Abstract
BackgroundThe lifestyle of filamentous fungi depends on the secretion of hydrolytic enzymes into the surrounding medium, which degrade polymeric substances into monomers that are then taken up to sustain metabolism. This feature has been exploited in biotechnology to establish platform strains with high secretory capacity including Aspergillus niger. The accepted paradigm is that proteins become mainly secreted at the tips of fungal hyphae. However, it is still a matter of debate if the amount of growing hyphal tips in filamentous fungi correlates with an increase in secretion, with previous studies showing either a positive or no correlation.ResultsHere, we followed a systematic approach to study protein secretion in A. niger. First, we put the glaA gene encoding for glucoamylase (GlaA), the most abundant secreted protein of A. niger, under control of the tunable Tet-on system. Regulation of glaA gene expression by omitting or adding the inducer doxycycline to cultivation media allowed us to study the effect of glaA under- or overexpression in the same isolate. By inducing glaA expression in a fluorescently tagged v-SNARE reporter strain expressing GFP-SncA, we could demonstrate that the amount of post-Golgi carriers indeed depends on and correlates with glaA gene expression. By deleting the racA gene, encoding the Rho-GTPase RacA in this isolate, we generated a strain which is identical to the parental strain with respect to biomass formation but produces about 20% more hyphal tips. This hyperbranching phenotype caused a more compact macromorphology in shake flask cultivations. When ensuring continuous high-level expression of glaA by repeated addition of doxycycline, this hyperbranching strain secreted up to four times more GlaA into the culture medium compared to its parental strain.ConclusionThe data obtained in this study strongly indicate that A. niger responds to forced transcription of secretory enzymes with increased formation of post-Golgi carriers to efficiently accommodate the incoming cargo load. This physiological adaptation can be rationally exploited to generate hypersecretion platforms based on a hyperbranching phenotype. We propose that a racA deletion background serves as an excellent chassis for such hypersecretion strains.
Highlights
The lifestyle of filamentous fungi depends on the secretion of hydrolytic enzymes into the surrounding medium, which degrade polymeric substances into monomers that are taken up to sustain metabolism
By using a GFP-SncA labelled reporter strain as background strain, we show here that (i) more post-Golgi carriers accumulate at hyphal tips in both ΔracA and its parental strain upon Tet-on driven overexpression of the glaA gene, and that (ii) this leads to an increased glucoamylase secretion in the hyperbranching ΔracA strain
Apical distribution of secretory vesicles at hyphal tips is driven by secretory cargo in both wildtype and hyperbranching ΔracA strain In order to study protein secretion in A. niger in a systematic manner, we selected our previously described reporter strain FG7 [30] (Table 1), which expresses the fluorescently tagged v-SNARE SncA (GFP-SncA) in an otherwise wildtype background as ancestor strain
Summary
The lifestyle of filamentous fungi depends on the secretion of hydrolytic enzymes into the surrounding medium, which degrade polymeric substances into monomers that are taken up to sustain metabolism. This feature has been exploited in biotechnology to establish platform strains with high secretory capacity including Aspergillus niger. The accepted paradigm is that proteins become mainly secreted at the tips of fungal hyphae It is still a matter of debate if the amount of growing hyphal tips in filamentous fungi correlates with an increase in secretion, with previous studies showing either a positive or no correlation. Given the outstanding secretory capacities of filamentous species such as A. niger and their promises as superior platform strains cultivable on renewable lignocellulosic feedstocks, it is of great interest to fully understand and exploit the link between polarised growth and secretion
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