for strains with enhanced, concomitant utilisation of alternative non-carbohydrates and polysaccharides could assist in the capacity to synthesize and secrete lignocellulolytic enzymes during periods of carbon limitation. Comparisons of numerous filamentous fungal genomes, including saprophytes and plant pathogens, has revealed the conservation and divergence in their lignocellulolytic potential and regulatory components, reflecting their occupied niche. In addition to CAZymes, numerous genes of unknown function are coordinately up-regulated during growth on lignocellulose, including novel secreted proteins. The study of these unknowns may subsequently result in the identification of new accessory proteins involved in lignocellulose deconstruction. Comparative analyses of the wealth of descriptive ‘‘omic’’ data sets, describing the response to growth on various lignocellulosic feedstocks, will permit the design of enzymatic cocktails fitting to a specific substrate, subsequently increasing hydrolysis efficiencies. In addition, transfer of discovered, and yet to be discovered, mechanisms for polysaccharide utilisation in filamentous fungi into industrial S. cerevisiae will facilitate the development of strains capable of dually fermenting cellobiose and xylobiose oligomers (Galazka et al., 2010; Ha et al., 2011; Kim et al., 2014). Thus, the fundamental knowledge of how filamentous fungi utilise lignocellulose will benefit green chemistries and the bioethanol industry two fold by improving lignocellulose hydrolysis and lignocellulosic hydrolysate fermentation. Author contributions NAB, and GHG collaborated in the concept and preparation of the manuscript. LR contributed to the preparation of the manuscript. Acknowledgments We would like to thank the Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de



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