Department Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.
BMC Genomics. 2012 Dec 13;13:701. doi: 10.1186/1471-2164-13-701.
Filamentous fungi such as Aspergillus niger are well known for their exceptionally high capacity for secretion of proteins, organic acids, and secondary metabolites and they are therefore used in biotechnology as versatile microbial production platforms. However, system-wide insights into their metabolic and secretory capacities are sparse and rational strain improvement approaches are therefore limited. In order to gain a genome-wide view on the transcriptional regulation of the protein secretory pathway of A. niger, we investigated the transcriptome of A. niger when it was forced to overexpression the glaA gene (encoding glucoamylase, GlaA) and secrete GlaA to high level.
An A. niger wild-type strain and a GlaA over-expressing strain, containing multiple copies of the glaA gene, were cultivated under maltose-limited chemostat conditions (specific growth rate 0.1 h-1). Elevated glaA mRNA and extracellular GlaA levels in the over-expressing strain were accompanied by elevated transcript levels from 772 genes and lowered transcript levels from 815 genes when compared to the wild-type strain. Using GO term enrichment analysis, four higher-order categories were identified in the up-regulated gene set: i) endoplasmic reticulum (ER) membrane translocation, ii) protein glycosylation, iii) vesicle transport, and iv) ion homeostasis. Among these, about 130 genes had predicted functions for the passage of proteins through the ER and those genes included target genes of the HacA transcription factor that mediates the unfolded protein response (UPR), e.g. bipA, clxA, prpA, tigA and pdiA. In order to identify those genes that are important for high-level secretion of proteins by A. niger, we compared the transcriptome of the GlaA overexpression strain of A. niger with six other relevant transcriptomes of A. niger. Overall, 40 genes were found to have either elevated (from 36 genes) or lowered (from 4 genes) transcript levels under all conditions that were examined, thus defining the core set of genes important for ensuring high protein traffic through the secretory pathway.
We have defined the A. niger genes that respond to elevated secretion of GlaA and, furthermore, we have defined a core set of genes that appear to be involved more generally in the intensified traffic of proteins through the secretory pathway of A. niger. The consistent up-regulation of a gene encoding the acetyl-coenzyme A transporter suggests a possible role for transient acetylation to ensure correct folding of secreted proteins.
丝状真菌，如黑曲霉，因其具有极高的分泌蛋白质、有机酸和次生代谢物的能力而闻名，因此被用作多功能微生物生产平台在生物技术中使用。然而，它们的代谢和分泌能力的系统级别的见解是稀疏的，因此合理的菌株改进方法受到限制。为了获得黑曲霉蛋白质分泌途径转录调控的全基因组视角，我们研究了当黑曲霉被迫过表达 glaA 基因（编码葡糖淀粉酶，GlaA）并高水平分泌 GlaA 时的转录组。
在麦芽糖限制恒化器条件下（比生长速率为 0.1 h-1）培养黑曲霉野生型菌株和含有多个 glaA 基因的 GlaA 过表达菌株。在过表达菌株中，glaA mRNA 和细胞外 GlaA 水平升高，与野生型菌株相比，772 个基因的转录水平升高，815 个基因的转录水平降低。使用 GO 术语富集分析，在上调基因集中确定了四个高级类别：i）内质网（ER）膜易位，ii）蛋白质糖基化，iii）囊泡运输，iv）离子稳态。其中，约 130 个基因具有通过 ER 传递蛋白质的预测功能，这些基因包括介导未折叠蛋白反应（UPR）的 HacA 转录因子的靶基因，例如 bipA、clxA、prpA、tigA 和 pdiA。为了确定那些对黑曲霉高水平分泌蛋白质重要的基因，我们将黑曲霉 GlaA 过表达菌株的转录组与其他六个黑曲霉的相关转录组进行了比较。总体而言，在所有检查的条件下，有 40 个基因的转录水平升高（来自 36 个基因）或降低（来自 4 个基因），从而定义了确保蛋白质通过分泌途径高效运输的核心基因集。
我们已经定义了对 GlaA 分泌升高有反应的黑曲霉基因，并且，我们已经定义了一个核心基因集，这些基因似乎更普遍地参与了黑曲霉分泌途径中蛋白质的强化运输。乙酰辅酶 A 转运体编码基因的一致上调表明，瞬时乙酰化可能在确保分泌蛋白正确折叠方面发挥作用。
