Nitrate is a dominant form of inorganic nitrogen (N) in soils and can be efficiently assimilated by bacteria, fungi and plants. We studied here the transcriptome of the short-term nitrate response using assimilating and non-assimilating strains of the model ascomycete Aspergillus nidulans. Among the 72 genes positively responding to nitrate, only 18 genes carry binding sites for the pathway-specific activator NirA. Forty-five genes were repressed by nitrate metabolism. Because nirA- strains are N-starved at nitrate induction conditions, we also compared the nitrate transcriptome with N-deprived conditions and found a partial overlap of differentially regulated genes between these conditions. Nitric oxide (NO)-metabolizing flavohaemoglobins were found to be co-regulated with nitrate assimilatory genes. Subsequent molecular characterization revealed that the strongly inducible FhbA is required for full activity of nitrate and nitrite reductase enzymes. The co-regulation of NO-detoxifying and nitrate/nitrite assimilating systems may represent a conserved mechanism, which serves to neutralize nitrosative stress imposed by an external NO source in saprophytic and pathogenic fungi. Our analysis using membrane-permeable NO donors suggests that signalling for NirA activation only indirectly depends on the nitrate transporters NrtA (CrnA) and NrtB (CrnB).

SummaryThe GATA factor AreA is a wide-domain regulator in Aspergillus nidulans with transcriptional activation and chromatin remodelling functions. AreA interacts with the nitrate-specific Zn 2 -C 6 cluster protein NirA and both proteins cooperate to synergistically activate nitrate-responsive genes. We have previously established that NirA in vivo DNA binding site occupancy is AreA dependent and in this report we provide a mechanistic explanation for our previous findings. We now show that AreA regulates NirA at two levels: (i) through the regulation of nitrate transporters, AreA affects indirectly the subcellular distribution of NirA which rapidly accumulates in the nucleus following induction; (ii) AreA directly stimulates NirA in vivo target DNA occupancy and does not act indirectly by chromatin remodelling. Simultaneous overexpression of NirA and the nitrate transporter CrnA bypasses the AreA requirement for NirA binding, permits utilization of nitrate and nitrite as sole N-sources in an areA null strain and leads to an AreA-independent nucleosome loss of positioning.

No abstract

Filamentous fungi are well-established expression hosts often used to produce extracellular proteins of use in the food and pharmaceutical industries. The expression systems presently used in Aspergillus species rely on either strong constitutive promoters, e.g., that for glyceraldehyde-3-phosphate dehydrogenase, or inducible systems derived from metabolic pathways, e.g., glaA (glucoamylase) or alc (alcohol dehydrogenase). We describe for Aspergillus nidulans and Aspergillus niger a novel expression system that utilizes the transcriptional activation of the human estrogen receptor by estrogenic substances. The system functions independently from metabolic signals and therefore can be used with low-cost, complex media. A combination of positive and negative regulatory elements in the promoter drives the expression of a reporter gene, yielding a linear dose response to the inducer. The off status is completely tight, yet the system responds within minutes to induction and reaches a level of expression of up to 15% of total cell protein after 8 h. Both Aspergillus species are very sensitive to estrogenic substances, and low-cost inducers function in the picomolar concentration range, at which estrogenic substances also can be found in the environment. Given this high sensitivity to estrogens, Aspergillus cells carrying estrogen-responsive units could be used to detect xenoestrogens in food or in the environment.In their natural environment, fungi use extracellular enzymes to gain access to complex, often water-insoluble carbon and nitrogen sources. Extracellular protein production by filamentous fungi usually is very efficient for homologous proteins, for which levels of grams per liter can be obtained. Aspergillus and Trichoderma strains have been reported to secrete up to 30 g of homologous proteins per liter in fermentation processes (e.g., glucoamylases) (11). The production of recombinant proteins of mammalian origin can be up to 4 orders of magnitude lower even when the same expression signals are used.The reason for this difference is not completely understood (2). Along with mRNA stability, codon usage, and translational efficiency, overloading the secretory machinery with a protein that has a "foreign" structure appears to be a critical bottleneck (16,34). High concentrations of such heterologous proteins can result in incorrect folding-leading to translational feedback inhibition by phosphorylation of the ␣ subunit of eukaryotic initiation factor 2 (23) and subsequent intracellular degradation of the misfolded protein. Thus, strong but highly regulatable expression systems are needed to control the flow of proteins through the secretory pathway.The use of strong promoters derived from housekeeping genes, such as those of the various fungal glyceraldehyde-3-phosphate dehydrogenase genes, has the disadvantage of continuous, growth-related expression levels (31). This problem may reduce yields of proteins susceptible to degradation or lead to cell death if the overexpressed foreign proteins are toxic to the ...

The production by filamentous fungi of therapeutic glycoproteins intended for use in mammals is held back by the inherent difference in protein N-glycosylation and by the inability of the fungal cell to modify proteins with mammalian glycosylation structures. Here, we report protein N-glycan engineering in two Aspergillus species. We functionally expressed in the fungal hosts heterologous chimeric fusion proteins containing different localization peptides and catalytic domains. This strategy allowed the isolation of a strain with a functional ␣-1,2-mannosidase producing increased amounts of N-glycans of the Man 5 GlcNAc 2 type. This strain was further engineered by the introduction of a functional GlcNAc transferase I construct yielding GlcNAcMan 5 GlcNac 2 N-glycans. Additionally, we deleted algC genes coding for an enzyme involved in an early step of the fungal glycosylation pathway yielding Man 3 GlcNAc 2 N-glycans. This modification of fungal glycosylation is a step toward the ability to produce humanized complex N-glycans on therapeutic proteins in filamentous fungi.