Deletion of the middle region of the transcription factor ClrB in Penicillium oxalicum enables cellulase production in the presence of glucose

Enzymes that degrade lignocellulose to simple sugars are of great interest in research and for biotechnology because of their role in converting plant biomass to fuels and chemicals. The synthesis of cellulolytic enzymes in filamentous fungi is tightly regulated at the transcriptional level, with the transcriptional activator ClrB/CLR-2 playing a critical role in many species. In Penicillium oxalicum, clrB overexpression could not relieve the dependence of cellulase expression on cellulose as an inducer, suggesting that clrB is controlled post-transcriptionally. In this study, using a reporter gene system in yeast, we identified the C-terminal region of ClrB/CLR-2 as a transcriptional activation domain. Expression of clrBID, encoding a ClrB derivative in which the DNA-binding and transcriptional activation domains are fused together to remove the middle region, led to cellulase production in the absence of cellulose in P. oxalicum. Strikingly, the clrBID-expressing strain produced cellulase on carbon sources that normally repress cellulase expression, including glucose and glycerol. Results from deletion of the carbon catabolite repressor gene creA in the clrBID-expressing strain suggested that the effect of clrBID is independent of CreA's repressive function. A similar modification of clrB in Aspergillus niger resulted in the production of a mannanase in glucose medium. Taken together, these results indicate that ClrB suppression under noninducing conditions involves its middle region, suggesting a potential strategy to engineer fungal strains for improved cellulase production on commonly used carbon sources.

Enzymes degrading lignocellulose to simple sugars are of great interest in research due to their critical role in converting plant cell wall biomass to fuels and chemicals (1,2). In industry, cellulases are mainly produced as secreted proteins by filamentous fungi such as Trichoderma reesei (3) and Penicillium species (4,5). Therefore, understanding the biological processes related to cellulase production in these fungi is important for engineering strains for higher production capacities (6,7).
Many species belonging to the Penicillium and Aspergillus genera, which are closely related in phylogeny (18), are efficient producers of cellulolytic enzymes. Some cellulases produced by Penicillium and Aspergillus species showed excellent catalytic efficiencies (19,20) or inhibitor tolerances (21,22), which are valuable in the bioconversion of lignocellulosic materials. So far, several Penicillium species, such as P. oxalicum (23) and Penicillium funiculosum (24), have been used for the production of commercial cellulase preparations. Whereas many mechanisms for the regulation of cellulase expression are conserved, some Penicillium and Aspergillus species have evolved unique characteristics in cellulase production. For example, lactose as an effective cellulase inducer in T. reesei does not induce cellulase expression in Penicillium echinulatum (25) and P. oxalicum (26). Also, different from the case in N. crassa, overexpression of clrB could not relieve the dependence of cellulase expression on inducer in P. oxalicum and A. nidulans, although their expression on cellulose was improved (12,14,27). Therefore, current cellulase production by Penicillium/Aspergillus species relies on insoluble cellulosic materials as the inducer, which is not favorable for submerged fermentation due to operation difficulty, enzyme adsorption, and toxicity problems (28). Expression of a gene encoding a chimeric transcriptional activator in P. oxalicum led to cellulase production under noninducing conditions, but cellulase expression in the resulting strains was still repressed by glucose (27).
In this study, we explored the possibility of enabling P. oxalicum to produce cellulase on glucose by mutating the sequence of ClrB. An internal deletion mutant of ClrB was constructed based on the identification of its transcriptional activation domain, and a strain expressing this mutant was able to produce cellulase on glucose and glycerol. The global transcriptional change in the mutant strain relative to reference strain was studied and compared with that in a creA-deletion mutant. These results deepen our understanding of cellulase gene expression in filamentous fungi and provide a novel strategy for engineering cellulase-producing strains.

The C-terminal region in ClrB/CLR2 is a transcriptional activation domain
Overexpression of the clrB gene in P. oxalicum using a constitutive gpdA promoter significantly improved cellulase production on cellulose, whereas cellulase activity was still hard to detect in the absence of cellulose (Fig. 1). We hypothesized that the transcriptional activation ability of ClrB was inhibited at the protein level under noninducing conditions. If that is the case, inactivation or removal of the regulatory region responsible for this inhibition is expected to cause constitutive transcriptional activation by ClrB.
For many Zn 2 Cys 6 -type transcription factors, the C-terminal region is responsible for transcriptional activation, and the middle region containing a "middle homology region" is involved in regulating their activities (29). Direct fusion of the transcriptional activation domain to the DNA-binding domain by removing the middle regulatory region could render several regulators constitutively active (30,31). If a similar mechanism exists in ClrB, fusion of its N-terminal DNA-binding domain (12,32) (Fig. S1) and transcriptional activation domain might lead to cellulose-independent cellulase expression. However, the location of transcriptional activation domain in ClrB remains unknown. Therefore, we started with the identification of transcriptional activation domain in ClrB using a yeast reporter system.
The full-length sequence of P. oxalicum ClrB (780 amino acids) and its truncated versions were fused to the C terminus of the DNA-binding domain of Saccharomyces cerevisiae transcription factor Gal4 ( Fig. 2A). S. cerevisiae cells expressing functional chimeric transcriptional activators could activate the expression of four reporter genes containing Gal4-binding sites on their promoters. Fusion of the DNA binding domainremoved ClrB (amino acids 123-780), but not the full-length ClrB, to Gal4-binding domain successfully activated the expression of reporter genes. Subsequently, the 123-780 region  ClrB. B, transcriptional activation function of the C-terminal region in N. crassa CLR-2. Yeast cells expressing the indicated fusion proteins were cultivated on "test" (SD/ϪTrp/ϪHis/ϩaureobasidin A/ϩX-␣-gal) and "control" (SD/ϪTrp) agar plates. For each construct, two independent yeast transformants were inoculated to agar plates. Adenine hemisulfate was added to the control plate in B, resulting in the white color colony of control strain containing empty vector.

ClrB mutation derepresses cellulase expression
of ClrB was divided into different fragments for the construction of chimeric transcription factors. The C-terminal 685-780 fragment was found to enable the expression of reporter genes when fused to Gal4-binding domain, suggesting that this region contains the transcriptional activation domain. Further dividing this region into two smaller fragments (residues 685-727 and 728 -780) revealed that each fragment had a transcriptional activation ability. None of the fragments from the middle region of ClrB (residues 123-684) resulted in the expression of reporter genes, suggesting that the C-terminal region is likely the only transcriptional activation domain in ClrB.
The C-terminal region is less conserved than the rest of the protein among ClrB homologs (see Fig. S2 for details). Actually, the transcriptional activation domain of P. oxalicum ClrB shows only random sequence similarity with the C-terminal region of N. crassa CLR-2 (Fig. S2). To test whether the transcriptional activation function of the C-terminal region is conserved in ClrB homologs, the 684 -812 fragment of CLR-2, as well as its two subfragments, were fused to the Gal4-binding domain. All three fusion proteins could activate the expression of reporter genes (Fig. 2B), suggesting the conserved functional domain architecture of ClrB/CLR-2 homologs.

Expression of an internal deletion mutant of clrB eliminated the dependence of cellulase production on cellulose
The N-terminal DNA-binding domain and C-terminal transcriptional activation domain of ClrB were fused together and tested for its function under cellulase-noninducing conditions. Compared with full-length ClrB, the predicted fusion protein ClrB ID (named for internal deletion) lacks the amino acids from positions 173-684 (Fig. 3A). To avoid the interference of native ClrB, the ClrB ID -encoding gene clrB ID driven by the gpdA promoter was introduced into a clrB-deletion mutant, resulting in strain gClrB ID . The gClrB ID strain was able to produce cellulase in the medium without the addition of a carbon source ( Fig. 3B and Fig. S3A). As a control, cellulase production was barely detected in reference strain M12. Consistently, proteins with molecular masses of major cellulases (33) were detected in the culture supernatant of gClrB ID but not in M12 (Fig. 3C), and the transcript abundances of major cellulase genes (cel7A-2, cel7B, and cel5B) were elevated by more than 150fold in gClrB ID relative to M12 (Fig. 3D). Moreover, the cellulase activity of gClrB ID in no-carbon medium was comparable with that of M12 in cellulose medium (Fig. 1), suggesting that the dependence of cellulase synthesis on cellulose was relieved in gClrB ID .

The gClrB ID strain produced cellulase on glucose and glycerol
Although cellulase expression in strain gClrB ID does not require cellulose as the inducer, the process might still be repressed by preferred carbon sources, such as glucose and glycerol. Thus, the cellulase activity of gClrB ID in the medium with glucose as the sole carbon source was further examined. Within the first 12 h after shifting the mycelia to glucose medium, residual glucose with a concentration higher than 10 g/liter could be detected in the culture (Fig. 4A). During this time, the reference strain M12 and clrB-deletion mutant ⌬clrB showed no cellulase activity ( Fig. 4 (B and C) and Fig. S3B). However, gClrB ID produced significant amounts of cellulase in the presence of glucose, which was more than 10 times higher than that of M12 in cellulose medium (Fig. 1). Strikingly, the transcript abundances of major cellulase genes on glucose increased by more than 800-fold in gClrB ID relative to M12 (Fig.  4D). The cellulase-producing phenotype was also observed in the medium with glycerol as a carbon source (Fig. 4F). Taken together, the expression of clrB ID could activate cellulase expression under classical cellulase-repressing conditions.
To verify the existence of full-length and mutated ClrB proteins in the cells, clrB and clrB ID genes with enhanced GFP (EGFP) 3 -encoding sequence fused to the downstream region were expressed in the clrB-deletion mutant ⌬clrB. The obtained strains were named gClrB-EGFP and gClrB ID -EGFP, respectively. The expression of clrB-egfp restored cellulase production on cellulose, and clrB ID -egfp expression led to cellulase production on glucose (Fig. S4), indicating that the tag did not affect the activity of ClrB and ClrB ID . The results of fluorescence microscopy and Western blotting revealed the presence of full-length and internally mutated ClrB when the strains were grown in glucose medium (Fig. 5). Interestingly, fluorescence signal was observed throughout mycelium in the gClrB-

ClrB mutation derepresses cellulase expression
EGFP strain but only in nuclei in gClrB ID -EGFP. Thus, it is likely that the internal deletion affected the activity of ClrB at levels beyond protein abundance.

Global changes of gene transcription on glucose caused by clrB ID expression
To investigate the genome-wide effect of clrB ID expression on gene expression, the transcriptome of gClrB ID on glucose was compared with that of M12 by RNA-Seq. A total of 658 genes were significantly up-regulated, whereas 1143 genes were significantly down-regulated, in gClrB ID relative to M12 (Fig.  6A). It is worth noting that the number of up-regulated genes with high -fold changes (Ͼ50-or 100-fold) was higher than that of down-regulated genes (Fig. 6B), which was consistent with the transcriptional activating function of ClrB. As expected, gene ontology (GO) terms related to the degradation of cellulose, pectin, and xylan were enriched for the significantly upregulated genes (Table S1). Actually, the 658 up-regulated genes included 14 of the 18 cellulases encoded by the P. oxalicum genome (34). In addition, 19 of the 51 hemicellulases were also significantly up-regulated in gClrB ID (Table S2). GO terms for transmembrane transport (including amino acid transport) and oxidation-reduction process were enriched for the downregulated genes (Table S1). The reason and physiological effect of the down-regulation of these genes remain unclear.
In our previous work, the regulon of ClrB in P. oxalicum has been studied by comparing the transcriptomes of WT and a clrB-deletion mutant on cellulose (12). By including these previous data, a hierarchical clustering analysis of cellulase genes was performed, which classified the genes into three groups (Fig. 6C). Group II genes (the biggest group) were significantly up-regulated by clrB ID expression on glucose and were induced by cellulose in a clrB-dependent manner. This group included genes encoding the most abundant cellulases detected in the secretome of P. oxalicum (e.g. Cel7A-2/PDE_07945, Cel6A/ PDE_07124, Cel5B/PDE_09226 and Cel7B/PDE_07929 (33)). Group I genes were also significantly up-regulated by clrB ID

ClrB mutation derepresses cellulase expression
expression on glucose, but were not efficiently induced by cellulose at the time of sampling. Group III genes were celluloseinduced, but their expression was either not enhanced or less enhanced by the expression of clrB ID . Interestingly, all of the four lytic polysaccharide monooxygenases, which degrade cellulose using an oxidative mechanism (35), were clustered into group III, implying the requirement of regulators other than ClrB for full induction of their expression. In summary, clrB ID expression triggered the transcription of a major portion of cellulase genes on glucose.

clrB ID expression had an additive effect with creA deletion on carbon catabolite derepression of cellulase genes
Gene deletion or mutation of carbon catabolite repressor CreA/Cre1/CRE-1 is reported to result in derepressed cellulase expression in several fungal species (36 -38). To investigate whether derepressed cellulase production by strain gClrB ID is related to the alleviation of CreA-mediated carbon catabolite repression, cellulase expression in gClrB ID was compared with those in creA-deletion mutants. The creA gene was deleted in reference strain M12 and gClrB ID , respectively, resulting in strains ⌬creA and gClrB ID ⌬creA. When glucose was used as the sole carbon source, no cellulase production was detected for strain ⌬creA before glucose depletion (Fig. 4, B and C). The transcript abundances of major cellulase genes and clrB were significantly elevated in ⌬creA relative to M12, but the times of increase were lower than those in gClrB ID by more than an order of magnitude (Fig. 4, D and E). Therefore, although the deletion of creA results in carbon catabolite derepression of cellulase genes, this relief was not sufficient for high-level cellulase production on glucose. In addition, strain gClrB ID ⌬creA showed further increased cellulase production compared with gClrB ID before glucose depletion (Fig. 4, B and C), suggesting that CreA still repressed cellulase expression in strain gClrB ID .
A typical phenotype of carbon catabolite repression-resistant cellulase-expressing strains is the growth on cellulose medium supplemented with 2-deoxyglucose (2-DOG), a nonmetabolizable analog of glucose (17,39). WT strains cannot grow on such medium because 2-DOG represses the expression of cellulase genes and therefore the synthesis of cellulases. For mutants in which catabolite repression was impaired (e.g. creAdeletion mutant), cellulase production was not repressed by 2-DOG, and therefore the strain could utilize cellulose (17,40). As expected, gClrB ID , ⌬creA, and the double mutant gClrB ID ⌬creA, but not M12, could grow and produce hydrolysis halos on the medium containing cellulose and 0.01 g/liter 2-DOG (Fig. 7). Increasing the concentration of 2-DOG to 0.03 g/liter showed that gClrB ID had a higher resistance to this inhibitor than ⌬creA. This confirmed that clrB ID expression had a greater effect on cellulase derepression than creA deletion (Fig. 4D). In the presence of 0.05 g/liter 2-DOG, only gClrB ID ⌬creA, and not the two single mutants, could grow on cellulose (Fig. 7). In other words, clrB ID expression and creA deletion have an additive effect on releasing the 2-DOG repression of cellulase expression, which is consistent with the highest cellulase production level of strain gClrB ID ⌬creA in liquid glucose medium (Fig. 4, B and C).
Moreover, clrB ID expression and creA deletion affected the expression of overlapping but distinct sets of genes. CreA/Cre1 was reported to repress a broad range of alternative carbon source-utilizing genes in filamentous fungi (15,41). In N. crassa, deletion of cre-1 resulted in dramatic up-regulation of genes encoding amylases and a high-affinity glucose transporter NCU04963 on sucrose (16). Therefore, the transcript abundances of the homologues of these genes in P. oxalicum, including amy13A/PDE_01201 (encoding ␣-amylase), amy15A/ PDE_09417 (glucoamylase) (42), hgtB/PDE_01388 (high affinity glucose transporter), and amylase transcriptional activator gene amyR/PDE_03964 (12), were determined. All four genes were up-regulated in ⌬creA strain relative to M12 on glucose (Fig. 8), suggesting that they are also targets of CreA-mediated catabolite repression in P. oxalicum. Interestingly, the expression of these four genes (especially amyR and hgtB) was lower in gClrB ID than that in M12, which was consistent with the RNA-Seq result (Table S3). Taken together, comparative analysis of the effects caused by clrB ID expression and creA deletion sug-  (68), and enzyme activity (CBH, cellobiohydrolase; EG, endo-␤-1,4-glucanase) are shown for each gene. Proteins detected in the secretome of P. oxalicum in a previous study (33) are shown in boldface type. The ⌬clrB strain in C was constructed by deleting the clrB gene in WT strain 114-2 (12).

ClrB mutation derepresses cellulase expression
gested that the derepressed cellulase expression in gClrB ID is not due to a relief from CreA-mediated carbon catabolite repression.

clrB ID expression moderately enhanced cellulase production on cellulose
In cellulose medium as an inducing condition, ClrB is expected to be turned to a functional transcriptional activator. In addition, the expression of clrB is induced by cellulose, followed by a reduction at a latter stage of cultivation (43). Whether the expression of clrB ID could further improve cellulase production under this condition was studied. Compared with M12, gClrB ID showed 1.6 -8.3-fold increases in cellulase production at different time points (Fig. 9 (A and B) and Fig.  S3C). Transcript abundance determination also showed significant up-regulation of major cellulase genes in gClrB ID (Fig.  9C). It should be noted that the cellulase activity of gClrB ID (around 0.02 p-nitrophenyl cellobiosidase (pNPCase) units/ml at 24 h) was similar to that of the gClrB strain, which overexpresses the intact clrB using the same promoter (Fig. 1). Therefore, the internal sequence deletion of clrB seemed not to play an important role in improving cellulase production on cellulose.
Deletion of creA has been previously demonstrated to remarkably improve cellulase production on cellulose by P. oxalicum (12). The cellulase activity of ⌬creA was much higher than that of gClrB ID on cellulose, particularly at the later stage of fermentation (Fig. 9, A and B). Even at the early stage (4 h after shifting mycelia to cellulose medium), cellulase gene expression in ⌬creA was more than 2-fold higher than that in gClrB ID (Fig. 9C). The expression of clrB was slightly higher in ⌬creA relative to reference strain M12 (Fig. 9D), suggesting that the enhancement of cellulase expression by creA deletion was not mediated by the up-regulation of clrB. Similar to that in M12 background, deletion of creA in gClrB ID resulted in a large increase in cellulase production (Fig. 9, A and B). These results showed that CreA strongly inhibits cellulase expression on cellulose regardless of the regulatory strength of ClrB.
The changes in gene transcription caused by clrB ID expression and creA deletion on cellulose were further studied by RNA-Seq. A total of 452 and 485 genes were significantly upregulated and down-regulated in gClrB ID strain relative to M12, respectively. Thus, clrB ID expression affected the transcription of fewer genes on cellulose than on glucose (Fig. 6B). Comparison of the significantly up-regulated genes in gClrB ID relative to M12 between glucose medium and cellulose medium revealed 138 genes up-regulated under both conditions (Fig.  10A). These genes included 13 cellulase genes and 12 hemicellulase genes and were significantly enriched for GO terms involved in lignocellulose degradation (Table S1). The expres-   The key for D is the same as that for C.

ClrB mutation derepresses cellulase expression
sion of 13 hemicellulase genes was up-regulated in gClrB ID on cellulose but not on glucose (Fig. 10A), suggesting that their transcription might require the cooperation of ClrB and other regulators that were active on cellulose. No specific GO term was enriched for the 520 genes up-regulated in gClrB ID uniquely on glucose, whereas heme-binding proteins and oxidoreductases were enriched for the 314 genes up-regulated uniquely on cellulose.
Deletion of creA resulted in 825 and 834 genes significantly up-regulated and down-regulated on cellulose, respectively. The up-regulated gene set included 162 genes that were also up-regulated in gClrB ID , of which 13 encode cellulases and 22 encode hemicellulases (Fig. 10B). In addition, 12 hemicellulase genes (e.g. xyl3A/PDE_00049 encoding a ␤-xylosidase) were uniquely up-regulated in ⌬creA (Table S2), suggesting a broader regulatory function of CreA than ClrB. GO terms related to ribosome biogenesis and conidiation were enriched for the 663 genes up-regulated in ⌬creA but not gClrB ID (Table  S1), indicating affected growth and development in the ⌬creA strain. When all the gene expression data were separated on a principal component (PC) analysis plot, the 15 samples (involving three strains and two conditions run in triplicates) were clustered into three big groups by the first and second PCs (Fig.  10C). The gClrB ID strain was mildly separated from M12 to the same direction along the PC2 axis on glucose and cellulose, suggesting an overlapped effect of clrB ID expression between the two conditions. On cellulose, the ⌬creA strain was clearly separated from M12 and gClrB ID on both axes, again supporting the strong regulatory role of CreA under this condition.

Expression of clrB ID analogue in Aspergillus niger led to derepressed lignocellulolytic enzyme production
To examine whether the effect of clrB ID expression on cellulase production is conserved in the family Aspergillaceae, a similar manipulation of clrB as that in gClrB ID was performed in A. niger, which is widely used for enzyme production in industry (44). The amino acid sequence of putative ClrB in A. niger (An12g01870, named AnClrB) has an identity of 58% with P. oxalicum ClrB. According to the result of sequence alignment (Fig. S5), the sequences encoding N-terminal (amino acids 1-158) and C-terminal (amino acids 679 -777) regions of AnClrB were fused together and overexpressed in strain N593. Two transformants, gAnClrB ID -1 and gAnClrB ID -2, were obtained and compared with the parent strain. As a control, a mutant overexpressing the intact AnClrB-encoding gene using the same promoter was constructed and named gAnClrB.
In the medium with glucose as the sole carbon source, all of the recombinant strains showed cellulase production before glucose depletion, which was not observed for the parent strain (Fig. 11, A and B). SDS-PAGE followed by MS analysis of the culture supernatants revealed the production of endoglucanase EglA (An14g02760) by the three recombinant strains but not the parental strain ( Fig. 11C and Table S4). Thus, overexpression of the AnClrB-encoding gene is sufficient to trigger some

ClrB mutation derepresses cellulase expression
cellulase expression on glucose in A. niger. In contrast, a protein band identified as ␤-mannanase Man5A (An05g01320) was uniquely detected in the culture supernatants of gAnClrB ID -1 and gAnClrB ID -2 but not gAnClrB (Fig. 11C and Table S4), suggesting that internal deletion of clrB resulted in derepressed expression of man5A.

Discussion
The crucial and conserved function of ClrB/CLR-2 in the transcriptional activation of cellulase genes have been welldocumented in several filamentous fungi (11)(12)(13)45). However, the understanding of how this protein functions as a transcriptional regulator is still limited. Previously, the N-terminal regions of ClrB in P. oxalicum (amino acids 1-163) and A. nidulans (residues 1-118) were shown to bind to cellulase gene promoters in vitro (12,32), and CLR-2 in N. crassa was found to act as a homodimer (13). These characteristics are typical for Zn 2 Cys 6 -type transcription factors (29,46). Another feature of many members in this family is the transcriptional activation function of the C-terminal region (46). In contrast to the DNA-binding domain, transcriptional activation domains do not have a well-defined structure and are hard to predict by computational methods (47). In this study, the C-terminal 96 amino acids in P. oxalicum ClrB and 129 amino acids in N. crassa CLR-2 were identified to constitute transcriptional activation domains, both of which seemed to contain smaller functional units (Fig. 2). The transcriptional activation domains from the two species share only random similarity, indicating the rapid evolution of this region. In addition, none of the two domains are rich in acidic amino acids, although this was observed for many other transcriptional activation domains. Whether the activation domains in ClrB/CLR-2 work in known manners to activate transcription (e.g. recruitment of RNA polymerase, Mediator, and/or nucleosome modifiers) could be studied in the future.
In N. crassa, overexpression of full-length CLR-2 was sufficient to cause cellulase production in the medium without a carbon source, but this phenomenon could not be observed in A. nidulans with the same manipulation (14). A result similar to that in A. nidulans was obtained in P. oxalicum (Fig. 1) (27). Therefore, the transcriptional activation ability of ClrB in A. nidulans and P. oxalicum is likely to be post-transcriptionally inhibited in the absence of cellulose signal. Here, the cellulose-independent cellulase-producing phenotype of strain gCl-rB ID suggested that the middle region accounting for 65.6% of the length of ClrB is dispensable for its activity (Fig. 3). Instead, the region is responsible for the regulation of ClrB in response to upstream signal. In the absence of cellulose, the middle region itself or some other inhibitory molecules interacting with this region (like the case of Gal80 in S. cerevisiae (48)) might participate in suppressing the activity of ClrB. The suppression seems not to act through mediating its degradation, as EGFP-tagged ClrB could be detected in the cell in glucose medium, despite the inactive cellulase expression under this condition ( Fig. 5 and Fig. S4B).
The results of clrB manipulation in A. niger have some differences from those in P. oxalicum (Fig. 11). Overexpression of full-length clrB in A. niger resulted in the production of cellobiohydrolase and endoglucanase EglA on glucose, which was not observed in P. oxalicum. However, derepressed production of mannanase Man5A in A. niger was only detected in the strain expressing internally deleted AnclrB, which was similar to the case of cellulase in P. oxalicum. The different responses of cellulase and mannanase to ClrB mutation in A. niger might be due to the existence of distinct regulatory modes of ClrB on different target genes, which has been reported in A. nidulans with regard to the regulation of cellulolytic and mannanolytic genes by ClrB (32). Together, the results in A. niger reveal that the mechanism for regulating lignocellulolytic enzyme production by ClrB is partially conserved across fungal species.
Several reports have suggested that inactive cellulase expression under repressing conditions is mainly due to insufficient transcriptional activation and not CreA/CRE1-mediated carbon catabolite repression. As mentioned above, overexpression of clr-2 in N. crassa led to cellulase production on repressing carbon source (sucrose) at a level similar to that on cellulose (14). In T. reesei, overexpression of the cellulase transcriptional activator gene xyr1 by a copper-responsive promoter also resulted in full relief of cellulase production from repression on glucose (49). In this study, the similar result was observed after mutating the sequence of clrB (Figs. 4 and 6). Of note, the cre-1/cre1/creA gene was not disrupted in the above three cases. In contrast, single deletion of creA in P. oxalicum was not sufficient for cellulase production on glucose despite the up-regulation of cellulase gene expression (Fig. 4, B-D). This consequence of creA deletion appears similar, although somewhat variable in severity among different fungal species. In T. reesei, deletion of cre1 led to detectable cellulase production on glu- Figure 11. The effect of AnclrB manipulation on extracellular enzyme production in A. niger. The parent strain N593, full-length AnclrB-overexpressing strain gAnClrB, and two independent transformants expressing internally deleted AnclrB were analyzed in 2% (w/v) glucose medium. A, concentrations of residual glucose in culture supernatants of strains. B, pNPCase activities of culture supernatants. C, SDS-PAGE analysis of culture supernatants 12 h after shifting mycelia to glucose medium. Silver nitrate was used for gel staining. The two protein bands identified by MS are indicated. In A and B, data represent mean Ϯ S.D. from triplicate cultivations. The key for B is the same as that for A.

ClrB mutation derepresses cellulase expression
cose, but the level was much lower than that under inducing conditions (36). In N. crassa, even deletion of cre-1 did not increase the expression of cellulase genes on sucrose (16). Taken together, the primary reason for cellulase gene repression on the preferred carbon source is the insufficient transcriptional activation due to low abundance and/or low activity of transcriptional activator, whereas CreA/Cre1/CRE-1 plays an additional role in repression.
Internal deletion of clrB resulted in only moderately enhanced cellulase production on cellulose relative to reference strain M12 (Fig. 9, A-C). This is not surprising because under inducing conditions, the activity of native ClrB is already at high levels. In contrast, deletion of creA had a markedly enhancing effect on cellulase expression on cellulose (Fig. 9, A-C). Similar results were previously reported in T. reesei (36) and N. crassa (16), where cre1/cre-1 deletion significantly increased cellulase expression under inducing conditions. It is worth noting that creA deletion did not affect cellulase expression in the medium without carbon source (Fig. 3, B-D). Therefore, CreA/Cre1/ CRE-1 seems to repress cellulase expression as long as the cell senses the signal from glucose (either exogenously added or gradually released from cellulose) or other preferred carbon sources. This mechanism might prevent excessive synthesis of cellulases during the growth on cellulose.
In conclusion, this study describes a novel clrB gain-of-function mutant whose expression led to cellulase production under repressing conditions in P. oxalicum. The result provides new insights into the understanding of the control of cellulase gene expression in filamentous fungi. The detailed mechanism, including the sensing of cellulose signal and its transduction to ClrB, should be clarified in the future. From the view of industrial application, soluble simple sugars like glucose and sucrose are more suitable for large-scale cellulase production in bioreactors (28). Combining the expression of clrB ID and engineering of other targets is expected to yield industrial Penicillium strains with high-level cellulase production ability on glucose.

Strains
A uracil auxotrophic strain M12 of P. oxalicum (previously classified as Penicillium decumbens) (50) was used as a parent for strain construction in this study (Table 1). To construct the clrB-overexpressing strain gClrB, the gpdA promoter from A. nidulans, the coding and terminator regions of P. oxalicum clrB, and selection marker gene AnpyrG from A. nidulans (51) were fused together through double-joint PCR (52) to obtain the overexpression cassette. To delete clrB, the upstream sequence of clrB, selection marker gene AnpyrG, and the down-stream sequence of clrB were fused together to obtain the gene deletion cassette. To express clrB ID , the A. nidulans gpdA promoter, the fragment of the clrB gene encoding the N-terminal 172 amino acids, the region encoding C-terminal 96 amino acids followed by terminator, and selection marker gene bar (53) were fused together. To express EGFP-tagged proteins, the sequence encoding (GGGS) 3 linker and EGFP was fused downstream of the target gene, and the hph gene (54) was used as the selection marker. To delete creA, the upstream sequence of creA, selection marker gene hph, and the downstream sequence of creA were fused together to obtain the gene deletion cassette. The gene deletion or overexpression cassettes were transformed to parental strains via protoplast transformation (55) as indicated in Table 1. Similarly, the AnclrB-overexpressing cassette fused to a ptrA marker gene (56) and AnclrB ID -expressing cassette fused to hph were constructed and transformed to A. niger strain N593 (a kind gift from Adrian Tsang, Concordia University, Montreal, Canada). Agar plates containing Vogel's salt (57), 2% (w/v) glucose, and 1 M sorbitol supplemented with 2.5 mg/ml glufosinate ammonium (for bar marker), 0.5 g/ml pyrithiamine (for ptrA marker), or 350 g/ml hygromycin B (for hph marker) were used for transformant screening. The transformants were purified by plate streaking, and the obtained strains were identified via PCR and DNA sequencing. All of the primers used for gene manipulation cassette construction are listed in Table S5.

Identification of transcriptional activation domain in ClrB/CLR-2
The clrB/clr-2 gene fragments of different lengths were amplified from the cDNA of P. oxalicum or genomic DNA of N. crassa and fused to vector pGBKT7 (Takara Bio, Shiga, Japan) using the ClonExpress II One Step Cloning Kit (Vazyme Biotech, Nanjing, China). To create sequence homology between the PCR product and vector, 16-bp-long sequences matching the ends of pGBKT7 linearized with EcoRI and BamHI were added to the 5Ј ends of primers. All of the primers used for vector construction are listed in Table S6. The resulted recombinant plasmids extracted from Escherichia coli cells were confirmed by Sanger sequencing and then separately transformed to S. cerevisiae strain Y2HGold (MATa, trp1-901, leu2-3,112, ura3-52, his3-200, gal4⌬, gal80⌬, LYS2::GAL1 UAS -Gal1 TATA -His3, GAL2 UAS -Gal2 TATA -Ade2, URA3::MEL1 UAS -Mel1 TATA -AUR1-C, MEL1; Takara Bio USA, Inc.). Transformants were screened on SD/ϪTrp medium. The recombinant yeast strains were then inoculated to agar plates of different compositions to test the expression of reporter genes. The test plate contained (per liter) 6.7 g of yeast

ClrB mutation derepresses cellulase expression
nitrogen base (Solarbio, Beijing, China), 20 g of glucose, 1.3 g of yeast synthetic drop-out medium supplements without histidine, leucine, tryptophan, and uracil (Sigma-Aldrich), 63 mg of leucine, 0.5 mg of aureobasidin A, and 40 mg of X-␣-gal. The control plate was different from the test plate by replacing aureobasidin A and X-␣-gal with 20 mg/liter histidine. Transcriptional activation of reporter genes AUR1-C, HIS3, ADE2, and MEL1 resulted in resistance to aureobasidin A, growth on histidine-deficient medium, formation of white colony, and hydrolysis of X-␣-gal (blue color), respectively. S. cerevisiae cells expressing the empty pGBKT7 vector were inoculated as a negative control.

Cultivation of P. oxalicum and A. niger strains
The fungal strains were cultivated on wheat bran extract slants for 4 days to collect conidia. For cultivation in liquid media, the conidia were inoculated to and precultivated in Vogel's salts supplemented with 2% (w/v) glucose with a final concentration of 10 6 conidia/ml. 0.1% (w/v) peptone was added to the medium for precultivation of A. niger strains. The 300-ml flasks containing 50-ml cultures were incubated on a rotary shaker at 200 rpm at 30°C. After 22 h of precultivation, the mycelia were collected on filter paper by vacuum filtration. Mycelia were resuspended in Vogel's salts supplemented with indicated carbon sources with a concentration of 6.0 g (for glucose and cellulose media) or 10.0 g (for the medium without carbon source) wet cell weight per liter. The cultivation was continued for 4 -96 h for the analyses of gene transcription, cellulase activity, and extracellular proteins. For cultivation on agar plates, 1.5 l of conidial suspension at a concentration of 10 7 /ml were spotted to Vogel's medium supplemented with 1.5% (w/v) agar powder and 2-DOG at different concentrations as indicated. Photographs of plates were taken after 48 h of cultivation at 30°C. Uracil at a concentration of 2 g/liter was added to the medium for all cultivations.

Cellulase assays, SDS-PAGE, and protein identification
The liquid cultures were taken from shake flasks and centrifuged at 4°C for 10 min. The supernatants were used for the determination of cellulase activities and SDS-PAGE analyses. For the measurement of cellobiohydrolase (a major type of cellulase) activity, 50 l of 1 mg/ml p-nitrophenyl-D-cellobioside (pNPC; Sigma-Aldrich) in 0.2 M acetate buffer (pH 4.8) supplemented with 10 mg/ml D-glucono-1,5-␦-lactone was mixed with 100 l of diluted culture supernatant and incubated at 50°C for 30 min. Then the reaction was stopped by adding 150 l of 10% Na 2 CO 3 , and the absorbance of the reaction system at 420 nm was determined. Filter paper activity was measured as described previously (55). One unit of enzyme activity was defined as the amount of enzyme required to release one mol of product (p-nitrophenyl or glucose equivalent) from the substrate per minute under the assayed conditions. Polyacrylamide gel at a concentration of 12.5% (w/v) was used for protein separation, and Coomassie Brilliant Blue R250 (Sangon, Shanghai, China) or silver (58) (for A. niger samples) was used for gel staining. Protein bands of interest were cut from SDS-polyacrylamide gels and analyzed on a MALDI-TOF/TOF 5800 mass spectrometer (AB SCIEX) by Shanghai Applied Protein Technology Co. Ltd. The MS data were processed using Data Explorer 4.5 (AB SCIEX). Then the extracted peak lists were searched using the Mascot search engine (version 2.2, Matrix Science) against the Uniprot database (December 7, 2018) restricted to the taxonomy A. niger (59,116 sequences). Search parameters included use of trypsin to generate peptides, a maximum of one missed cleavage permitted, carbamidomethyl (C) as fixed modification, oxidation (M) as variable modification, 100-ppm peptide mass tolerance, 0.4-Da fragment mass tolerance, and p Ͻ 0.05 as threshold score for accepting individual spectra.

Quantitative RT-PCR (qRT-PCR)
The mycelia were collected on filter paper by vacuum filtration, and then ground to powder in liquid nitrogen. Total RNA extraction and cDNA synthesis were performed using the RNAiso Plus reagent (Takara Bio) and PrimeScript RT reagent kit with genomic DNA eraser (Takara Bio) according to the manufacturer's instructions. The 20-l qRT-PCR mixture was prepared using SYBR Premix Ex Taq (Perfect Real Time, Takara Bio), and the amplification was carried out on a Light-Cycler 480 system with software version 4.0 (Roche Applied Science). The PCR program included 95°C for 2 min for initial denaturation, 40 cycles of 95°C for 10 s followed by 61°C for 30 s, and a dissociation stage in which the temperature increased from 65 to 95°C with a gradient of 0.1°C/s. Fluorescence signal was gathered at the end of each extension step at 80°C. The copy number of transcripts was calculated by comparing the Cp value with the standard curve of each gene, respectively. The transcript level of actin gene (Gene ID: PDE_01092) was used as an internal reference for data normalization. The primers used for qRT-PCR are shown in Table S7.

Microscopy analysis
The conidia were inoculated to Vogel's salts supplemented with 2% (w/v) glucose and cultivated for 9 h. The mycelia were stained with Hoechst 33342 (Sigma-Aldrich) with a final concentration of 10 g/ml for 20 min, washed, and resuspended in 2% (w/v) glucose medium. Images were acquired with a laserscanning confocal microscope (LSM 880, Zeiss).

Western blotting
The mycelia precultivated in Vogel's salts supplemented with 2% (w/v) glucose for 22 h were transferred to the same medium, further cultivated for 4 h, and ground to powder in liquid nitrogen. Total protein was extracted from the mycelia by mixing with the extraction buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, pH 7.5) on ice for 30 min and then centrifugation at 12,000 rpm at 4°C for 10 min to collect the supernatant. Equal amounts of total protein were separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane. After blocking with 5% (w/v) skim milk, the membrane was incubated with GFP tag antibody (rabbit polyclonal, Proteintech Group; 1:1000 dilution) and then with horseradish peroxidase-conjugated Affinipure goat antirabbit IgG(HϩL) (Proteintech Group; 1:5000 dilution). Target bands were visualized using an enhanced chemiluminescent reagent kit (Wuhan Sanying).

RNA-Seq
High-throughput sequencing of RNA samples was performed by Whbioacme Co. Ltd. (Wuhan, China). mRNA libraries for sequencing were prepared using the KAPA mRNA capture kit and KAPA Stranded RNA-Seq kit (Roche Applied Science) according to the manufacturer's instructions. Pairedend sequencing was performed on Illumina Hiseq X Ten with a read length of 150 bp. All clean reads obtained after raw data processing were mapped to the reference genome of 114-2 (34) using hisat2 2.0.0-beta (59) with default parameters for pairedend reads. Raw counts of mapped reads on gene level were quantified using featureCounts version 1.5.0-p1 (60). FPKM (fragments per kilobase per million mapped fragments) was used to represent the gene expression values. Deseq2 (61) was used to compare the gene expression levels between samples and perform statistical analysis. Genes of significantly differential expressions were identified with combined thresholds (FDR Ͻ0.001 and -fold change Ͼ2). Genesis 1.8.1 (62) was used for hierarchical clustering analysis of genes after adjusting FPKM values by log 2 transformation and within-gene normalization. Blast2GO (63) was used for GO enrichment analysis of gene sets with a threshold of FDR Ͻ0.05. PC analysis was performed using the prcomp function in R 3.4.4, and the result was visualized using ggbiplot.