The carbon starvation response of Aspergillus niger during submerged cultivation: insights from the transcriptome and secretome

Abstract

Background: Filamentous fungi are confronted with changes and limitations of their carbon source during growth in their natural habitats and during industrial applications. To survive life-threatening starvation conditions, carbon from endogenous resources becomes mobilized to fuel maintenance and self-propagation. Key to understand the underlying cellular processes is the system-wide analysis of fungal starvation responses in a temporal and spatial resolution. The knowledge deduced is important for the development of optimized industrial production processes.

Results: This study describes the physiological, morphological and genome-wide transcriptional changes caused by prolonged carbon starvation during submerged batch cultivation of the filamentous fungus Aspergillus niger. Bioreactor cultivation supported highly reproducible growth conditions and monitoring of physiological parameters. Changes in hyphal growth and morphology were analyzed at distinct cultivation phases using automated image analysis. The Affymetrix GeneChip platform was used to establish genome-wide transcriptional profiles for three selected time points during prolonged carbon starvation. Compared to the exponential growth transcriptome, about 50% (7,292) of all genes displayed differential gene expression during at least one of the starvation time points. Enrichment analysis of Gene Ontology, Pfam domain and KEGG pathway annotations uncovered autophagy and asexual reproduction as major global transcriptional trends. Induced transcription of genes encoding hydrolytic enzymes was accompanied by increased secretion of hydrolases including chitinases, glucanases, proteases and phospholipases as identified by mass spectrometry.

Conclusions: This study is the first system-wide analysis of the carbon starvation response in a filamentous fungus. Morphological, transcriptomic and secretomic analyses identified key events important for fungal survival and their chronology. The dataset obtained forms a comprehensive framework for further elucidation of the interrelation and interplay of the individual cellular events involved.

Figure 1

Physiology and expression profiles of aging carbon limited batch cultures. (A) Growth curve combined with profiles for extracellular protease activity and extracellular protein concentrations. (B) Summary of physiological parameters including dissolved oxygen tension (DOT), titrant addition, O₂ consumption and CO₂ production rates. (C) Northern analysis for the gamma-actin encoding gene actA (An15g00560), the β-N-acetylglucosaminidase nagA (An09g02240) and the regulator of conidiation brlA (An01g10540).

Figure 2

Hyphal morphology during four distinct cultivation phases. (A) Intact hyphae from the exponential growth phase with an average diameter of approximately 3 μm. (B) 16 hours after carbon depletion empty hyphal compartments emerged (white triangles) and new hyphae with a significantly reduced average diameter of approximately 1 μm appeared (black triangles). (C) 60 hours after carbon depletion, the number of empty hyphal compartments increased and thin hyphae elongated in a non-branching manner. First reproductive structures emerged (white-edged triangles). Thin hyphae even grew cryptically inside empty hyphal ghosts (black-edged triangles). (D) Even 140 hours after carbon depletion, surviving compartments were present (black pentagon) often bearing morphologically reduced reproductive structures (white-edged triangle). The mycelial network consisted largely of empty hyphal ghosts but hyphal fragmentation was rarely observed. The scale bar refers to 5 μm.

Figure 3

Hyphal population dynamics. For six distinct time points, probability density curves of hyphal diameters are shown. 2 hours prior to carbon depletion, a single population of hyphae with a mean diameter of approximately 3 μm was detected. After carbon depletion, a second population with a significantly reduced mean diameter of approximately 1 μm started to emerge. Throughout the course of starvation, the ratio of thin/thick hyphae gradually increased, indicating secondary growth on the expense of dying compartments.

Figure 4

Venn diagram. Venn diagram showing numbers of up- and downregulated genes in black and grey, respectively. Differential expression (FDR q-value < 0.005) was assessed by comparison of expression profiles from day 1, 3 and 6 of carbon depletion with expression profiles from the exponential growth phase

Figure 5

Summary of GO enrichment results. Summary of GO enrichment results for the up- and downregulated gene sets of day 1, 3 and 6 of carbon starvation. Statistically significant overrepresentation (FDR < 0.05) is indicated in black.

Figure 6

Model for the carbon starvation response in A. niger Schematic representation of major early, intermediate and late processes during prolonged submerged carbon starvation. During the early phase of starvation, secondary growth fueled by carbon recycling is initiated as characterized by the formation of thin hyphae. Two mechanisms resulting in empty hyphal compartments are depicted. On the left side, apoptotic/necrotic signals lead to cell death of compartments. Cytoplasmic content leaks into the culture broth. Surviving compartments are protected by autophagic processes isolating/inactivating cell death signals. On the right side, endogenous recycling of neighboring compartments by autophagic processes leads to the formation of empty hyphal ghosts. Cytoplasmic content does not leak into the culture broth. During the intermediate phase, earlier processes continue and first reproductive structures emerge. Towards later phase, these processes proceed resulting in few surviving compartments often bearing reproductive structures and elongating thin hyphae. Depending on strain (e.g. ΔcreA) and cultivation conditions (e.g. elevated pH), a largely empty non fragmented mycelial network remains (left side) or fragmentation of empty hyphal ghosts occurs by hydrolytic weakening of cell walls (right side).