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

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.

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

BACKGROUND: Filamentous fungi such as Aspergillus niger 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 A. niger exposed to secretion stress. RESULTS: 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 hacA 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. CONCLUSION: 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.

Production of bifunctional proteins by Aspergillus awamori: llama variable heavy chain antibody fragment (V(HH)) R9 coupled to Arthromyces ramosus peroxidase (ARP).

The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5'- or 3'-terminal ends of the gene encoding llama variable heavy chain antibody fragment V(HH) R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which produced up to 30 mgl(-1) fusion protein in the culture medium. Both fusion proteins showed peroxidase activity in an ABTS activity test. Considerable amounts of fusion protein were detected intracellularly, suggesting that the fungus encounters problems in secreting these kind of proteins. ELISA experiments showed that ARP-R9 was less able to bind its antigen, the azo-dye RR6, as compared to R9-ARP. Furthermore, in contrast to R9-ARP, ARP-R9 bound to RR6 did not show peroxidase activity anymore. These results indicate that fusion of ARP to the C-terminus of the antibody fragment V(HH) R9 (R9-ARP) is the preferred orientation.

Promoter analysis of the avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum in the model filamentous fungus Aspergillus nidulans.

The promoter of avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum contains 12 sequences within a region of 0.6 kb that are reminiscent of the binding sequences of the GATA-type regulator involved in nitrogen utilisation of the filamentous fungi Aspergillus nidulans and Neurospora crassa. Mutational analysis of this 0.6-kb promoter region, fused to the beta-glucuronidase reporter gene, revealed that two regions, each containing two TAGATA boxes in inverted orientation and overlapping by two base pairs, are important for induction of Avr9 promoter activity in A. nidulans. Each overlapping TAGATA box differentially affected Avr9 promoter activity when shifted apart by nucleotide insertions. The other regions, which do not contain two overlapping TAGATA boxes have no, or only a limited, contribution to the inducibility of promoter activity.