Deletion of pH Regulator pac-3 Affects Cellulase and Xylanase Activity during Sugarcane Bagasse Degradation by Neurospora crassa

Abstract

Microorganisms play a vital role in bioethanol production whose usage as fuel energy is increasing worldwide. The filamentous fungus Neurospora crassa synthesize and secrete the major enzymes involved in plant cell wall deconstruction. The production of cellulases and hemicellulases is known to be affected by the environmental pH; however, the regulatory mechanisms of this process are still poorly understood. In this study, we investigated the role of the pH regulator PAC-3 in N. crassa during their growth on sugarcane bagasse at different pH conditions. Our data indicate that secretion of cellulolytic enzymes is reduced in the mutant Δpac-3 at alkaline pH, whereas xylanases are positively regulated by PAC-3 in acidic (pH 5.0), neutral (pH 7.0), and alkaline (pH 10.0) medium. Gene expression profiles, evaluated by real-time qPCR, revealed that genes encoding cellulases and hemicellulases are also subject to PAC-3 control. Moreover, deletion of pac-3 affects the expression of transcription factor-encoding genes. Together, the results suggest that the regulation of holocellulase genes by PAC-3 can occur as directly as in indirect manner. Our study helps improve the understanding of holocellulolytic performance in response to PAC-3 and should thereby contribute to the better use of N. crassa in the biotechnology industry.

Fig 1. Total protein content (A) and pH changes (B) after growth of N. crassa for eight days on sugarcane bagasse.

Strains 74A, Δmus-52, and Δpac-3, were cultivated on sugarcane bagasse at pH 3.0, 5.0, 7.0, 8.0, and 10.0 (initial pH). The final growth and pH was measured on the eighth day. The error bar indicates the standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 2. PAC-3 influences holocellulase activity in N. crassa.

Total cellulolytic (FPase) (A), endoglucanase (CMCase) (B), and xylanolytic activities (C) were assayed in the supernatant of the strains 74A, Δmus-52, and Δpac-3 after their cultivation for eight days on sugarcane bagasse. Values show the mean of three replicates. The error bar indicates the standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3. Western blot analysis shows a reduced amount of cellulase (CBHI) at both neutral and alkaline pH.

The analysis was performed using supernatants collected from the N. crassa 74A, Δmus-52, and Δpac-3 strains after growth for eight days on sugarcane bagasse at pH 3.0, 5.0, 7.0, 8.0, and 10.0. Anti-cellulase antibody from Trichoderma viride were used to detect CBH-1.

Fig 4. Gene expression profiles of holocellulolytic enzymes in N. crassa.

The strains 74A, Δmus-52, and Δpac-3 grown on sugarcane bagasse at different pH (3.0, 5.0, 7.0, 8.0, and 10.0) were used for RT-qPCR experiments. (A) cbh-1 = Cellobiohydrolase 1 (exoglucanase) NCU07340; (B) gh7-1 = Endoglucanase 1 NCU05057; (C) gh3-2 = β-glucosidase 1 NCU08054; (D) gh11-1 = Endo-1,4-β-xylanase 1 NCU02855; (E) gh10-4 = Endo-1,4-β-xylanase 2 NCU07130; (F) gh43-5 = β-xylosidase NCU09652. Values show the mean of three replicates. The error bar indicates the standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5. Schematic representation of putative PAC-3 binding sites (5′ -BGCCVAGV-3′).

The analysis was performed using the region 1.0-kbp upstream of cellulolytic and xylanolytic genes. The position of the motifs is relative to the translation initiation codon (ATG). B = C or G or T; V = A or C or G, according to International Union of Pure and Applied Chemistry (IUPAC) norms.

Fig 6. Effect of pac-3 deletion on transcriptional factors gene expression.

The strains 74A, Δmus-52, and Δpac-3 grown on sugarcane bagasse at different pH (3.0, 5.0, 7.0, 8.0, and 10.0) were used for RT-qPCR experiments. (A) xlr-1 = Xylan degradation regulator-1 NCU06971; (B) cre-1 = Carbon catabolite regulator NCU08807; (C) clr-1 = Cellulose degradation regulator-1 NCU07705 and (D) clr-2 = Cellulose degradation regulator-2 NCU08042. Values show the mean of three replicates. The error bar indicates the standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 7. The model proposed for the regulation of holocellulolytic-encoding genes in N. crassa by PAC-3.

Changes in the pH environment lead to activation of PAC-3, which directly or indirectly regulates transcription of cellulases and xylanases as well as the transcription factors XLR-1, CLR-1, and CLR-2 genes. The transcription factor CLR-1 regulate transcription of both xlr-1 and clr-2 genes. These transcription factors can also regulate cellulases and xylanases. XLR-1 regulate transcription of gh3-1, gh11-1, and gh43-5 genes (purple arrows); CLR-1 regulate transcription of cbh-1 and gh7-1 genes (green arrows), and CLR-2 regulate transcription of cbh-1, gh7-1, and gh11-1 genes (red arrows). (?) Missing component in regulation of cre-1 by PAC-3.