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

Abstract

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 clrB^ID, 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 clrB^ID-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 clrB^ID-expressing strain suggested that the effect of clrB^ID 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.

Introduction

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).

The synthesis of cellulases in filamentous fungi is mainly regulated at the transcriptional level. In general, the transcription of a set of cellulase genes is repressed in the presence of easy-to-use carbon sources (e.g. glucose) and coordinately induced by cellulosic materials (8). Several transcription factors (e.g. XlnR/Xyr1/XLR-1, CLR-1, CLR-2/ClrB, Ace2, CreA/Cre1/CRE-1, Ace1, and VIB-1) have been identified to participate in this regulation, of which many are conserved in different fungal species (for reviews, see Refs. 9 and 10). The Zn₂Cys₆-type transcription factor CLR-2/ClrB is essential for cellulase induction in several ascomycete fungi, including Neurospora crassa, Aspergillus nidulans (11), and Penicillium oxalicum (12). CLR-2 directly binds to the promoter of 164 genes in N. crassa cultivated on cellulose, which include around 30 lignocellulolytic enzyme genes (13). Overexpression of clrB/clr-2 led to significantly enhanced cellulase production on cellulose (12, 14) and even constitutive cellulase production under noninducing conditions in N. crassa (14). On the other hand, the global carbon-catabolite repressor CreA/Cre1/CRE-1 represses cellulase expression in the presence of preferred carbon sources in many fungal species (15, 16). In addition to direct inhibition of cellulase gene transcription, CreA/Cre1/CRE-1 also represses the expression of cellulase activator genes, including clrB/clr-2 (12, 17).

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.

Results

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.

Figure 1.

Cellulase production of clrB-overexpressing strain gClrB and reference strain M12. The cellobiohydrolase (measured as pNPCase) activities of culture supernatants in 2% (w/v) cellulose medium, the medium without carbon source, or 2% (w/v) glucose medium are shown. For glucose medium, around 8 g/liter glucose was detected in the culture supernatants after 12 h. Data represent mean ± S.D. (error bars) from triplicate cultivations.

For many Zn₂Cys₆-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 domain-removed 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 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.

Figure 2.

Identification of transcriptional activation domains in ClrB and CLR-2. A, transcriptional activation function of different regions in P. oxalicum 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/−Trp/−His/+aureobasidin A/+X-α-gal) and “control” (SD/−Trp) 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.

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.

Figure 3.

Expression of clrB internal deletion mutant clrB^ID results in cellulose-independent cellulase production. A, diagram representing the domain architectures of ClrB and ClrB^ID. MHR, middle homology region domain. B, pNPCase activities of culture supernatants of strains in the medium without carbon source. C, SDS-PAGE analysis of culture supernatants 48 h after shifting mycelia to the medium without carbon source. Coomassie Brilliant Blue was used for gel staining. D, expression levels of cellulase genes determined by qRT-PCR. Transcript abundances (relative to strain M12) 4 h after shifting mycelia to the medium without carbon source are shown. In B and D, data represent mean ± S.D. (error bars) from triplicate cultivations.

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 150-fold 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 ΔclrB 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.

Figure 4.

Expression of clrB^ID results in cellulase production on glucose and glycerol. A, concentrations of residual glucose in culture supernatants of strains in 2% (w/v) glucose medium. B, pNPCase activities of culture supernatants in glucose medium. C, SDS-PAGE analysis of culture supernatants 12 h after shifting mycelia to glucose medium. Coomassie Brilliant Blue was used for gel staining. D, expression levels of cellulase genes determined by qRT-PCR. Transcript abundances (relative to strain M12) 4 h after shifting mycelia to glucose medium are shown. E, expression levels of cellulase regulator genes (relative to strain M12) determined by qRT-PCR. F, pNPCase activities of culture supernatants in 2% (w/v) glycerol medium. In A, B, and D–F, data represent mean ± S.D. (error bars) from triplicate cultivations. The keys for B and E are the same as for A and D, respectively.

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 ΔclrB. 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-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.

Figure 5.

Detection of full-length and internally deleted ClrB proteins in glucose medium. ClrB and ClrB^ID were tagged by EGFP at C termini. A, fluorescence microscopy analysis of ClrB-EGFP and ClrB^ID-EGFP. The parent strain ΔclrB was used as a control. The nuclei were stained with Hoechst 33342. Scale bar, 20 μm. B, Western blot analysis of ClrB-EGFP and ClrB^ID-EGFP. As a loading control, cell extracts were subjected to SDS-PAGE and Coomassie Brilliant Blue (CBB) staining. DIC, differential interference contrast.

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 (>50- 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 up-regulated 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 down-regulated genes (Table S1). The reason and physiological effect of the down-regulation of these genes remain unclear.

Figure 6.

Comparative transcriptome analysis of gClrB^ID strain and reference strain M12 on 2% (w/v) glucose. A, scatterplot of FPKM values for all genes in gClrB^ID and M12. Significantly up-regulated and down-regulated genes (FDR <0.001 and -fold change >2) are shown in red and blue, respectively. B, number of genes of significantly differential expression with the indicated -fold changes in comparison of gClrB^ID versus M12. C, hierarchical clustering analysis of cellulase genes according to their transcriptional profiles. Blue, white, and red, maximum negative expression, zero expression, and maximum positive expression after data normalization, respectively. The suffix of the gene ID (locus tag prefix: PDE), family number in the CAZy database (68), and enzyme activity (CBH, cellobiohydrolase; EG, endo-β-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 ΔclrB strain in C was constructed by deleting the clrB gene in WT strain 114-2 (12).

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 expression on glucose, but were not efficiently induced by cellulose at the time of sampling. Group III genes were cellulose-induced, 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 ΔcreA and gClrB^IDΔcreA. When glucose was used as the sole carbon source, no cellulase production was detected for strain ΔcreA before glucose depletion (Fig. 4, B and C). The transcript abundances of major cellulase genes and clrB were significantly elevated in ΔcreA 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^IDΔcreA 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. creA-deletion mutant), cellulase production was not repressed by 2-DOG, and therefore the strain could utilize cellulose (17, 40). As expected, gClrB^ID, ΔcreA, and the double mutant gClrB^IDΔcreA, 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 ΔcreA. 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^IDΔcreA, 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^IDΔcreA in liquid glucose medium (Fig. 4, B and C).

Figure 7.

The resistance of clrB and creA mutants to 2-deoxyglucose. Growth and cellulose hydrolysis by strains on agar plates containing 1% (w/v) ball-milled cellulose and increasing concentrations of 2-deoxyglucose are shown. The plates were incubated at 30 °C for 48 h.

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 α-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 ΔcreA 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 suggested that the derepressed cellulase expression in gClrB^ID is not due to a relief from CreA-mediated carbon catabolite repression.

Figure 8.

The effect of clrB^ID expression on the transcription of CreA target genes. Transcript abundances of genes (relative to reference strain M12) 4 h after shifting mycelia to 2% (w/v) glucose medium were determined by qRT-PCR. Data represent mean ± S.D. (error bars) from triplicate cultivations.

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.

Figure 9.

Contributions of clrB internal deletion and creA deletion to enhanced cellulase production on 2% (w/v) cellulose. A, pNPCase activities of culture supernatants of strains in cellulose medium. B, SDS-PAGE analysis of culture supernatants 96 h after shifting mycelia to cellulose medium. Coomassie Brilliant Blue was used for gel staining. C, expression levels of cellulase genes determined by qRT-PCR. Transcript abundances (relative to M12) 4 h after shifting mycelia to cellulose medium are shown. D, expression levels of cellulase regulator genes (relative to M12) determined by qRT-PCR. In A, C, and D, data represent mean ± S.D. (error bars) from triplicate cultivations. The key for D is the same as that for C.

Deletion of creA has been previously demonstrated to remarkably improve cellulase production on cellulose by P. oxalicum (12). The cellulase activity of ΔcreA 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 ΔcreA was more than 2-fold higher than that in gClrB^ID (Fig. 9C). The expression of clrB was slightly higher in ΔcreA 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 up-regulated 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 expression 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.

Figure 10.

Comparison of the effects of clrB^ID expression and creA deletion on the transcriptome. A, overlaps between genes up-regulated in gClrB^ID strain on glucose and on cellulose. B, overlaps between genes up-regulated in gClrB^ID strain and in ΔcreA on cellulose. C, PC analysis plot of transcriptome data. The two most significant variances among samples are shown. Each dot represents one biological triplicate.

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 β-xylosidase) were uniquely up-regulated in ΔcreA (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 ΔcreA but not gClrB^ID (Table S1), indicating affected growth and development in the ΔcreA 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 ΔcreA 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 cellulase expression on glucose in A. niger. In contrast, a protein band identified as β-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.

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 ± S.D. from triplicate cultivations. The key for B is the same as that for A.

Discussion

The crucial and conserved function of ClrB/CLR-2 in the transcriptional activation of cellulase genes have been well-documented in several filamentous fungi (11–13, 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 Zn₂Cys₆-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 gClrB^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 glucose, 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.

Experimental procedures

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 downstream 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 μg/ml pyrithiamine (for ptrA marker), or 350 μg/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.

Table 1.

P. oxalicum strains used in this study

Strain	Genotype or characteristics	Parent strain	Reference
M12	pyrG−	114-2	Ref. 50
gClrB	pyrG−, AngpdA(p)-clrB::AnpyrG	M12	This study
ΔclrB	pyrG−, ΔclrB::AnpyrG	M12	This study
gClrBID	pyrG−, ΔclrB::AnpyrG, AngpdA(p)-clrBID::bar	ΔclrB	This study
gClrB-EGFP	pyrG−, ΔclrB::AnpyrG, AngpdA(p)-clrB-egfp::hph	ΔclrB	This study
gClrBID-EGFP	pyrG−, ΔclrB::AnpyrG, AngpdA(p)-clrBID-egfp::hph	ΔclrB	This study
ΔcreA	pyrG−, ΔcreA::hph	M12	This study
gClrBIDΔcreA	pyrG−, ΔclrB::AnpyrG, AngpdA(p)-clrBID::bar, ΔcreA::hph	gClrBID	This study

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 5′ 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, gal4Δ, gal80Δ, 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/−Trp 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 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-α-gal. The control plate was different from the test plate by replacing aureobasidin A and X-α-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-α-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 10⁶ 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 μl of conidial suspension at a concentration of 10⁷/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 μl 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-δ-lactone was mixed with 100 μl of diluted culture supernatant and incubated at 50 °C for 30 min. Then the reaction was stopped by adding 150 μl of 10% Na₂CO₃, 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 μmol 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 < 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-μl qRT-PCR mixture was prepared using SYBR Premix Ex Taq (Perfect Real Time, Takara Bio), and the amplification was carried out on a LightCycler 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 μg/ml for 20 min, washed, and resuspended in 2% (w/v) glucose medium. Images were acquired with a laser-scanning 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 anti-rabbit IgG(H+L) (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. Paired-end 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 paired-end 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 <0.001 and -fold change >2). Genesis 1.8.1 (62) was used for hierarchical clustering analysis of genes after adjusting FPKM values by log₂ transformation and within-gene normalization. Blast2GO (63) was used for GO enrichment analysis of gene sets with a threshold of FDR <0.05. PC analysis was performed using the prcomp function in R 3.4.4, and the result was visualized using ggbiplot.

Protein sequence analysis

Conserved protein domains in ClrB were predicted using the InterProScan tool (http://www.ebi.ac.uk/interpro/)⁴ (64). The transcriptional activation domain was predicted using the 9aaTAD tool (http://www.med.muni.cz/9aaTAD/)⁴ (65) by choosing the moderately stringent pattern. Secondary structure was predicted using the PSIPRED server (http://bioinf.cs.ucl.ac.uk/psipred/)⁴ (66). For sequence comparison, ClrB in P. oxalicum and its orthologs in A. niger, A. nidulans (GenBank^TM accession number XP_660973.1), Aspergillus oryzae (BAM66380.1), and N. crassa (XP_962712.2) were aligned using ClustalX 2.1 (67).

Author contributions

L. G., Y. X., S. L., C. X., J. X., and G. L. investigation; L. G. and G. L. writing-original draft; X. S. and Y. Qu resources; X. S., Y. Qin, G. L., and Y. Qu data curation; X. S., Y. Qin, G. L., and Y. Qu supervision; X. S., G. L., and Y. Qu funding acquisition; X. S. and G. L. project administration; Y. Qin writing-review and editing; G. L. conceptualization.