Appl Microbiol Biotechnol (2014) 98:4829–4837 DOI 10.1007/s00253-014-5707-6

MINI-REVIEW

Complex regulation of hydrolytic enzyme genes for cellulosic biomass degradation in filamentous fungi Shuji Tani & Takashi Kawaguchi & Tetsuo Kobayashi

Received: 27 December 2013 / Revised: 17 March 2014 / Accepted: 17 March 2014 / Published online: 11 April 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Filamentous fungi produce cellulolytic and hemicellulolytic enzymes in response to small inducer molecules liberated from cellulosic biomass. Enzyme production is mainly regulated at the level of transcription. The first transcription factor identified as being involved in cellulosic biomass degradation was XlnR, which mediates D-xylose-triggered induction of xylanolytic and cellulolytic genes in Aspergillus. XlnR has played the leading role for over a decade in studies aimed at clarification of gene regulation related to cellulosic biomass degradation. Very recently, several new transcription factors were identified, namely Clr-1/2 in Neurospora; ManR, McmA, and ClbR in Aspergillus; and BglR in Trichoderma, all of which participate in the regulation of cellulolytic and/or hemicellulolytic enzyme production. Furthermore, as well as the carbon sources available, other factors such as light signaling and anti-sense RNA accumulation have been shown to contribute to this regulation. Here, we review the recent advancements demonstrating that multiple factors coordinately regulate the expression of cellulosic biomass degrading enzyme genes. Keywords CAZy genes . Gene regulation . Filamentous fungi . Transcription factor . Zn(II)2Cys6 binuclear cluster domain S. Tani (*) : T. Kawaguchi Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan e-mail: [email protected] T. Kobayashi Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan

Introduction Biorefining of lignocellulosic biomass is considered to be an important alternative to oil refining for sustainable development of the human economy and society (Liu et al. 2013). However, industrial production of fuels and useful chemicals from lignocellulose still requires extensive basic and applied work because of several problems yet to be solved. The major stumbling block is that lignocellulosic biomass is a recalcitrant substrate for bioconversion. The crystalline nature of cellulose makes enzymatic hydrolysis inefficient, leading to a requirement for unacceptably large amounts of enzymes. Lignin prevents access of enzymes to substrate carbohydrates; therefore, energetically expensive and corrosive chemical pretreatments are required to remove it (Somerville 2006). One approach to reduce the cost of cellulosic biomass decomposition is enhancement of hydrolysis efficiency and enzyme production. A typical example of the former can be seen in the improvement of the Trichoderma reesei (Hypocrea jecorina) cellulolytic enzyme mixture for efficient decomposition of pretreated corn stover. T. reesei is a renowned filamentous fungus that produces an exceptionally large amount of cellulases that are superior for decomposition of crystalline cellulose. The hydrolytic activity of the T. reesei cellulolytic enzyme mixture towards pretreated biomasses such as corn stover and rice straw was significantly enhanced by supplementation with crude enzyme preparations nominally enriched in xylanase, pectinase, and β-glucosidase activity (Berlin et al. 2007; Kawai et al. 2012; Nakazawa et al. 2012). To reduce the cost of enzyme production, it is rational to breed fungal strains based on an understanding of the molecular mechanisms controlling the CAZy genes. The regulatory mechanisms of these genes have been studied for over a decade in the model filamentous fungi Aspergillus nidulans and Neurospora crassa,

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and in the industrial strains Aspergillus oryzae, Aspergillus niger, and Aspergillus aculeatus. According to the currently accepted view of cellulase and hemicellulase induction mechanisms, low molecular weight compounds, such as monosaccharides or disaccharides, trigger enzyme production because polysaccharides are too large to enter the cells and hence be recognized as inducers (Kubicek et al. 1993). These inducers are produced by hydrolysis of cellulose and hemicellulose by small amounts of constitutively expressed cellulases and hemicellulases, and in some cases, they need to be further converted by transglycosylation prior to functioning as physiological inducers. Various mono- and disaccharides derived from cellulosic biomass are proposed to be inducers for cellulase production: D-xylose in A. niger (de Souza et al. 2011), cellobiose in A. oryzae (Marui et al. 2002), gentiobiose in Penicillium purpurogenum (Kurasawa et al. 1992), and sophorose in Aspergillus terreus and T. reesei (Hrmova et al. 1991; Mandels et al. 1962). Pure oligosaccharides, such as β-cellobiono-1,5-lactone, D-xylose, xylobiose, galactose, and lactose, have been reported to induce cellulase and hemicellulase production in T. reesei (Aro et al. 2005; Karaffa et al. 2006; Kubicek and Penttilä 1998; Morikawa et al. 1995; Stricker et al. 2006, 2007). However, how these physiological inducers control the activity of downstream transcription factors, or even what transcription factors are involved in cellulase and hemicellulase regulation, is not fully understood. XlnR in A. niger was the first transcription factor identified that controls cellulase and hemicellulase gene expression (van Peij et al. 1998), and possesses the Zn(II)2Cys6 binuclear cluster domain specifically found in fungal transcription factors (Todd and Andrianopoulos 1997). XlnR plays a key role in xylan-triggered induction of not only xylanases and the accessory enzymes involved in xylan degradation, but also cellulases (Stricker et al. 2007). Cellobiose as well as D-xylose triggers XlnR-dependent expression of xylanolytic and cellulolytic enzymes in A. oryzae (Marui et al. 2002; Noguchi et al. 2009). In addition, XlnR-independent signaling pathways for induction of cellulolytic genes have been reported in A. nidulans (Endo et al. 2008), A. oryzae (Marui et al. 2002), and Fusarium graminearum (Brunner et al. 2007). XlnR orthologs are highly conserved among ascomycetic filamentous fungi, implying that they share similar functions. However, Xyr1, the XlnR homolog in T. reesei, appears to have more diverse functions. It governs the expression of signals from various inducers (xylose, xylobiose, and sophorose) and regulates all modes of gene expression (basal, derepressed, and induced) of the major cellulase and hemicellulase encoding genes (Stricker et al. 2007). Just recently, research on the regulation of cellulase and hemicellulase expression entered a new stage with the identification of novel transcription factors involved in the XlnRindependent pathway in N. crassa, A. nidulans, A. aculeatus,

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and A. oryzae. In this mini-review, we summarize recent advancements in identification and characterization of new regulators and signaling pathways controlling the expression of cellulosic biomass degrading enzyme genes.

Newly identified transcription factors controlling the expression of cellulase, hemicellulase, and β-glucosidase genes in filamentous fungi Ninomiya et al. established a highly efficient gene-targeting technique using deletion mutants of the ku80 or ku70 genes involved in non-homologous end-joining pathways of DNA double-strand break repair (Ninomiya et al. 2004). This technique allowed us to rapidly construct gene knockout libraries of Neurospora and Aspergillus, leading to the identification of multiple transcription factors involved in cellulase and hemicellulase gene regulation (Colot et al. 2006; Ogawa et al. 2012). Coradetti et al. screened the Neurospora transcription factor deletion set for mutants with deficient growth on cellulose (Avicel). From this screening, they found not only transcription factors with known influence on cellulase production, including nit-2 (a homolog of the nitrogen regulator areA) (Lockington et al. 2002), pacC (pH sensing) (Tilburn et al. 1995), and cre-1 (carbon catabolite repression) (Sun and Glass 2011), but also novel regulators essential for cellulose degradation in N. crassa, namely clr-1 and clr-2 (Coradetti et al. 2012). Both CLR-1 and CLR-2 are positive transcription factors that contain a Zn(II)2Cys6 binuclear cluster domain. When inducing molecules such as Avicel exist around N. crassa, CLR-1 promotes the expression of a variety of genes including those encoding a cellodextrin transporter, proteins involved in protein secretion, cellulases, hemicellulases, and β-glucosidase, as well as the clr-2 gene. CLR-2 regulates only a small number of genes; these are mainly cellulase and hemicellulase genes. The expression profiles of 212 genes specifically induced on Avicel were classified into four modes based on the effect of clr-1 and clr-2 deletion on their expression, namely clr-1/2 dependent, clr-modulated, clr-1 dependent, and clr-independent. No genes were classified as clr-2 dependent, implying that CLR-1 senses the presence of inducing molecules such as cellobiose and stimulates induction of the Avicel regulon. CLR-2 may induce, in a CLR-1-dependent manner, a portion of the cellulase and hemicellulase genes, accelerating the liberation of inducing molecules in a positive feedback loop (Coradetti et al. 2012). The homologs of CLR-1 and CLR-2 in A. nidulans, designated ClrA and ClrB, are also involved in regulation of cellulolytic and hemicellulolytic genes. However, it seems the regulatory circuit is not fully conserved because deletion of clrA has a less significant effect on cellulase and hemicellulase gene expression compared with the deletion of clr-1 in N. crassa. ManR, a DNA-binding protein possessing the Zn(II)2Cys6 binuclear cluster domain, was identified as required for

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endo-β-mannanase production by screening an A. oryzae RIB40 gene-disruptant library that covered about 400 genes involved in transcriptional regulation (Ogawa et al. 2012). It controls the expression not only of mannanolytic enzyme genes but also of cellulolytic enzyme genes, based on DNA microarray analysis followed by quantitative PCR (Ogawa et al. 2012, 2013). ManR is orthologous to ClrB, suggesting the involvement of ClrB in mannanolytic gene regulation. ManR-dependent gene expression is induced by β-mannan and β-mannobiose, and also by CMC and Avicel. Gene regulation by ManR is analogous to that by XlnR, considering that XlnR regulates the expression of xylanolytic and cellulolytic genes and that their expression is triggered not only by xylan and D-xylose but also by Avicel and cellobiose, at least in A. oryzae (Marui et al. 2002; Noguchi et al. 2009; Stricker et al. 2008). ManR and XlnR regulate overlapping cellulolytic genes, including the cellobiohydrolase genes (celC, celD, cbhD) and the β-glucosidase gene (bgl5). However, they regulate different endoglucanase genes; while the eglA gene is the target of ManR, the celA and celB genes are under the control of XlnR (Fig. 1) (Ogawa et al. 2013). Exhaustive mutational analysis of the eglA promoter in A. nidulans revealed only a single cis-element (cellulose responsive element, CeRE) essential for induction, with similar sequences distributed among various cellulase promoters (Endo et al. 2008). CeRE contains a consensus sequence for

Repressing condition

the binding of SRF-type MADS box proteins, CC(A/T)6GG (Endo et al. 2008; Shore and Sharrocks 1995), and the sole SRF-type MADS box protein McmA in A. nidulans does bind to CeRE (Yamakawa et al. 2013). Furthermore, A. nidulans carrying the mcmAI70A gene, which encodes a mutant McmA with an isoleucine 70 to alanine substitution, displays impaired induction of eglA, eglB, and cbhA genes, implying that McmA regulates the cellulase genes by binding directly to their promoters. In general, MADS box proteins regulate gene expression through interaction with various cofactors. For example, Saccharomyces cerevisiae Mcm1p interacts with various proteins such as α1, α2, Ste12p, Yox1p, Yhp1p, Fkh2p, Arg80p, and Arg81p, and regulates different sets of genes depending on the interacting protein (Messenguy and Dubois 2003). Its functional variation is achieved by mutually exclusive interactions of the Mcm1p homodimer with a transcriptional activator and repressor (Darieva et al. 2010). As described above, McmA regulates at least eglA, eglB, and cbhA, which are also under the control of ClrB. It is plausible that McmA interact with ClrB and its orthologs such as ManR to control cellulase gene expression in Aspergillus, and in fact, McmA and ClrB cooperatively bind to the CeRE of the eglA promoter (Kobayashi et al., 27th Fungal Genetics Conference) (Fig. 1). Cellobiose response regulator (ClbR), a DNA-binding protein possessing the Zn(II)2Cys6 binuclear cluster domain, was identified by screening a T-DNA insertion mutant library that

Inducing condition Mannan

Cellulose

Xylan

-1,4-Mannobiose Cellobiose

Glucose

D-Xylose

ClbR

CreA

ManR

XlnR

McmA

(B)

(C)

(D)

AAA

(A)

AAA AAA

AAA AAA

AAA AAA

AAA AAA

AAA

AAA

Fig. 1 Models of regulation mechanisms for cellulosic biomass degrading enzyme genes in Eurotiomycetes. In the presence of glucose (repressing conditions), CreA represses gene expression. Short lengths of anti-sense RNA tend to be accumulated in a CreA-dependent manner. In the presence of inducing molecules (inducing conditions), inducers activate Zn(II)2Cys6-type transcription factors resulting in the induction of

(B)

AAA

AAA

(A)

(D)

AAA

AAA AAA

AAA AAA

(C)

AAA

AAA

AAA

gene expression. Signaling pathways triggering gene expression can be classified into at least four types: (a) ManR and McmA dependent, (b) ManR dependent, (c) ManR and XlnR dependent, and (d) XlnR dependent. Sense RNA rather than anti-sense RNA increases, which increases enzyme production followed by accelerated liberation of inducing molecules by a positive feedback loop

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was constructed via Agrobacterium-mediated transformation in A. aculeatus (Kunitake et al. 2011, 2013). In A. aculeatus, at least two signaling pathways control gene expression in response to cellulosic biomass, namely the XlnR-dependent and XlnR-independent signaling pathways (Tani et al. 2012). The cellobiose- and cellulose-responsive induction of the FIIIavicelase (cbhI), FII-carboxymethyl cellulase (cmc2), and FIa-xylanase (xynIa) genes is under the control of the XlnRindependent signaling pathway, while the cellulose-, D-xylose-, and arabinose-responsive induction of the FIcarboxymethyl cellulase (cmc1) and FIb-xylanase (xynIb) genes is under the control of the XlnR-dependent signaling pathway. ClbR participates in cellulose-inductive expression regardless of XlnR dependency, but not in xylose- or arabinose-inductive expression. Cellobiose-inductive and XlnR-independent expression, which was confirmed to be under the control of ManR (Kunitake et al., 27th Fungal Genetics Conference), is also under the control of ClbR. Although XlnR-dependent cellobiose induction has been reported in A. oryzae, A. aculeatus XlnR does not respond to cellobiose. This difference is probably because of the 1-deoxynojirimycin added to inhibit β-glucosidase activity in the culture supernatant of A. aculeatus (Tani et al. 2012) (Fig. 1). T. reesei has a long history of strain improvement aimed at hyper-production of cellulases through repeated conventional mutagenesis. The availability of a number of mutants with a mutant family tree makes the organism suitable for discovery of genes affecting cellulase production. The Zn(II)2Cys6 transcription factor BglR (β-glucosidase regulator) was identified by a comparative genomic analysis between the mutant strain PC-37 and its parent KDG-12 (Nitta et al. 2012). One of the 19 SNPs identified by comparison of the genome sequences was located within the DNA-binding region of bglR, and its negative function on cellulase production was confirmed by complementation and deletion analyses. Deletion of bglR causes elevated production of endoglucanases and also reduction of cellobiose hydrolyzing activity. Increased cellulase production may be due to carbon catabolite derepression considering that decreased βglucosidase activity would lead to lower glucose concentrations (Nitta et al. 2012). Alternatively, it might be caused by decreased hydrolysis of inducing molecules such as sophorose. Although Xyr1 governs the regulation of cellulase and hemicellulase gene expression in T. reesei, the β-glucosidase genes seem to be regulated by a different signaling pathway.

Regulatory mechanisms underlying the expression of cellulolytic and hemicellulolytic genes Until recently, research on the regulation of plant cell wall degrading enzymes mainly focused on XlnR orthologs. Progress in identification of novel transcription factors as

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mentioned above has established the basis for a deep understanding of the complex regulatory mechanisms involved in cellulolytic and hemicellulolytic genes. One question that has arisen is how these transcription factors are activated in the presence of inducing molecules. The activity of transcription factors can be regulated at various levels, including transcription, mRNA stability, nuclear import/export, DNA-binding, post-translational modification, protein–protein interaction, and so on. Most of the transcription factors described above are classified into the fungal-specific zinc binuclear cluster superfamily characterized by the Zn(II)2Cys6-type DNA binding motif. Gal4p, which regulates the GAL regulon for galactose catabolism in S. cerevisiae, is the best-characterized transcription factor of the superfamily. Gal4p-dependent transcription is repressed under noninducing conditions because the co-repressor Gal80p binds to the transcriptional activation domain of Gal4p (Johnston 1987; Keegan et al. 1986; Ma and Ptashne 1987). When galactose and ATP are present, they bind to the sensor protein Gal3p, leading to the formation of the Gal3p-Gal80p complex. This in turn causes dissociation of the Gal4p-Gal80p complex, leading to Gal4p-dependent transcription (Lavy et al. 2012; Sil et al. 1999). NADP also destabilizes the interaction between Gal4p and Gal80p and appears to be the initial trigger for activation (Kumar et al. 2008; Li et al. 2010). However, the mechanisms composed of sensor, co-repressor, and transcriptional activator are not applicable to other Zn(II)2Cys6-type transcription factors; the regulatory mechanisms differ from factor to factor even in the same superfamily. In the case of Leu3p, which regulates the leucine biosynthetic pathway in S. cerevisiae, the middle region of the protein represses the function of the Cterminal activation domain by intra-molecular interaction in the absence of α-isopropylmalate (α-ΙΡΜ), a metabolic intermediate of leucine biosynthesis, and this self-masking is released in the presence of α-ΙΡΜ (Poulou et al. 2010; Sze et al. 1992; Goosen et al. 1987; Wang et al. 1999) (Ligand Binding Domain) (Malleret et al. 2001; Poulou et al. 2010). Hap1p is involved in gene regulation in response to heme and oxygen levels. It exists within a large complex containing chaperones such as Hsp90, Hsp70, and Hsp40 (Lan et al. 2004). Binding of heme to Hap1p causes a conformational change of the complex leading to binding to its target sites on various promoters. These S. cerevisiae factors localize to the nuclei regardless of the presence or absence of inducing stimuli, while NirA and AmyR in A. nidulans, which are involved in utilization of nitrate and starch, respectively, accumulate in the nuclei in response to nitrate and isomaltose (Bernreiter et al. 2007; Makita et al. 2009; Murakoshi et al. 2012). Because of the diversity in regulatory mechanisms, it is very important to more deeply understand the characteristic features of each transcription factor to fine-tune its function for industrial use. However, only very limited information is currently available even in the case of XlnR, which was

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discovered over a decade ago. Analysis of various XlnR mutants in A. niger has revealed that a putative coiled-coil region spanning residues 635 to 668 is involved in nuclear import because XlnR1–635 is cytoplasmic and XlnR1–668 is nuclear in the presence of D-xylose. However, it is yet to be clarified whether nuclear accumulation of XlnR is triggered by D -xylose. XlnR 1–668 confers higher productivity on xylanases compared to the wild type. In addition, while Y864F substitution in XlnR causes drastically decreased xylanase productivity, V756F substitution leads to increased productivity with D-glucose as a carbon source. Based on analysis of a number of xlnR mutants, a model of functional domain architecture was proposed, in which the C-terminal part of XlnR downstream of residue 668 contains the activation domain regulated by self-masking and a D-glucose inhibitory region (Hasper et al. 2004). Although it still remains unclear how XlnR activity is regulated, the inducer-triggered phosphorylation of A. oryzae XlnR may provide a clue for clarification of the regulatory mechanisms at the molecular level. XlnR exists as a mixture of variously phosphorylated isoforms in A. oryzae in the absence of D-xylose. Once Dxylose is added, it triggers additional phosphorylation of the protein within 5 min, prior to the accumulation of the xylanase G2 mRNA (Noguchi et al. 2011). This inducer-triggered phosphorylation is reversible; removal of D-xylose causes dephosphorylation back to the mixture found in the absence of the inducer. This implies that the additionally phosphorylated isoforms function in the activation of target genes. Phosphorylation modulates the activity of transcription factors in various ways. It may affect the stability, subcellular localization, DNA binding activity, or transcriptional activity of a transcription factor. Phosphorylation may facilitate protein– protein interaction and oligomerization, or may mark a transcription factor for inactivation as a consequence of its ability to activate transcription (Holmberg et al. 2002). Noguchi et al. indicated that the additional phosphorylation does not affect XlnR stability, and also suggested that it would not regulate the nuclear-cytoplasmic trafficking (Noguchi et al. 2011). Clarification of the precise physiological roles of the multisite phosphorylation of XlnR may provide fundamental and essential information not only for a deep understanding of the complex regulation of xylanolytic and cellulolytic genes but also for innovative applications in the field of biotechnology, considering that multisite phosphorylation provides sophisticated regulation of transcription factors (Holmberg et al. 2002).

Other factors controlling cellulase gene expression in filamentous fungi The expression of cellulosic biomass degrading enzyme genes is regulated by various global regulators. The protein

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methyltransferase LaeA is a global regulator of secondary metabolite gene clusters such as sterigmatocystin formation and controls sexual and asexual developmental processes in Ascomycota. LAE1 in T. reesei, which is the ortholog of LaeA, regulates the expression of cellulases, polysaccharide hydrolases, and xyr1 (Seiboth et al. 2012). However, LAE1 does not rescue sterigmatocystin formation of the laeA-null mutant of A. nidulans. Therefore, the biological roles of A. nidulans LaeA and T. reesei LAE1 are much less conserved than hitherto thought. Transcriptome analysis revealed that antisense transcripts (AS) predominate on glucose medium (repressing conditions for cellulase production), while the sense transcript (S) is expressed on wheat straw medium (inducing conditions) in A. niger (Delmas et al. 2012). Examples of such genes include transporters and permeases, CAZy enzymes, and a putative lipase. Many genomic loci contain transcription units on both strands; therefore, two oppositely oriented transcripts can overlap. Often, one strand codes for a protein, whereas the transcript from the other strand is non-encoding. Such natural antisense transcripts (NATs) can negatively regulate the conjugated sense transcripts. NATs are highly prevalent in a wide range of species—for example, around 15 % of human protein-encoding genes have an associated NAT (Lapidot and Pilpel 2006). Interestingly, sense transcription is seen in both glucose and straw conditions in the ΔcreA strain, suggesting that the AS/S ratio switch is regulated either directly or indirectly by CreA (Delmas et al. 2012). Light is also a cue that controls the expression of the cellulosic biomass degrading enzyme genes in T. reesei and N. crassa. The photoreceptors White Collar 1 and 2 (WC-1 and WC-2), transcription factors of the fungal GATA zinc finger family containing PAS domains, and VVD, a third photoreceptor also containing a PAS domain, are involved in the regulation of cellulase gene expression in N. crassa (Schmoll et al. 2012). The cyclic AMP (cAMP) pathway, a central signaling cascade with crucial functions in all organisms, regulates cellulase gene expression in T. reesei (Schuster et al. 2012). Specifically, transcripts of cellulase genes considerably increase in darkness compared with those in light when grown on lactose, and such light responsiveness is strongly enhanced in mutant strains lacking cAMPdependent protein kinase A (PKA) catalytic subunit 1 (PKAC1) and adenylate cyclase (ACY1). ACY1 has a consistently positive effect on cellulase gene expression in both light and darkness, while PKAC1 influences the transcript levels of cellulase genes positively in light but negatively in darkness. The transcript levels of XYR1 also show a similar pattern in the mutant strains. Therefore, the regulatory output of the cAMP pathway may be established via adjustment of XYR1 abundance in T. reesei (Schuster et al. 2012).

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Improving enzyme production by enhancing the expression of regulators Transcription machinery engineering is a sophisticated means of improving arbitrary enzyme production (Alper et al. 2006; Alper and Stephanopoulos 2007). Overexpression of a pathway-specific transcriptional activator, xlnR, in A. oryzae resulted in elevated xylanolytic and cellulolytic activities in the culture supernatant, in which nearly 40 secreted proteins were increased. At the transcriptional level, nearly one third of the CAZy genes were confirmed to be upregulated by overexpression of xlnR, including hydrolytic genes for degradation of β1,4-xylan, arabinoxylan, cellulose, and xyloglucan, and catabolic genes for the conversion of D-xylose to xylulose-5phosphate (Noguchi et al. 2009). Overexpression of manR in A. oryzae also successfully increased mannanolytic and cellulolytic enzyme production and the expression of genes under the control of ManR (Ogawa et al. 2013). Overexpression of clr-2 in N. crassa and clrB in A. nidulans increased the expression of almost 50 % of the celluloseresponsive genes, including a subset of 28 genes encoding cellulolytic and hemicellulolytic enzymes (Coradetti et al. 2013). However, the function of transcription factors is not always conserved in filamentous fungi. While overexpression of clr-2 causes elevated expression of cellulase genes even under non-inducing conditions, overexpression of clrB leads to sufficient expression of the genes only under inducing conditions in A. nidulans (Coradetti et al. 2013). Although how CLR-2 and ClrB respond to cellulose needs to be determined for further understanding of the regulatory mechanisms underlying cellulase gene expression, these differences could be yielded by various types of paralogs because their numbers in Aspergillus (Eurotiomycetes) and Neurospora (Sordariomycetes) are significantly different (Table 1). It has been reported that Xyr1 in T. reesei governs all modes of inducible expression of the CAZy

genes (Stricker et al. 2007), while AraR, which is 32 % identical to XlnR in A. niger, regulates the expression of the genes involved in pentose utilization (Battaglia et al. 2011a, b). Because gene duplication followed by evolution of new functions contributes to the building of new regulation circuits (Baker et al. 2013; Coyle et al. 2013), comprehensive analyses of paralogs are also necessary to establish rational systems to improve enzyme production.

Concluding remarks The regulation mechanisms of the CAZy genes involved in cellulosic biomass degradation have been investigated mainly in industrial strains such as A. niger, A. oryzae, A. aculeatus, and T. reesei as well as the model strains, A. nidulans and N. crassa. It is considered that the hydrolytic system of these fungi from cellulosic biomass to simple sugars is basically similar at an enzymatic level, although they are taxonomically different; Aspergillus belongs to the class Eurotiomycetes, while Trichoderma and Neurospora belong to the class Sordariomycetes. The critical difference of these classes is the number of genes encoding the CAZy enzymes and transcription factors. The genomes of Aspergillus encode significantly more CAZy enzymes (Martinez et al. 2008) and transcription factors (Table 1) than those of T. reesei and N. crassa. Although comprehensive and intensive analyses are required, recent progress in this field has revealed that the fungal strains possess regulatory mechanisms basically similar but distinct in some respects from each other for the expression of the cellulase and hemicellulase genes. The regulators probably cooperatively, or competitively in some cases, organize gene expression. Cooperative transcriptional regulation is a reasonable means of harmonizing the expression levels of the cellulase and hemicellulase genes to produce an optimal enzyme

Table 1 Distribution of transcription factors related to cellulosic biomass degradation and their orthologs/paralogs in ascomycetic filamentous fungi Representative name

BglR

ClbR

CLR-1/ClrA

ManR/ClrB/CLR-2

McmA

XlnR

Eurotiomycetes A. niger A. aculeatus A. oryzae A. nidulans P. chrysogenum

1 1 1 1 1

3 3 (ClbR) 3 3 3

1 1 1 2 (ClrA) 1

1 1 1 (ManR) 2 (ClrB) 2

1 1 1 1 (McmA) 1

2 (XlnR, AraR) 2 (XlnR) 2 (XlnR, AraR) 3 (XlnR, AraR, GalA) 3

Sordariomycetes N. crassa F. oxysporum T. reesei

1 1 1 (BglR)

1 1 1

1 (CLR-1) 1 0

1 (CLR-2) 1 1

1 1 1

1 (XLR-1) 1 1 (Xyr1)

The numbers of orthologous and paralogous factors in each species are shown. Factors with identified functions are indicated by name. Amino acid sequences with more than 20 % identity were defined as paralogs

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composition, considering the natural occurrence of their substrates as a mixture in plant cell walls under different physicochemical conditions. These sophisticated regulatory mechanisms may also function to optimize carbon and energy flux. To establish highly efficient enzymatic hydrolysis systems for cellulosic biomass, suitable pretreatments would be required depending on its origin. However, at the same time, an enzyme mixture of the best composition for each pretreated biomass is required. Therefore, the regulatory mechanisms need to be clarified in every industrial species to apply transcription machinery engineering as the most fruitful means of producing enzymes of the best composition with the highest yield.

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