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.

Proteomic analysis reflects an environmental alkalinization-coupled pH-dependent mechanism of regulating lignocellulases in Trichoderma guizhouense NJAU4742

“…The results here clearly showed that extracellular proteins, such as cellulases, hemicellulases and chitinases, were under the regulation of this possible system. Similarly, some cellulase and xylanase genes were also reported to be controlled by the ambient pH in T. reesei [35], Humicola grisea [36] and Neurospora crassa [19], and thus together showed the universality of pH regulation of lignocellulases in filamentous fungi. Actually, there are already some reports that the integrity of seven proteins, designated PacC, PalA, PalB, PalC, PalF, PalH and PalI, were responsible for gene regulation at different pH values [14].…”

“…For example, deletion of the pacC gene in T. reesei significantly increased the cellulolytic enzyme activities at neutral pH [18], but the transcriptome analysis showed only a few cellulase-and hemicellulase-encoding genes were clearly under PacC regulation [16]. In Aspergillus nidulans [37], PacC significantly upregulated the expression of cellulolytic genes at alkaline pH, but had a reverse observation in N. crassa [19]. The homologs of the seven proteins in the whole PacC-mediated system could be detected and encoded in the genome of NJAU4742 strain, and thus suggested that those pH-regulated lignocellulases were possibly under the control of this system.…”

“…Furthermore, deeper studies revealed that a pH regulatory system could be mediated by the transcription factor of PacC, which was responsible for differential protein expression at different pH values [14,17]. It was reported that knocking out the pacC gene could significantly affect both cellulase and xylanase activities in two model filamentous fungi, Trichoderma reesei and Neurospora crassa [18,19]. These previous results showed that a PacC-mediated mechanism likely exists as a general way of regulating lignocellulases in filamentous fungi.…”

Functional diversity in the pH signaling pathway: an overview of the pathway regulation in Neurospora crassa

“…In addition, we described that the CRZ-1 transcription factor cooperates with PAC-3 to regulate the metabolism of both these carbohydrates under pH stress (Virgilio et al 2017). The PAC-3 transcription factor was also described to regulate the expression of genes encoding holocellulolytic enzymes influencing the cellulase and xylanase activities (Campos Antoniêto et al 2017). The production of cellulolytic and xylanolytic enzymes is regulated by glucose through the action of the CreA (A. nidulans), CRE-1 (N. crassa) or CRE1 (T. reesei) transcription factors.…”

“…One of the first investigations regarding the role of the pH signaling pathway in N. crassa metabolism was related to the control of phosphatase secretion under regulation of the Pal/PacC signal transduction pathway (reviewed in Rossi et al 2013). More recently, the participation of PAC-3 in the regulation of the xylanase, cellulolytic and endoglucanase activities was also reported (Campos Antoniêto et al 2017). An important metabolic process regulated by the pH signaling pathway in N. crassa is the metabolism of the reserve carbohydrates glycogen and trehalose.…”