Effect of Introns and AT-Rich Sequences on Expression of the Bacterial Hygromycin B Resistance Gene in the Basidiomycete Schizophyllum commune

Previously, it was shown that introns are required for efficient mRNA accumulation in Schizophyllum commune and that the presence of AT-rich sequences in the coding region of genes can result in truncation of transcripts in this homobasidiomycete. Here we show that intron-dependent mRNA accumulation and truncation of transcripts are two independent events that both affect expression of the bacterial hygromycin B resistance gene in S. commune

Spatially Resolving the Secretome within the Mycelium of the Cell Factory <i>Aspergillus niger</i>

<i>Aspergillus niger</i> is an important cell factory for the industrial production of enzymes. These enzymes are released into the culture medium, from which they can be easily isolated. Here, we determined with stable isotope dimethyl labeling the secretome of five concentric zones of 7-day-old xylose-grown colonies of <i>A. niger</i> that had either or not been treated with cycloheximide. As expected, cycloheximide blocked secretion of proteins at the periphery of the colony. Unexpectedly, protein release was increased by cycloheximide in the intermediate and central zones of the mycelium when compared to nontreated colonies. Electron microscopy indicated that this is due to partial degradation of the cell wall. In total, 124 proteins were identified in cycloheximide-treated colonies, of which 19 secreted proteins had not been identified before. Within the pool of 124 proteins, 53 secreted proteins were absent in nontreated colonies, and additionally, 35 proteins were released ≥4-fold in the central and subperipheral zones of cycloheximide-treated colonies when compared to nontreated colonies. The composition of the secretome in each of the five concentric zones differed. This study thus describes spatial release of proteins in <i>A. niger</i>, which is instrumental in understanding how fungi degrade complex substrates in nature

Deletion of <i>flbA</i> Results in Increased Secretome Complexity and Reduced Secretion Heterogeneity in Colonies of <i>Aspergillus niger</i>

<i>Aspergillus niger</i> is a cell factory for the production of enzymes. This fungus secretes proteins in the central part and at the periphery of the colony. The sporulating zone of the colony overlapped with the nonsecreting subperipheral zone, indicating that sporulation inhibits protein secretion. Indeed, strain Δ<i>flbA</i> that is affected early in the sporulation program secreted proteins throughout the colony. In contrast, the Δ<i>brlA</i> strain that initiates but not completes sporulation did not show altered spatial secretion. The secretome of 5 concentric zones of xylose-grown Δ<i>flbA</i> colonies was assessed by quantitative proteomics. In total 138 proteins with a signal sequence for secretion were identified in the medium of Δ<i>flbA</i> colonies. Of these, 18 proteins had never been reported to be part of the secretome of <i>A. niger</i>, while 101 proteins had previously not been identified in the culture medium of xylose-grown wild type colonies. Taken together, inactivation of <i>flbA</i> results in spatial changes in secretion and in a more complex secretome. The latter may be explained by the fact that strain Δ<i>flbA</i> has a thinner cell wall compared to the wild type, enabling efficient release of proteins. These results are of interest to improve <i>A. niger</i> as a cell factory