Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus.

BACKGROUND: The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus. RESULTS: We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli. CONCLUSIONS: Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.

Human granulocyte colony stimulating factor (G-CSF) produced in the filamentous fungus Aspergillus niger.

For the first time, a fungal production system is described for expression and secretion of the medically important human protein G-CSF, in Aspergillus niger. A reliable strategy was chosen with in-frame fusion of G-CSF behind a KEX2 cleavage site downstream of the coding region of the highly secreted homologous glucoamylase. This provided secretion levels of 5-10 mg/l culture medium of correctly processed G-CSF, although the majority of the protein (>90%) was biologically inactive. Following denaturation/ concentration and chromatographic separation/ renaturation, the G-CSF proliferation activity increased considerably, and analytical immobilised metal affinity chromatography confirmed the monomeric and correctly folded protein. These data suggest that this human secretory protein secreted into the medium of A. niger was not correctly folded, and that it escaped the endoplasmic reticulum folding control systems. This is compared to the folding of G-CSF produced in bacteria and yeast.

A comparative genomic analysis of calcium and proton signaling/homeostasis in Aspergillus species.

A large number of proteins involved in calcium and intracellular pH signaling and homeostasis have previously been discovered and characterized in Saccharomyces cerevisiae, Homo sapiens and Arabidopsis thaliana,but relatively few have been identified in Aspergillus species. The aim of this study was to identify proteins regulating the intracellular concentration of calcium ions and protons in Aspergillus spp. and compare these with other fungi. For Aspergillus spp. we identified 46, 97 and 105 putative Ca2+-permeable channels, cation/proton transporters and P-ATPases, respectively, the majority of them previously unknown. The subunits composing V-type H+ ATPase and F0F1 ATP synthase (F-type ATPase) from Aspergillus spp. were identified. The greater redundancy of Ca2+-permeable channels, cation/proton exchangers and P-ATPases in filamentous fungi (between 28 putative proteins from A. clavatus and 34 from A. oryzae)compared to that of S. cerevisiae (19 proteins) reflects a more complex cellular organization and filamentous growth form. On the other hand the complexity of V-type H+ ATPase and F0F1 ATP synthase in filamentous fungi is comparable to that in ascomycetous yeast species indicating that both ATPase complexes are a basic universal requirement of the fungal cell.

Expression of human lymphotoxin α in Aspergillus niger.

A gene-fusion expression strategy was applied for heterologous expression of human lymphotoxin alpha (LTα) in the Aspergillus niger AB1.13 protease-deficient strain. The LTα gene was fused with the A. niger glucoamylase GII-form as a carrier-gene, behind its transcription control and secretion signals. Special attention was paid to the influence of different codon usage on secretion of protein. In the case of human tumor necrosis factor alpha (TNFα) a dramatic change of secretion has been observed when human cDNA sequence was used instead of synthetic E. coli biased codons. In the case of LTα such a change of codon usage brought improvement at the RNA level, however, no increase in the quantity of secreted protein was observed, due to the proteolitic activity of the host organism. The estimated yield of secretion of LTα from A. niger into the soya medium was 50 pg l-1 of culture.