Aspergillus nidulans protein kinase A plays an important role in cellulase production

Abstract

Background: The production of bioethanol from lignocellulosic feedstocks is dependent on lignocellulosic biomass degradation by hydrolytic enzymes. The main component of lignocellulose is cellulose and different types of organisms are able to secrete cellulases. The filamentous fungus Aspergillus nidulans serves as a model organism to study cellulase production and the available tools allow exploring more in depth the mechanisms governing cellulase production and carbon catabolite repression.

Results: In A. nidulans, microarray data identified the cAMP-dependent protein kinase A (PkaA) as being involved in the transcriptional modulation and the production of lignocellulolytic enzymes in the presence of cellulose. Deletion of pkaA resulted in increased hydrolytic enzyme secretion, but reduced growth in the presence of lignocellulosic components and various other carbon sources. Furthermore, genes involved in fungal development were increased in the ΔpkaA strain, probably leading to the increased hyphal branching as was observed in this strain. This would allow the secretion of higher amounts of proteins. In addition, the expression of SynA, encoding a V-SNARE synaptobrevin protein involved in secretion, was increased in the ΔpkaA mutant. Deletion of pkaA also resulted in the reduced nuclear localization of the carbon catabolite repressor CreA in the presence of glucose and in partial de-repression when grown on cellulose. PkaA is involved in the glucose signaling pathway as the absence of this protein resulted in reduced glucose uptake and lower hexokinase/glucokinase activity, directing the cell to starvation conditions. Genome-wide transcriptomics showed that the expression of genes encoding proteins involved in fatty acid metabolism, mitochondrial function and in the use of cell storages was increased.

Conclusions: This study shows that PkaA is involved in hydrolytic enzyme production in A. nidulans. It appears that this protein kinase blocks the glucose pathway, hence forcing the cell to change to starvation conditions, increasing hydrolytic enzyme secretion and inducing the usage of cellular storages. This work uncovered new regulatory avenues governing the tight interplay between the metabolic states of the cell, which are important for the production of hydrolytic enzymes targeting lignocellulosic biomass. Deletion of pkaA resulted in a strain with increased hydrolytic enzyme secretion and reduced biomass formation.

Keywords: Aspergillus nidulans; Carbon catabolite repression; Cellulose; Glucose metabolism; Protein kinase A.

Fig. 1

a Deletion of pkaA results in an earlier onset of glycoside hydrolase (GH) gene expression. Gene expression values are shown for various enzymes belonging to different GH families in the wild-type (WT) and ΔpkaA strains when grown in biological triplicates in cellulose-rich media for 8 and 24 h. b Genes encoding transcription factors important for cellulose, hemicellulose and fatty acid utilization as well as for mediating the carbon starvation response are up-regulated at an earlier time point in the ΔpkaA strain. Experiments were carried out in biological triplicates and all changes in the levels of gene expression listed here have a statistical significance of p < 0.01

Fig. 2

Deletion of pkaA results in an increase in secreted hydrolytic enzymes. a Cellulase activities, b β-glucosidase activities, c xylanase activities and d β-xylosidase activities in different strains. Mycelia were grown in complete media for 24 h before being transferred to minimal medium supplemented with 1 % Avicel (C, cellulose) or xylan (X) or to minimal medium supplemented with 2 % glucose and 1 % cellulose or xylan (G + C; G + X) for 5 or 3 days, respectively. Enzymatic activities were determined in culture supernatants. All enzyme activities were normalized by intracellular protein concentration. Experiments were carried out in biological triplicates and the statistical significance of (***) p < 0.001 between repressing (G + C; G + X) and de-repressing (C; X) conditions

Fig. 3

Deletion of pkaA results in increased hyphal branching. a Western blot of GFP::SynA. Mycelia were grown from spores in complete media for 24 h and then transferred to minimal media supplemented with 1 % cellulose for 3 and 5 days before proteins were extracted. For normalization, a gel was run with the total protein extract and subsequently stained with Coomassie blue. b Mycelia were grown from spores in complete media (YUU), minimal media supplemented with glucose (MM + Gluco) or minimal media supplemented with CMC carboxymethylcellulose (MM + CMC) for 3 days at 37 °C. Pictures were taken at a ×20 magnification (scale bar 100 μm)

Fig. 4

PkaA is involved in the response to carbon starvation. a Western blot, b PkaA activity and c fluorescence microscopy of pkaA::GFP. Mycelia were grown from spores in minimal media supplemented with 1 % glucose for 16 h at 22 °C, washed 2× with water before being transferred to minimal media without any carbon source (starvation) for 15, 30, 60 and 120 min

Fig. 5

PkaA is involved in the response to carbon starvation. a Western blot pkaA::GFP. The pkaA::GFP strain was grown in minimal media supplemented with 1 % glucose and then transferred to minimal media supplemented with 1 % avicel for the indicated amounts of time. b Fluorescence microscopy of PkaA::GFP grown in the same condition described above

Fig. 6

PkaA is involved in the response to carbon starvation. Fluorescence microscopy of pkaA:GFP when grown from spores in 1 % avicel for 24 h and then transferred to minimal media supplemented with 1 % glucose as sole carbon source

Fig. 7

PkaA is involved in glycolysis and controls the expression of genes required for using alternative carbon sources. a glucose uptake, b hexokinase/glucokinase activity, c glycerol levels, d pyruvate levels, e α-Ketoglutarate dehydrogenase activities and f trehalose utilization in the wild-type and ΔpkaA strains. Mycelia were grown from spores in complete media and then transferred to minimal media supplemented with glucose for 24 h or to glucose and 1 M sorbitol for 10, 30 and 60 min. g Strains were grown in complete media for 24 h and then transferred to minimal media supplemented with 1 % cellulose for 24 and 120 h before the ADP/ATP ratio was measured in mycelia cell extracts. Experiments were carried out in biological triplicates and the statistical significance of *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 8

A possible model for the interaction between A. nidulans PkaA and SnfA during carbon catabolite repression (CCR) repressing (glucose) and de-repressing (cellulose) conditions. In the presence of glucose, A. nidulans PkaA is activated and represses SnfA. CreA is transported into the nucleus via importins where it represses either directly or indirectly the expression of cellulase-encoding genes (e.g., eglA) and their corresponding positive regulators (e.g., clrB). In the presence of cellulose, PkaA is inactive whereas SnfA is activated and probably mediates the phosphorylation and re-localization of CreA into the cytoplasm, resulting in cellulose gene de-repression. It is unknown whether SnfA is transported into the nucleus or if there is another intermediary protein that is phosphorylated by SnfA and which is responsible for CreA removal from the nucleus. The role played by nuclear importins/exportins during these processes also remains unknown. PkaA activity affects hyphal morphology, protein secretion and glucose transport