Please use this identifier to cite or link to this item: http://dx.doi.org/10.14279/depositonce-7125
Main Title: Construction of an improved Aspergillus niger platform for enhanced glucoamylase secretion
Author(s): Fiedler, Markus R. M.
Barthel, Lars
Kubisch, Christin
Nai, Corrado
Meyer, Vera
Type: Article
Language Code: en
Abstract: Background The 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. Results Here, 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. Conclusion The 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.
URI: https://depositonce.tu-berlin.de//handle/11303/7963
http://dx.doi.org/10.14279/depositonce-7125
Issue Date: 16-Jun-2018
Date Available: 22-Jun-2018
DDC Class: 570 Biowissenschaften; Biologie
Subject(s): Aspergillus niger
Tet-on
protein secretion
GTPase RacA
v-SNARE
hyperbranching
post-Golgi carrier
glucoamylase
Sponsor/Funder: DFG, TH 662/19-1, Open Access Publizieren 2017 - 2018 / Technische Universität Berlin
License: https://creativecommons.org/licenses/by/4.0/
Journal Title: Microbial Cell Factories
Publisher: Biomed Central
Publisher Place: London
Volume: 17
Issue: 95
Publisher DOI: 10.1186/s12934-018-0941-8
Page Start: 1
Page End: 12
ISSN: 1475-2859
Appears in Collections:FG Angewandte und Molekulare Mikrobiologie » Publications

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