cAMP signaling factors regulate carbon catabolite repression of hemicellulase genes in Aspergillus nidulans

Effect of creA, pkaA, and ganB deletion on β-xylanase production

The effect of the deletions on growth is shown in Fig. 1a. On d-xylose, all of the strains including the reference strain grew similarly. On d-xylose plus d-glucose, the ΔcreAΔpkaA, ΔganB, and ΔcreAΔganB strains exhibited better growth than the others at 12 h, but the increase in the biomass was less than twofold.

Fig. 1figure 1

Effect of deletions of creA, pkaA, and ganB on growth and the production of β-xylanase. Precultured strains were further grown in MM containing d-xylose supplemented with or without d-glucose (xyl + glc or xyl) for 6 h and 12 h, and dry mycelial weight (a) and total β-xylanase activity in the culture supernatant (b) were measured. White bars, the reference strain; light gray bars, ΔcreA; dark gray bars, ΔpkaA; filled bars, ΔcreAΔpkaA; dashed bars, ΔganB; striped bars, ΔcreAΔganB. Error bars indicate the standard deviations of three independent experiments. Different letters indicate a significant difference at P < 0.05 by one-way analysis of variance (ANOVA) with Tukey’s post hoc test

The total β-xylanase activity per milligram dry mycelia produced in the culture supernatants is shown in Fig. 1b. In the overnight precultures with Bacto Peptone as the main carbon source, the β-xylanase activity was extremely low in all the strains, indicating that the deletions did not lead to a significant increase in basal level β-xylanase production. When only d-xylose was used as the carbon source (inducing conditions), all the deletants exhibited a notable increase in the production of β-xylanase activity as compared to the reference strain. The fold increases over the reference strain at 6 h in the ΔcreA, ΔpkaA, and ΔganB strains were 5.9, 5.2, and 4.6, respectively, while the ΔcreAΔpkaA and ΔcreAΔganB strains exhibited 14.6- and 15.7-fold increases. Higher β-xylanase activity in the double deletants compared to the single deletants was also observed at 12 h. As described previously, d-xylose not only functions as a β-xylanase inducer but also as a repressing carbon source in CreA-dependent CCR (de Vries et al. 1999; Orejas et al. 1999, 2001; Mach-Aigner et al. 2012), which accounts for the increase in the ΔcreA strain. Not only that, the increases in the other deletants indicate that PkaA and GanB are also involved in CCR by d-xylose, and furthermore, the higher activity in the double deletants indicates that CreA and PkaA/GanB independently participate in CCR. Under d-glucose-added conditions (repressing conditions), the production of the β-xylanase activity decreased in all the strains as compared to that in the inducing conditions. Therefore, mechanisms other than those based on CreA and PkaA/GanB are obviously present in CCR of β-xylanase expression. However, tolerance against d-glucose-derived repression was detected in the deletants. While a sevenfold decrease by d-glucose addition was detected in the reference strain at 6 h, the fold decreases in the deletants were 3.3 for ΔcreA, 2.3 for ΔpkaA, 2.3 for ΔcreAΔpkaA, 3.6 for ΔganB, and 2.9 for ΔcreAΔganB. Similar results were also obtained at 12 h.

Effect of creA, pkaA, and ganB deletion on the transcription of β-xylanase genes

In the β-xylanase productivity measurements described above, a high d-xylose concentration of 1% (67 mM) was applied to support growth. However, such a high concentration caused significant d-xylose-derived repression of the β-xylanase production. To minimize the d-xylose repression, a lower concentration of 3 mM d-xylose, which gave the highest expression of xlnA and xlnC in the pilot study to determine the optimal d-xylose concentration for induction (data not shown), was used in the transcription analysis of the β-xylanase genes. As a repressing carbon source, 30 mM d-glucose was added under the repressing conditions, which led to significant decrease in the β-xylanase gene expression in the reference strain as described below.

Increase in expression of β-xylanase genes was observed in all the deletants under d-xylose conditions, and the level of increase differed depending on the deleted gene as well as each xylanase gene (Fig. 2). While the increase confirms that d-xylose has the dual function as an inducer and a repressing carbon source, it also implies that PkaA and GanB are involved in d-xylose repression. d-Glucose addition caused a significant decrease of β-xylanase expression in the reference strain; the expression of the major β-xylanase genes, namely xlnA, xlnB, and xlnC, dropped 48-fold, 168-fold, and 63-fold, respectively, which accounts for 2.1%, 0.60%, and 1.6% of the expression levels in the absence of d-glucose (Fig. 3). In contrast, the decrease in expression levels caused by the d-glucose addition was much smaller in all the deletants except for ΔcreA. Specifically, the deletants other than ΔcreA exhibited expression of 29–48% for xlnA, 5.7–13% for xlnB, and 14–26% for xlnC as compared to those in the absence of d-glucose, while the percentages were only 4.5%, 0.94%, and 1.9% in the ΔcreA strain (Fig. 3). These results imply that the cAMP signaling factors PkaA and GanB play major roles, independently from CreA, in d-glucose-derived CCR of the β-xylanase genes.

Fig. 2figure 2

Transcriptional analysis of xlnA, xlnB, xlnC, and xlnR. Each strain was cultured under d-xylose-induced conditions (xyl) and under d-xylose plus d-glucose conditions (xyl + glc). Transcription levels of the genes before the addition of the monosaccharides are also included (0 h). The qPCR data were normalized with actA encoding γ-actin and are shown on a logarithmic scale of 2 to base (log2 (gene/actA × 104)). White bars, the reference strain; light gray bars, ΔcreA; dark gray bars, ΔpkaA; filled bars, ΔcreAΔpkaA; dashed bars, ΔganB; striped bars, ΔcreAΔganB. Error bars indicate the standard deviations of three independent experiments. Different letters indicate significant differences at P < 0.05 by one-way ANOVA with Tukey’s post hoc test

However, it should be noted that the ratios were still far below 100%, even in the double deletants, suggesting the presence of a CCR mechanism independent of creA as well as pkaA/ganB. We examined the expression of xlnR, which encodes a transcriptional activator of the β-xylanase genes. The expression was repressed by d-glucose in the reference strain, but not completely released by deletion of creA, pkaA, and ganB (Figs. 2, 3).

Fig. 3figure 3

Relative expression levels of the β-xylanase genes on d-xylose plus d-glucose conditions as compared to d-xylose-induced conditions. The de-repression ratio in the vertical axis was calculated by dividing the expression level under the d-xylose plus d-glucose conditions (repressing conditions) by that under the d-xylose conditions (inducing conditions), so that 100% de-repression means the same expression level as that without the repressing carbon source. Asterisks indicate significant differences at P < 0.05 by one-way ANOVA with Dunnett’s post hoc test

Effects of creA, pkaA, and ganB deletions on β-mannanase production

To investigate the effect of d-glucose on β-mannanase production in the creA, pkaA, and ganB deletants, precultured mycelia were transferred to MM media containing 0.5% LBG (inducing conditions) or 0.5% LBG plus 1% d-glucose (repressing conditions) and cultivated for 6 or 12 h. Growth of the strains did not differ greatly (Fig. 4a). Although the addition of d-glucose enhanced their growth, reaching 1.7- to 2.3-fold higher biomass at 12 h compared to that without d-glucose, this was simply due to the increase in the amount of the carbon source (Fig. 4a). The double deletants under the repressing conditions grew slightly better (1.3- to 1.4-fold) compared to the reference strain.

Fig. 4figure 4

Effect of deletions of creA, pkaA, and ganB on growth and the production of β-mannanase. Precultured strains were further grown in MM containing LBG with or without d-glucose (LBG + glc or LBG) for 6 h and 12 h, and dry mycelial weight (a) and total β-mannanase activity in the culture supernatant (b) were measured. White bars, the reference strain; light gray bars, ΔcreA; dark gray bars, ΔpkaA; filled bars, ΔcreAΔpkaA; dashed bars, ΔganB; striped bars, ΔcreAΔganB. Error bars indicate the standard deviations of three independent experiments. Different letters indicate significant differences at P < 0.05 by one-way ANOVA with Tukey’s post hoc test

β-Mannanase activity in the culture supernatants is shown in Fig. 4b. While β-mannanase activity was extremely low or not detectable in the precultures, the activity of approximately 0.2 to 1.0 and 0.3 to 0.8 U/mg dry mycelia at 6 h and 12 h, respectively, was produced under the inducing conditions with 0.5% LBG. It should be noted that all the deletants displayed increased β-mannanase activity compared to the reference strain, reaching 4.3-fold higher activity in the case of the double deletants at 6 h. The addition of d-glucose caused a significant decrease in the activity. The activity was barely detectable in the reference strain or in the single deletants at 6 h, while the double deletants displayed partial tolerance against the d-glucose-derived decrease. At 12 h, not only the double deletants but also the ΔcreA strain produced the same level of β-mannanase. These results are nearly identical to those for cellulase production (Kunitake et al. 2019), suggesting that the mechanisms that negatively regulate β-mannanase production are similar to those for cellulase.

Effects of creA, pkaA, and ganB deletions on the transcription of β-mannanase genes

High-molecular-weight substrates such as LBG that induce polysaccharide-degrading enzymes are generally not direct inducers. Small molecules produced by their degradation, typically mono- or di-saccharides, function as the direct inducers that lead to a rapid increase in transcription of the genes encoding the enzymes. In Aspergillus oryzae, 1,4-β-mannobiose acts as an inducer of expression of β-mannanase genes (Ogawa et al. 2012). Here, the disaccharide also induced the expression of the β-mannanase genes manB, manC, manE, and manF in A. nidulans; their transcripts accumulated at 1.5 h after the addition of 3 mM 1,4-β-mannobiose (Fig. 5).

Fig. 5figure 5

Expression of manB, manC, manE, and manF. Each strain was cultured under β-mannobiose-inducing conditions (mb) and d-glucose (mb + glc)- or d-xylose (mb + xyl)-added conditions. Relative expression levels of the genes were normalized with actA and are shown on a logarithmic scale (log2 (gene/actA × 105). White bars, the reference strain; light gray bars, ΔcreA; dark gray bars, ΔpkaA; filled bars, ΔcreAΔpkaA; dashed bars, ΔganB; striped bars, ΔcreAΔganB. Error bars indicate the standard deviations of three independent experiments. Different letters indicate significant differences at P < 0.05 by one-way ANOVA with Tukey’s post hoc test

To evaluate the involvement of the creA, pkaA, and ganB genes in CCR in regulating the β-mannanase genes, the effect of adding d-glucose or d-xylose on the expression of the β-mannanase genes was examined. The addition of d-glucose and d-xylose caused a 3- to 8-fold and a 5- to 50-fold decrease, respectively, in the reference strain (Fig. 5). The expression levels of the β-mannanase genes under such repressed conditions increased in all the deletants as compared to those in the reference strain, and furthermore, it appeared that the contributions of CreA, PkaA, and GanB differed depending on the repressing carbon source (Fig. 6). In the case of d-glucose repression, the expression of β-mannanase genes was partially de-repressed in the creA, pkaA, and ganB deletants. And while the expression levels were 13–29% in the reference strain under the repressed conditions, they increased to 30–66% in the deletants. Furthermore, the repression was nearly fully negated in the double deletants (ΔcreAΔpkaA and ΔcreAΔganB). Although de-repression of the manF gene was still partial in the double deletants as a single exception, these results indicated that d-glucose-derived CCR of the β-mannanase genes is regulated by the independent actions of CreA and PkaA/GanB (cAMP signaling). In contrast, CreA predominantly appeared to function in CCR by d-xylose, because de-repression caused by pkaA and ganB deletion was much weaker than that caused by creA deletion (decreased to 2.0–19% in the reference strain, 5.3–37% in ΔpkaA or ΔganB, and 51–87% in ΔcreA). Thus, the contribution of the GanB/PkaA signaling pathway appeared to be very minor in CCR by d-xylose.

Fig. 6figure 6

Relative expression levels of the β-mannanase genes on β-mannobiose plus d-glucose (a) or d-xylose (b) conditions as compared to β-mannobiose-induced conditions. The de-repression ratio in the vertical axis was calculated by dividing the expression level under the repressing conditions by that under the inducing conditions. Asterisks indicate significant differences at P < 0.05 by ANOVA with Dunnett’s post hoc test

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