Construction of an improved Aspergillus niger platform for enhanced glucoamylase secretion

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.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0941-8) contains supplementary material, which is available to authorized users.

From three‐dimensional morphology to effective diffusivity in filamentous fungal pellets

“…The A. niger hyperbranching strain MF22.4, which has been shown to be a better protein‐secretion strain than the wild‐type strain (due to deletion of the racA gene; Fiedler, Barthel, Kubisch, Nai, & Meyer, ), was used in this study. Pellets were obtained by submerged cultivation of MF22.4 for 48 hr and freeze‐drying following the previously described protocol (Schmideder et al, ).…”

Genetic Engineering of Filamentous Fungi for Efficient Protein Expression and Secretion

“…In A. niger, the Rho GTPase RacA regulates the polymerization and depolymerization of actin at the tip of mycelium (Kwon et al, 2011). When racA was deleted, the mycelial tip increased by about 20%, the number of secreted vesicles increased, and the secretion of glucoamylase increased 4 times as compared to the wild type strain (Fiedler et al, 2018). Similarly, deletion of racA resulted in a hyperbranched phenotype and three folds increase of cellulase activity in T. reesei (Fitz et al, 2019).…”

Improving cytosolic aspartate biosynthesis increases glucoamylase production in Aspergillus niger under oxygen limitation

“…Xin et al [21] also demonstrated that overexpression of glucoamylase encoding gene glaA and α-amylase encoding gene amyA substantially increases the expression and secretion of glucoamylase in A. niger. Furthermore, researchers were also focused on the improvement of protein secretion ability to increase glucoamylase production [22,23]. However, introduction of excessive glaA copies may overload host cells and fails to increase glucoamylase production.…”