The Aspergillus nidulans Pyruvate Dehydrogenase Kinases Are Essential To Integrate Carbon Source Metabolism

Abstract

The pyruvate dehydrogenase complex (PDH), that converts pyruvate to acetyl-coA, is regulated by pyruvate dehydrogenase kinases (PDHK) and phosphatases (PDHP) that have been shown to be important for morphology, pathogenicity and carbon source utilization in different fungal species. The aim of this study was to investigate the role played by the three PDHKs PkpA, PkpB and PkpC in carbon source utilization in the reference filamentous fungus Aspergillus nidulans, in order to unravel regulatory mechanisms which could prove useful for fungal biotechnological and biomedical applications. PkpA and PkpB were shown to be mitochondrial whereas PkpC localized to the mitochondria in a carbon source-dependent manner. Only PkpA was shown to regulate PDH activity. In the presence of glucose, deletion of pkpA and pkpC resulted in reduced glucose utilization, which affected carbon catabolite repression (CCR) and hydrolytic enzyme secretion, due to de-regulated glycolysis and TCA cycle enzyme activities. Furthermore, PkpC was shown to be required for the correct metabolic utilization of cellulose and acetate. PkpC negatively regulated the activity of the glyoxylate cycle enzyme isocitrate lyase (ICL), required for acetate metabolism. In summary, this study identified PDHKs important for the regulation of central carbon metabolism in the presence of different carbon sources, with effects on the secretion of biotechnologically important enzymes and carbon source-related growth. This work demonstrates how central carbon metabolism can affect a variety of fungal traits and lays a basis for further investigation into these characteristics with potential interest for different applications.

Keywords: Aspergillus nidulans; carbon catabolite repression; carbon source utilization and regulation; pyruvate dehydrogenase kinases.

Figure 1

PkpC is important for growth on various carbon sources. A. The wild-type (TN02a3), ΔpkpA, ΔpkpB and ΔpkpC strains were grown on complete medium (YUU) or minimal medium supplemented with 1% (w/v or v/v) of all carbon sources except for laureate (2.5 mM) and oleic acid (2.5 mM). Plates were inoculated with 10⁵ spores of each strain. B-C. Complementation of the ΔpkpA and ΔpkpC strains rescues the observed growth defect in the presence of allyl alcohol (AA). The wild-type (WT), protein kinase deletion and complemented strains as well as the double deletion strains were grown on minimal medium supplemented with glucose and increasing concentrations of AA for 5 days at 30°C before (B.) representative pictures were taken and (C.) radial diameter was measured. Error bars indicate standard deviations of biological triplicates (*P-value < 0.05; **P-value < 0.005; ***P-value < 0.0005 as determined by a one-tailed, paired student t-test).

Figure 2

PkpC localises to the mitochondria in a carbon source-dependent manner and is not important for PDH activity. A. Western blot of the cytosolic and mitochondrial fractions of the wild-type, PkpA::GFP, PkpB::GFP and PkpC::GFP strains when grown for 24 hr in minimal medium supplemented with glucose and after transfer to acetate-rich minimal medium for 1 h. Mycelia were harvested and subjected to cell fractionation. 50 µg of protein from each cellular extract was run on a 12% SDS-PAGE gel and subsequently electroblotted to a membrane. Pkp proteins were detected by using anti-GFP antibody, whereas anti- S. cerevisiae Pgk1 and anti-cytochrome C antibodies were used as a fractionation controls for the cytosolic and mitochondrial enrichment, respectively (CE: crude extract, C: cytosolic extract, M: mitochondrial extract). B. Activity of the pyruvate dehydrogenase complex (PDH) in the wild-type and ΔpkpA, ΔpkpB and ΔpkpC strains when transferred from minimal medium supplemented with casamino acids to glucose-rich medium for 6 h. Standard deviations present the average of 3 biological replicates (***P-value < 0.0005 as determined by a one-tailed, paired student t-test).

Figure 3

PkpA and PkpC are involved in hydrolytic enzyme secretion. A., B., C. Silver-stained protein gels showing biological triplicates of 20 μl total protein from 10 × concentrated culture supernatants of the wild-type and the pyruvate dehydrogenase protein kinase (PDHK) deletion strains when grown for 24 h in minimal medium supplemented with casamino acids and after transfer to cellulose-rich or cellulose- and glucose-rich medium for 5 days. Total extracellular secreted protein (TESP) concentrations, as determined by Bradford assay, for each biological triplicate are also shown. D., E. Cellulase and xylanase activities in the supernatants of the wild-type and PDHK deletion strains when grown in the same above specified conditions. Enzyme activities were normalized by intracellular protein concentration. Standard deviations present the average of 3 biological replicates (*P-value < 0.05; **P-value < 0.005; ***P-value < 0.0005 as determined by a one-tailed, paired student t-test).

Figure 4

Genetic interaction between the pyruvate dehydrogenase protein kinase-encoding genes. A. Growth of the wild-type (TN02a3), ΔpkpA, ΔpkpB, ΔpkpC and the double deletion strains in the presence of 1% w/v of different carbon sources. B. Glucose consumption, measured indirectly by assessing the concentration of extracellular glucose, of the WT single and double deletion strains when grown directly in minimal medium supplemented with glucose for 72 h. Standard deviations represent the average of 3 biological replicates (**P-value < 0.01; ***P-value < 0.001 as determined by a two-way ANOVA test and Bonferroni post-tests).

Figure 5

PkpA and PkpC are important for glucose uptake, carbon catabolite repression and metabolism. A. Glucose sensing is intact in the pkp deletion mutants. Spore diameter of the wild-type and protein kinase deletion strains after 0 h (control), 2 h, 4 h and 6 h incubation in minimal medium supplemented with 1% w/v glucose at 37°C, 160 rpm. Standard deviations present the average size of 100 spores of three biological replicates. B. Dryweight of the strains grown in the above specified conditions is also shown. C. alpha-ketoglutarate dehydrogenase activity and D. intracellular pyruvate levels. All strains were grown for 24 h in casamino acid-rich medium and then transferred to minimal medium supplemented with glucose for 1 h. E. Hexokinase (HXK) and F. isocitrate lyase (ICL) activities in the wild-type and pkp deletion mutants when grown in the same conditions as specified under C. and D. with the exception that strains were incubated for 6 h in these conditions. Standard deviations represent the average of 3 biological replicates (*P-value < 0.05; **P-value < 0.005; ***P-value < 0.0005 as determined by a one-tailed, paired student t-test).

Figure 6

Primary metabolism is significantly altered in the pkpA and pkpC deletion mutants in the presence of glucose and cellulose. A., C. Hierarchical clustering and principal component analysis of the levels of metabolites identified in the wild-type (WT) and pyruvate dehydrogenase kinase (pkp) deletion strains when incubated for 16 h and 48 h in minimal medium supplemented with glucose or cellulose respectively. B. Heat map showing the Log2-fold change of metabolite levels that were significantly different between the WT and ΔpkpA and ΔpkpC strains after 16 h incubation in glucose-rich conditions. Arrows indicate the intracellular storage compounds glycerol and trehalose whereas pentose phosphate pathway (PPP) and TCA (tricarboxylic acid) cycle intermediates are also indicated. D. Growth of the wild-type and pkp deletion mutants in the presence of TCA cycle precursors. Medium was supplemented with 50 mM of each amino acids and plates were inoculated with 10⁵ spores.

Figure 7

Levels of identified metabolites and the pathways in which they are produced in the wild-type and pyruvate dehydrogenase kinase deletion strains. A., B. Maps of primary metabolism (glycolysis, TCA cycle and pentose phosphate pathway) showing the average levels of identified pathway-specific metabolites of 4 biological replicates in the wild-type (WT), ΔpkpA, ΔpkpB and ΔpkpC strains when grown for A. 16 h in glucose-rich or for B. 48 h in cellulose-rich medium.

Figure 8

Schematic overview of enzymes and cellular processes regulated by PkpA, PkpB and PkpC when growing on different carbon sources. A. In the presence of glucose, PkpA positively regulates the activity of the PDH (pyruvate dehydrogenase complex) either directly or indirectly, allowing glucose consumption, glycolysis (HXK – hexokinase), the TCA (tricarboxylic acid) cycle (α-KGDH – α-ketoglutarate dehydrogenase) and carbon catabolite repression (CCR) to occur. In the absence of this protein kinase, the aforementioned metabolic pathways are reduced, resulting in faulty CCR and increased protease secretion. The targets of PkpB are currently unknown, but deletion of the corresponding gene resulted in increased hydrolytic enzyme secretion in the simultaneous presence of glucose and cellulose (G + C). PkpC is predicted to regulate the activity of the ATP citrate synthase AclB, therefore indirectly ensuring glucose consumption, glycolysis and TCA cycle progression, and CCR. In the absence of pkpC, the aforementioned metabolic pathways are reduced, resulting in faulty CCR and increased protease and hydrolytic enzyme secretion in the presence of G + C. Additional protein targets are likely to exist that were not detected in the here defined conditions. B. In the presence of cellulose, the specific targets of PkpA, PkpB and PkpC remain undefined. Deletion of pkpA and pkpC resulted in increased hydrolytic enzyme secretion in this carbon source, with the latter strain presenting different concentrations of TCA cycle intermediates. C. In the presence of acetate, PkpA and PkpB protein targets remain unkown, whereas PkpC negatively regulates the activity of the glyoxylate cycle enzyme, isocitrate lyase (ICL) AcuD. Furthermore, putative interactions of PkpC with additional glyoxylate cycle enzymes and an acetate permease are predicted; this protein kinase therefore being crucial for acetate metabolism.