Department Applied and Molecular Microbiology, Institute of Biotechnology, Technische Universität Berlin, Gustav-Meyer-Allee 25, 13355, Berlin, Germany.
Microb Cell Fact. 2018 Jun 16;17(1):95. doi: 10.1186/s12934-018-0941-8.
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.
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.
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.
丝状真菌的生活方式依赖于将水解酶分泌到周围介质中，将聚合物物质降解为单体，然后被吸收以维持代谢。这一特性已在生物技术中得到利用，建立了具有高分泌能力的平台菌株，包括黑曲霉。公认的模式是，蛋白质主要在真菌菌丝的尖端分泌。然而，丝状真菌中生长的菌丝尖端数量与分泌增加是否相关，仍然存在争议，以前的研究表明存在正相关或无相关性。
在这里，我们采用系统的方法研究了黑曲霉中的蛋白质分泌。首先，我们将编码葡糖淀粉酶（GlaA）的 glaA 基因置于可调控的 Tet-on 系统的控制之下。GlaA 基因表达的调节通过在培养物中省略或添加诱导剂土霉素，使我们能够在同一分离株中研究 glaA 过表达或低表达的影响。通过诱导在荧光标记的 v-SNARE 报告株中表达 GFP-SncA 的 glaA 表达，我们可以证明高尔基体后载体的数量确实取决于和与 glaA 基因表达相关。通过在该分离株中删除编码 RacA 的 racA 基因，我们产生了一种与亲本菌株在生物量形成方面相同但产生约 20%更多菌丝尖端的菌株。这种超分枝表型导致摇瓶培养物中更紧凑的宏观形态。当通过重复添加土霉素来确保 glaA 的持续高水平表达时，与亲本菌株相比，这种超分枝菌株将多达四倍的 GlaA 分泌到培养基中。
本研究获得的数据强烈表明，黑曲霉对分泌酶的强制转录作出反应，增加了高尔基体后载体的形成，以有效地容纳传入的货物负荷。这种生理适应可以被合理地利用来产生基于超分枝表型的超分泌平台。我们提出，racA 缺失背景可作为此类超分泌菌株的理想底盘。
