Dataset Information

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.

SUBMITTER: Jorgensen TR

PROVIDER: S-EPMC2639373 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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

Jørgensen Thomas R TR Goosen Theo T Hondel Cees A M J J van den CA Ram Arthur F J AF Iversen Jens J L JJ

BMC genomics 20090123

<h4>Background</h4>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 know ...[more]

PMID: 19166577

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Project description:BACKGROUND: Filamentous fungi such as Aspergillus niger are well known for their exceptionally high capacity for secretion of proteins, organic acids, and secondary metabolites and they are therefore used in biotechnology as versatile microbial production platforms. However, system-wide insights into their metabolic and secretory capacities are sparse and rational strain improvement approaches are therefore limited. In order to gain a genome-wide view on the transcriptional regulation of the protein secretory pathway of A. niger, we investigated the transcriptome of A. niger when it was forced to overexpression the glaA gene (encoding glucoamylase, GlaA) and secrete GlaA to high level. RESULTS: An A. niger wild-type strain and a GlaA over-expressing strain, containing multiple copies of the glaA gene, were cultivated under maltose-limited chemostat conditions (specific growth rate 0.1 h-1). Elevated glaA mRNA and extracellular GlaA levels in the over-expressing strain were accompanied by elevated transcript levels from 772 genes and lowered transcript levels from 815 genes when compared to the wild-type strain. Using GO term enrichment analysis, four higher-order categories were identified in the up-regulated gene set: i) endoplasmic reticulum (ER) membrane translocation, ii) protein glycosylation, iii) vesicle transport, and iv) ion homeostasis. Among these, about 130 genes had predicted functions for the passage of proteins through the ER and those genes included target genes of the HacA transcription factor that mediates the unfolded protein response (UPR), e.g. bipA, clxA, prpA, tigA and pdiA. In order to identify those genes that are important for high-level secretion of proteins by A. niger, we compared the transcriptome of the GlaA overexpression strain of A. niger with six other relevant transcriptomes of A. niger. Overall, 40 genes were found to have either elevated (from 36 genes) or lowered (from 4 genes) transcript levels under all conditions that were examined, thus defining the core set of genes important for ensuring high protein traffic through the secretory pathway. CONCLUSION: We have defined the A. niger genes that respond to elevated secretion of GlaA and, furthermore, we have defined a core set of genes that appear to be involved more generally in the intensified traffic of proteins through the secretory pathway of A. niger. The consistent up-regulation of a gene encoding the acetyl-coenzyme A transporter suggests a possible role for transient acetylation to ensure correct folding of secreted proteins.

Project description:Fungi utilize polysaccharide substrates through extracellular digestion catalyzed by secreted enzymes. Thus far, protein secretion by the filamentous fungus Aspergillus niger has mainly been studied at the level of individual proteins and by genome and transcriptome analyses. To extend these studies, a complementary proteomics approach was applied with the aim to investigate the changes in secretome and microsomal protein composition resulting from a shift to a high level secretion condition. During growth of A. niger on D-sorbitol, small amounts of D-maltose or D-xylose were used as inducers of the extracellular amylolytic and xylanolytic enzymes. Upon induction, protein compositions in the extracellular broth as well as in enriched secretory organelle (microsomal) fractions were analyzed using a shotgun proteomics approach. In total 102 secreted proteins and 1,126 microsomal proteins were identified in this study. Induction by D-maltose or D-xylose resulted in the increase in specific extracellular enzymes, such as glucoamylase A on D-maltose and ?-xylosidase D on D-xylose, as well as of microsomal proteins. This reflects the differential expression of selected genes coding for dedicated extracellular enzymes. As expected, the addition of extra D-sorbitol had no effect on the expression of carbohydrate-active enzymes, compared to addition of D-xylose or D-maltose. Furthermore, D-maltose induction caused an increase in microsomal proteins related to translation (e.g., Rpl15) and vesicular transport (e.g., the endosomal-cargo receptor Erv14). Millimolar amounts of the inducers D-maltose and D-xylose are sufficient to cause a direct response in specific protein expression levels. Also, after induction by D-maltose or D-xylose, the induced enzymes were found in microsomes and extracellular. In agreement with our previous findings for D-xylose induction, D-maltose induction leads to recruitment of proteins involved in proteasome-mediated degradation.

Project description:Background:Understanding how fungi degrade lignocellulose is a cornerstone of improving renewables-based biotechnology, in particular for the production of hydrolytic enzymes. Considerable progress has been made in investigating fungal degradation during time-points where CAZyme expression peaks. However, a robust understanding of the fungal survival strategies over its life time on lignocellulose is thereby missed. Here we aimed to uncover the physiological responses of the biotechnological workhorse and enzyme producer Aspergillus niger over its life time to six substrates important for biofuel production. Results:We analysed the response of A. niger to the feedstock Miscanthus and compared it with our previous study on wheat straw, alone or in combination with hydrothermal or ionic liquid feedstock pretreatments. Conserved (substrate-independent) metabolic responses as well as those affected by pretreatment and feedstock were identified via multivariate analysis of genome-wide transcriptomics combined with targeted transcript and protein analyses and mapping to a metabolic model. Initial exposure to all substrates increased fatty acid beta-oxidation and lipid metabolism transcripts. In a strain carrying a deletion of the ortholog of the Aspergillus nidulans fatty acid beta-oxidation transcriptional regulator farA, there was a reduction in expression of selected lignocellulose degradative CAZyme-encoding genes suggesting that beta-oxidation contributes to adaptation to lignocellulose. Mannan degradation expression was wheat straw feedstock-dependent and pectin degradation was higher on the untreated substrates. In the later life stages, known and novel secondary metabolite gene clusters were activated, which are of high interest due to their potential to synthesize bioactive compounds. Conclusion:In this study, which includes the first transcriptional response of Aspergilli to Miscanthus, we highlighted that life time as well as substrate composition and structure (via variations in pretreatment and feedstock) influence the fungal responses to lignocellulose. We also demonstrated that the fungal response contains physiological stages that are conserved across substrates and are typically found outside of the conditions with high CAZyme expression, as exemplified by the stages that are dominated by lipid and secondary metabolism.

Dataset Information

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

Background

Results

Conclusion

Publications

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

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