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

<p>Abstract</p> <p>Background</p> <p>The filamentous fungus, <it>Aspergillus niger</it>, 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 <it>A. niger </it>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 <it>N</it>-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 <it>N</it>-glycosylation.</p> <p>Results</p> <p>This study compares the physiology and transcriptome of <it>A. niger </it>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, <it>N</it>-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.</p> <p>Conclusion</p> <p>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.</p

Genomic analysis of the secretion stress response in the enzyme-producing cell factory Aspergillus niger

<p>Abstract</p> <p>Background</p> <p>Filamentous fungi such as <it>Aspergillus niger </it>have a high capacity secretory system and are therefore widely exploited for the industrial production of native and heterologous proteins. However, in most cases the yields of non-fungal proteins are significantly lower than those obtained for fungal proteins. One well-studied bottleneck appears to be the result of mis-folding of heterologous proteins in the ER during early stages of secretion, with related stress responses in the host, including the unfolded protein response (UPR). This study aims at uncovering transcriptional and translational responses occurring in <it>A. niger </it>exposed to secretion stress.</p> <p>Results</p> <p>A genome-wide transcriptional analysis of protein secretion-related stress responses was determined using Affymetrix DNA GeneChips and independent verification for selected genes. Endoplasmic reticulum (ER)-associated stress was induced either by chemical treatment of the wild-type cells with dithiothreitol (DTT) or tunicamycin, or by expressing a human protein, tissue plasminogen activator (t-PA). All of these treatments triggered the UPR, as shown by the expression levels of several well-known UPR target genes. The predicted proteins encoded by most of the up-regulated genes function as part of the secretory system including chaperones, foldases, glycosylation enzymes, vesicle transport proteins, and ER-associated degradation proteins. Several genes were down-regulated under stress conditions and these included several genes that encode secreted enzymes. Moreover, translational regulation under ER stress was investigated by polysomal fractionation. This analysis confirmed the post-transcriptional control of <it>hacA </it>expression and highlighted that differential translation also occurs during ER stress, in particular for some genes encoding secreted proteins or proteins involved in ribosomal biogenesis and assembly.</p> <p>Conclusion</p> <p>This is first genome-wide analysis of both transcriptional and translational events following protein secretion stress. Insight has been gained into the molecular basis of protein secretion and secretion-related stress in an effective protein-secreting fungus, and provides an opportunity to identify target genes for manipulation in strain improvement strategies.</p

Venn diagrams of the numbers of overlapping and non-overlapping induced (≥ 1

<p><b>Copyright information:</b></p><p>Taken from "Genomic analysis of the secretion stress response in the enzyme-producing cell factory "</p><p>BMC Genomics 2007;8():158-158.</p><p>Published online 11 Jun 2007</p><p>PMCID:PMC1894978.</p><p></p>5 fold) or repressed (≤ 1.5 fold) genes on the array after exposure to DTT or tunicamycin (Tun) and in the t-PA producing strain (t-PA)

Model of the secretory pathway under different ER stress conditions (t-PA secretion, tunicamycin and DTT) together with examples of genes that are transcriptionally induced or repressed

<p><b>Copyright information:</b></p><p>Taken from "Genomic analysis of the secretion stress response in the enzyme-producing cell factory "</p><p>BMC Genomics 2007;8():158-158.</p><p>Published online 11 Jun 2007</p><p>PMCID:PMC1894978.</p><p></p> The gene designation is provided where previously known or, otherwise, the gene name is provided. Red, genes up-regulated by 3 conditions; orange, genes up-regulated by 2 conditions; yellow, genes up-regulated by 1 condition; light blue, genes down-regulated by 1 condition; blue, genes down-regulated by 2 conditions. N, nucleus; ER, endoplasmic reticulum; E, endosome; V, vacuole; G, Golgi. ERAD is ER-associated degradation

GeneChip results were confirmed for some genes using Northern blotting and RT-PCR

<p><b>Copyright information:</b></p><p>Taken from "Genomic analysis of the secretion stress response in the enzyme-producing cell factory "</p><p>BMC Genomics 2007;8():158-158.</p><p>Published online 11 Jun 2007</p><p>PMCID:PMC1894978.</p><p></p> Examples are provided for both Northern blots (A) and RT-PCR (B). Note that the RT-PCR for the mRNA was designed to indicate enhanced splicing of the mRNA intron under stress conditions (DTT, tunicamycin and production of t-PA). This is shown as a relative increase in the amount of the processed (lower band) form of the mRNA compared to the unprocessed higher band (). Probing or PCR for an actin gene was used as a non-stress-responsive control transcript

Hierarchical clustering of records in a dendrogram (tree graph) based on the similarity of the signal log ratios obtained in each of the duplicate stress studies

<p><b>Copyright information:</b></p><p>Taken from "Genomic analysis of the secretion stress response in the enzyme-producing cell factory "</p><p>BMC Genomics 2007;8():158-158.</p><p>Published online 11 Jun 2007</p><p>PMCID:PMC1894978.</p><p></p> Records (188) were selected based on differential expression in the tPA comparisons. This tree is representative for multiple clusterings performed using signals or signal log ratios. The genes have been rearranged into their cluster order and are represented on the vertical axis. The experiments are represented on the horizontal axis. The significance of the colour scale is indicated

(A) Representative absorbance profile for RNA separated by velocity sedimentation through a 15–60% sucrose gradient

<p><b>Copyright information:</b></p><p>Taken from "Genomic analysis of the secretion stress response in the enzyme-producing cell factory "</p><p>BMC Genomics 2007;8():158-158.</p><p>Published online 11 Jun 2007</p><p>PMCID:PMC1894978.</p><p></p> Fractions are numbered from the top to the bottom of the gradient. (B) RNA was extracted from each fraction and subjected to electrophoresis through a formaldehyde gel. The ribosomal RNA distribution profile (25S, 18S and 5S rRNA; indicated by arrowheads) enables the the assignment of ODpeaks, corresponding to the 40S and 60S ribosomal subunits and to intact ribosomes (80S). (C) RT-PCR analysis with primers were performed from each fraction of collected gradients from treated and non-treated cells. The full length mRNA (arrow) as well as low-molecular-weight version of (arrowhead) can be visualized