Carbon Catabolite Repression in Filamentous Fungi Is Regulated by Phosphorylation of the Transcription Factor CreA

Abstract

Filamentous fungi of the genus Aspergillus are of particular interest for biotechnological applications due to their natural capacity to secrete carbohydrate-active enzymes (CAZy) that target plant biomass. The presence of easily metabolizable sugars such as glucose, whose concentrations increase during plant biomass hydrolysis, results in the repression of CAZy-encoding genes in a process known as carbon catabolite repression (CCR), which is undesired for the purpose of large-scale enzyme production. To date, the C2H2 transcription factor CreA has been described as the major CC repressor in Aspergillus spp., although little is known about the role of posttranslational modifications in this process. In this work, phosphorylation sites were identified by mass spectrometry on Aspergillus nidulans CreA, and subsequently, the previously identified but uncharacterized site S262, the characterized site S319, and the newly identified sites S268 and T308 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was investigated. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 was not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. All sites were shown to be important for glycogen and trehalose metabolism. This study highlights the importance of CreA phosphorylation sites for the regulation of CCR. These sites are interesting targets for biotechnological strain engineering without the need to delete essential genes, which could result in undesired side effects. IMPORTANCE In filamentous fungi, the transcription factor CreA controls carbohydrate metabolism through the regulation of genes encoding enzymes required for the use of alternative carbon sources. In this work, phosphorylation sites were identified on Aspergillus nidulans CreA, and subsequently, the two newly identified sites S268 and T308, the previously identified but uncharacterized site S262, and the previously characterized site S319 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was characterized. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 is not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. This work characterized novel CreA phosphorylation sites under carbon catabolite-repressing conditions and showed that they are crucial for CreA protein turnover, control of carbohydrate utilization, and biotechnologically relevant enzyme production.

Parallel proteomic and phosphoproteomic analyses reveal cellobiose‐dependent regulation of lignocellulase secretion in the filamentous fungus Neurospora crassa

“…Multiple phosphorylation sites of Cre1/CreA have been identified in N . crassa (Figure S1) and other filamentous fungi (de Assis et al, 2021; Horta et al, 2019) and characterized. However, only dephosphorylation on S6 and S211 was identified in response to cellobiose and Avicel (Table 1), and none of them were characterized in previous studies.…”

Induction and Repression of Hydrolase Genes in Aspergillus oryzae

“…Six serine residues (S262, S277, S288, S289, S312, and S319) in A. nidulans CreA were identified as phosphorylation sites by LC-MS analysis, and three of them (S289, S312, and S319) were phosphorylated only under conditions of growth in a glucose medium (Alam et al, 2017). Another recent study revealed six additional phosphorylation sites in A. nidulans CreA (S176, S268, S281, S284, T308, and S406; de Assis et al, 2021). The replacement of T308 with alanine significantly inhibited the nuclear accumulation of CreA-GFP.…”

“…The replacement of T308 with alanine significantly inhibited the nuclear accumulation of CreA-GFP. In contrast, the replacement of S262 or S268 with alanine increased the nuclear localization of CreA-GFP under de-repressing conditions, although these CreA-GFP mutants were not detectable by western blotting under such conditions ( de Assis et al, 2021 ). Phosphorylation at S319 is lost upon the deletion of pkaA , which encodes a cAMP-dependent protein kinase, whereas CreA is not a direct target of this protein kinase ( Ribeiro et al, 2019 ).…”

“…Phosphorylation at S319 is lost upon the deletion of pkaA , which encodes a cAMP-dependent protein kinase, whereas CreA is not a direct target of this protein kinase ( Ribeiro et al, 2019 ). Although the replacement of S319 with alanine has no significant effect on CreA subcellular localization, this mutation and the T308A mutation significantly increased CreA-GFP protein levels under repressing conditions ( de Assis et al, 2021 ). However, none of the substitutions of these phosphorylation sites with alanine had a significant effect on CreA degradation under de-repressing conditions ( de Assis et al, 2021 ).…”

“…Although the replacement of S319 with alanine has no significant effect on CreA subcellular localization, this mutation and the T308A mutation significantly increased CreA-GFP protein levels under repressing conditions ( de Assis et al, 2021 ). However, none of the substitutions of these phosphorylation sites with alanine had a significant effect on CreA degradation under de-repressing conditions ( de Assis et al, 2021 ). A recent study showed that replacement of S388 at the T. reesei Cre1 C-terminus with valine releases CCR ( Han et al, 2020 ).…”