Transcriptomic comparison of Aspergillus niger growing on two different sugars reveals coordinated regulation of the secretory pathway

Abstract

Background: The filamentous fungus, Aspergillus niger, responds to nutrient availability by modulating secretion of various substrate degrading hydrolases. This ability has made it an important organism in industrial production of secreted glycoproteins. The recent publication of the A. niger genome sequence and availability of microarrays allow high resolution studies of transcriptional regulation of basal cellular processes, like those of glycoprotein synthesis and secretion. It is known that the activities of certain secretory pathway enzymes involved N-glycosylation are elevated in response to carbon source induced secretion of the glycoprotein glucoamylase. We have investigated whether carbon source dependent enhancement of protein secretion can lead to upregulation of secretory pathway elements extending beyond those involved in N-glycosylation.

Results: This study compares the physiology and transcriptome of A. niger growing at the same specific growth rate (0.16 h(-1)) on xylose or maltose in carbon-limited chemostat cultures. Transcription profiles were obtained using Affymetrix GeneChip analysis of six replicate cultures for each of the two growth-limiting carbon sources. The production rate of extracellular proteins per gram dry mycelium was about three times higher on maltose compared to xylose. The defined culture conditions resulted in high reproducibility, discriminating even low-fold differences in transcription, which is characteristic of genes encoding basal cellular functions. This included elements in the secretory pathway and central metabolic pathways. Increased protein secretion on maltose was accompanied by induced transcription of > 90 genes related to protein secretion. The upregulated genes encode key elements in protein translocation to the endoplasmic reticulum (ER), folding, N-glycosylation, quality control, and vesicle packaging and transport between ER and Golgi. The induction effect of maltose resembles the unfolded protein response (UPR), which results from ER-stress and has previously been defined by treatment with chemicals interfering with folding of glycoproteins or by expression of heterologous proteins.

Conclusion: We show that upregulation of secretory pathway genes also occurs in conditions inducing secretion of endogenous glycoproteins - representing a more normal physiological state. Transcriptional regulation of protein synthesis and secretory pathway genes may thus reflect a general mechanism for modulation of secretion capacity in response to the conditional need for extracellular enzymes.

Figure 1

Morphology of mycelium in chemostat cultures of A. niger. (A) Steady state on xylose (50 h). (B) Steady state on maltose (80 h).

Figure 2

Growth profiles of triplicate A. niger AB94-85 (A) and ABGT1026 (B) chemostat cultures, Dry weight biomass concentration (gDW kg^-1) as a function of time (h) illustrates growth of three replicate cultures (open square, circle and triangle). Dot-line indicates start of continuous cultivation – exit from batch culture. Dash-line represents the switch to maltose as carbon source after 5 RT with xylose as the growth-limiting substrate. Arrows indicate time-points, where mycelium was harvested for transcriptomic analysis.

Figure 3

Signal intensity variation among replicate cultures. Variation is expressed as coefficient of variation (CV) of mean signal intensities of independent triplicate measurements, and shown for steady-state gene-expression of xylose- (A, C) and maltose-limited (B, D) cultures of AB94-85 and ABGT1026. Only genes with Detection call Marginal or Present in at least one of three measurements are shown (expressed genes). Blue and red circles and identifiers indicate maltose-expressed genes involved in xylose/xylan catabolism and clustered fumonisin biosynthesis gene homologs, respectively.

Figure 4

Functional classification of differentially expressed genes (open bars indicate number of genes with higher transcript levels on xylose; hatched bars represent genes higher expressed on maltose). (A) Representation of major functional categories (Funcat) among differentially expressed genes. (B) Subcellular localization of differentially expressed genes. Unclassified ORFs: high on xylose, 40% (213/528); high on maltose, 30% (214/712).

Figure 5

Differentially expressed genes in sub-Funcats of Fig. 4A. (A) 06 – Protein fate. (B) 08 – Cellular transport and transport mechanisms. (C) 05 – Protein synthesis.