ABSTRACT

A simple intracellular proteomic study was conducted to investigate the biological activities of Aspergillus niger during industrial enzyme production. A strain actively secreting a heterologous enzyme was compared to a reference strain. In total, 1824 spots on 2-D gels were analyzed using MALDI-TOF MS, yielding 343 proteins. The elevated levels of UPR components, BipA, PDI, and calnexin, and proteins related to ERAD and ROS reduction, were observed in the enzyme-producer. The results suggest the occurrence of these responses in the enzyme-producers. Major glycolytic enzymes, Fba1, EnoA, and GpdA, were abundant but at a reduced level relative to the reference, indicating a potential repression of the glycolytic pathway. Interestingly, it was observed that a portion of over-expressed heterologous enzyme accumulated inside the cells and digested during fermentation, suggesting the secretion capacity of the strain was not enough for completing secretion. Newly identified conserved-proteins, likely in signal transduction, and other proteins were also investigated.

Abbreviations: 2-D: two-dimensional; UPR: unfolded protein response; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; PDI: protein disulfide-isomerase; ROS: reactive oxygen species; RESS: Repression under Secretion Stress; CSAP: Conserved Small Abundant Protein; TCTP: translationally controlled tumor protein.

摘要

进行了简单的细胞内蛋白质组学研究，以研究黑曲霉的生物学活性在工业酶生产过程中。将主动分泌异源酶的菌株与参考菌株进行比较。总共使用MALDI-TOF MS分析了2-D凝胶上的1824个斑点，得到343种蛋白质。在酶生产者中观察到UPR成分，BipA，PDI和钙联接蛋白以及与ERAD和ROS减少有关的蛋白质水平升高。结果表明这些反应在酶生产者中发生。主要的糖酵解酶Fba1，EnoA和GpdA丰富，但相对于参考水平降低，表明糖酵解途径可能受到抑制。有趣的是，观察到一部分过表达的异源酶在细胞内积累并在发酵过程中被消化，这表明该菌株的分泌能力不足以完成分泌。

缩写：2-D：二维；UPR：展开的蛋白质反应；ER：内质网；ERAD：与ER相关的蛋白质降解；PDI：蛋白质二硫键异构酶；ROS：活性氧；RESS：分泌压力下的抑制；CSAP：保守的小丰度蛋白质；TCTP：翻译控制的肿瘤蛋白。