Abstract
BackgroundIn filamentous ascomycete fungi, the utilization of alternate carbon sources is influenced by the zinc finger transcription factor CreA/CRE-1, which encodes a carbon catabolite repressor protein homologous to Mig1 from Saccharomyces cerevisiae. In Neurospora crassa, deletion of cre-1 results in increased secretion of amylase and β-galactosidase.Methodology/Principal FindingsHere we show that a strain carrying a deletion of cre-1 has increased cellulolytic activity and increased expression of cellulolytic genes during growth on crystalline cellulose (Avicel). Constitutive expression of cre-1 complements the phenotype of a N. crassa Δcre-1 strain grown on Avicel, and also results in stronger repression of cellulolytic protein secretion and enzyme activity. We determined the CRE-1 regulon by investigating the secretome and transcriptome of a Δcre-1 strain as compared to wild type when grown on Avicel versus minimal medium. Chromatin immunoprecipitation-PCR of putative target genes showed that CRE-1 binds to only some adjacent 5′-SYGGRG-3′ motifs, consistent with previous findings in other fungi, and suggests that unidentified additional regulatory factors affect CRE-1 binding to promoter regions. Characterization of 30 mutants containing deletions in genes whose expression level increased in a Δcre-1 strain under cellulolytic conditions identified novel genes that affect cellulase activity and protein secretion.Conclusions/SignificanceOur data provide comprehensive information on the CRE-1 regulon in N. crassa and contribute to deciphering the global role of carbon catabolite repression in filamentous ascomycete fungi during plant cell wall deconstruction.
Highlights
Many microorganisms, especially filamentous fungi, secrete hydrolytic enzymes that play a key role in the degradation of plant cell wall polymers [1,2], which consist mainly of cellulose, hemicellulose, and lignin
Some aspects of carbon catabolite repression (CCR) that affect production of hydrolytic enzymes have been evaluated in the industrial species, such as Hypocrea jecorina (Trichoderma reesei) and Aspergilli
When grown on 2% Avicel medium as a sole carbon source, the Dcre-1 strain consumed Avicel faster than WT (e.g. 3–4 days vs 5–6 days), secreted 30% more extracellular protein and showed 50% higher endoglucanase activity (Figure 1C and D)
Summary
Especially filamentous fungi, secrete hydrolytic enzymes that play a key role in the degradation of plant cell wall polymers [1,2], which consist mainly of cellulose, hemicellulose, and lignin. Of these 75 genes, five showed a greater than 20-fold increase in expression level in the Dcre-1 mutant under MM conditions, including one direct target of CRE-1 identified in other systems, NCU09805 (aamylase A) [39,40], a predicted target of CreA in Aspergilli (glucoamylase; NCU01517) [39], a high affinity glucose transporter (NCU04963), a protein related to b-fructofuranosidase (NCU04265), and a starch binding protein (NCU08746) (Figure 6A).
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