Functional characterisation of the non-essential protein kinases and phosphatases regulating Aspergillus nidulans hydrolytic enzyme production

Abstract

BackgroundDespite recent advances in the understanding of lignocellulolytic enzyme regulation, less is known about how different carbon sources are sensed and the signaling cascades that result in the adaptation of cellular metabolism and hydrolase secretion. Therefore, the role played by non-essential protein kinases (NPK) and phosphatases (NPP) in the sensing of carbon and/or energetic status was investigated in the model filamentous fungus Aspergillus nidulans.ResultsEleven NPKs and seven NPPs were identified as being involved in cellulase, and in some cases also hemicellulase, production in A. nidulans. The regulation of CreA-mediated carbon catabolite repression (CCR) in the parental strain was determined by fluorescence microscopy, utilising a CreA::GFP fusion protein. The sensing of phosphorylated glucose, via the RAS signalling pathway induced CreA repression, while carbon starvation resulted in derepression. Growth on cellulose represented carbon starvation and derepressing conditions. The involvement of the identified NPKs in the regulation of cellulose-induced responses and CreA derepression was assessed by genome-wide transcriptomics (GEO accession 47810). CreA::GFP localisation and the restoration of endocellulase activity via the introduction of the ∆creA mutation, was assessed in the NPK-deficient backgrounds. The absence of either the schA or snfA kinase dramatically reduced cellulose-induced transcriptional responses, including the expression of hydrolytic enzymes and transporters. The mechanism by which these two NPKs controlled gene transcription was identified, as the NPK-deficient mutants were not able to unlock CreA-mediated carbon catabolite repression under derepressing conditions, such as carbon starvation or growth on cellulose.ConclusionsCollectively, this study identified multiple kinases and phosphatases involved in the sensing of carbon and/or energetic status, while demonstrating the overlapping, synergistic roles of schA and snfA in the regulation of CreA derepression and hydrolytic enzyme production in A. nidulans. The importance of a carbon starvation-induced signal for CreA derepression, permitting transcriptional activator binding, appeared paramount for hydrolase secretion.

The Genomes of Three Uneven Siblings: Footprints of the Lifestyles of Three Trichoderma Species

“…Metabolic pathways regulating the detection of different carbon sources and hydrolytic enzyme production include protein phosphatases as regulators (817). Seven protein phosphatases were found to influence cellulase and hemicellulase production in A. nidulans.…”

Enhanced glycosyl hydrolase production in Aspergillus nidulans using transcription factor engineering approaches

“…Analysis of its genome [15] in combination with proteomic studies [9,16] show it to have a similar polysaccharide degradation potential to that of the industrial fungi, and in addition the regulatory mechanisms involved in controlling the expression of plant cell wall degrading activities are mostly conserved between it and industrially important fungi [17][18][19][20]. Finally, the use of A. nidulans as a production vehicle could provoke less concern regarding mycotoxin synthesis since its genome encodes the metabolic pathway for the less toxic compound sterigmatocystin compared to certain industrial strains which still retain the capacity to produce very harmful mycotoxins [21,22].…”

Transcriptional Regulation and Responses in Filamentous Fungi Exposed to Lignocellulose

“…In filamentous fungi, CCR is mediated by the CreA/CRE1 protein [28], which is a functional homologue of Mig1p. CreA/CRE1 is localized in the cytoplasm under non-repressing conditions and is shuttled to the nucleus under repressing conditions [37,38]. Its localization is regulated by its phosphorylation state [37], similar to Mig1p.…”

“…CreA/CRE1 is localized in the cytoplasm under non-repressing conditions and is shuttled to the nucleus under repressing conditions [37,38]. Its localization is regulated by its phosphorylation state [37], similar to Mig1p. As noted by Brown et al [15], the phosphorylation state of CreA/CRE1 has a different effect in T. reesei compared to other filamentous fungi where in T. reesei phosphorylation of CRE1 leads to repression (instead of derepression) of genes [39].…”

“…Brown et al [37] studied two non-essential protein kinases SnfA (the homologue of yeast Snf1p) and SchA in A. nidulans. They demonstrated that the deletion of snfA or schA decreased the production of hydrolytic enzymes by decreasing the ability of A. nidulans to unlock the CreA repression mechanism under de-repressing conditions (either starvation or Avicel cellulose).…”