Aguiar Tatiana Q, Ribeiro Orquídea, Arvas Mikko, Wiebe Marilyn G, Penttilä Merja, Domingues Lucília
CEB - Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal.
BMC Genomics. 2014 Dec 18;15(1):1137. doi: 10.1186/1471-2164-15-1137.
Ashbya gossypii is a filamentous Saccharomycete used for the industrial production of riboflavin that has been recently explored as a host system for recombinant protein production. To gain insight into the protein secretory pathway of this biotechnologically relevant fungus, we undertook genome-wide analyses to explore its secretome and its transcriptional responses to protein secretion stress.
A computational pipeline was used to predict the inventory of proteins putatively secreted by A. gossypii via the general secretory pathway. The proteins actually secreted by this fungus into the supernatants of submerged cultures in minimal and rich medium were mapped by two-dimensional gel electrophoresis, revealing that most of the A. gossypii secreted proteins have an isoelectric point between 4 and 6, and a molecular mass above 25 kDa. These analyses together indicated that 1-4% of A. gossypii proteins are likely to be secreted, of which less than 33% are putative hydrolases. Furthermore, transcriptomic analyses carried out in A. gossypii cells under recombinant protein secretion conditions and dithiothreitol-induced secretion stress unexpectedly revealed that a conventional unfolded protein response (UPR) was not activated in any of the conditions, as the expression levels of several well-known UPR target genes (e.g. IRE1, KAR2, HAC1 and PDI1 homologs) remained unaffected. However, several other genes involved in protein unfolding, endoplasmatic reticulum-associated degradation, proteolysis, vesicle trafficking, vacuolar protein sorting, secretion and mRNA degradation were up-regulated by dithiothreitol-induced secretion stress. Conversely, the transcription of several genes encoding secretory proteins, such as components of the glycosylation pathway, was severely repressed by dithiothreitol
This study provides the first insights into the secretion stress response of A. gossypii, as well as a basic understanding of its protein secretion potential, which is more similar to that of yeast than to that of other filamentous fungi. Contrary to what has been widely described for yeast and fungi, a conventional UPR was not observed in A. gossypii, but alternative protein quality control mechanisms enabled it to cope with secretion stress. These data will help provide strategies for improving heterologous protein secretion in A. gossypii.
棉阿舒囊霉是一种丝状酵母菌，用于工业生产核黄素，最近被开发为重组蛋白生产的宿主系统。为深入了解这种具有生物技术相关性的真菌的蛋白质分泌途径，我们进行了全基因组分析，以探究其分泌组及其对蛋白质分泌应激的转录反应。
使用一个计算流程来预测棉阿舒囊霉可能通过一般分泌途径分泌的蛋白质清单。通过二维凝胶电泳对该真菌在基本培养基和丰富培养基中实际分泌到深层培养上清液中的蛋白质进行图谱分析，结果表明，棉阿舒囊霉分泌的大多数蛋白质的等电点在4至6之间，分子量大于25 kDa。这些分析共同表明，棉阿舒囊霉中1%-4%的蛋白质可能会被分泌，其中不到33%是假定的水解酶。此外，在重组蛋白分泌条件下以及二硫苏糖醇诱导的分泌应激下对棉阿舒囊霉细胞进行的转录组分析意外发现，在任何条件下均未激活传统的未折叠蛋白反应（UPR），因为几个著名的UPR靶基因（如IRE1、KAR2、HAC1和PDI1同源物）的表达水平未受影响。然而，二硫苏糖醇诱导的分泌应激上调了其他几个参与蛋白质解折叠、内质网相关降解、蛋白水解、囊泡运输、液泡蛋白分选、分泌和mRNA降解的基因。相反，二硫苏糖醇严重抑制了几个编码分泌蛋白的基因的转录，如糖基化途径的成分。
本研究首次深入了解了棉阿舒囊霉的分泌应激反应，并对其蛋白质分泌潜力有了基本认识，其与酵母的蛋白质分泌潜力比与其他丝状真菌更为相似。与酵母和真菌中广泛描述的情况相反，在棉阿舒囊霉中未观察到传统的UPR，但替代的蛋白质质量控制机制使其能够应对分泌应激。这些数据将有助于提供提高棉阿舒囊霉中异源蛋白分泌的策略。
