Experiment on marine fungus Microascus brevicaulis strain

The marine fungus Microascus brevicaulis strain LF580 is a non-model secondary metabolite producer with high yields of the two secondary metabolites scopularide A and B, which exhibit distinct activities against tumour cell lines. A mutant strain was obtained using UV mutagenesis, showing besides higher production levels faster growth and differences in pellet formation. Comparative proteomics were applied to gain deeper understanding of the regulation of production and of the physiology of this fungus and its mutant. For this purpose, an optimised protein extraction protocol was established. Here, we show the first proteome study of a marine fungus. In total, 4759 proteins were identified. The central metabolic pathway of LF580 could be mapped by using KEGG pathway analysis and GO annotation. Using iTRAQ labelling, 318 proteins were shown to be significantly regulated in the mutant strain: 189 were down- and 129 upregulated. Proteomics are a powerful tool for the understanding of regulatory aspects: The differences on proteome level could be attributed to a limited nutrient availability in wild type strain due to a strong pellet formation. This information can be applied to optimisation on strain and process level. The linkage between nutrient limitation and pellet formation in the non-model fungus M. brevicaulis is in consensus with the knowledge on model organisms like Aspergillus niger and Penicillium chrysogenum.海洋真菌短柄小囊壳菌（Microascus brevicaulis）菌株LF580是一类非模式次生代谢产物生产菌，可高产两种次生代谢产物scopularide A与scopularide B，二者对肿瘤细胞系展现出显著的生物活性。本研究通过紫外诱变（UV mutagenesis）获得该菌株的突变株，该突变株不仅次生代谢产物产量更高，同时生长速率更快，且菌丝球形成特征存在差异。为深入解析该真菌及其突变株的次生代谢调控机制与生理特性，本研究采用比较蛋白质组学（comparative proteomics）技术开展研究，并建立了优化的蛋白质提取方案。本研究首次完成了海洋真菌的蛋白质组学分析，共鉴定得到4759种蛋白质。通过京都基因与基因组百科全书（KEGG）通路分析与基因本体（GO）注释，成功绘制了LF580的核心代谢通路图谱。采用同重同位素相对与绝对定量标记（iTRAQ）技术，本研究发现突变株中共有318种蛋白质的表达量存在显著差异，其中189种蛋白质表达量下调，129种上调。蛋白质组学是解析代谢调控机制的有力工具：野生型菌株因菌丝球大量形成而导致营养可利用性受限，这一现象可解释其蛋白质组水平的表达差异。该研究结果可用于菌株与发酵工艺的优化。短柄小囊壳菌（M. brevicaulis）作为非模式真菌，其营养限制与菌丝球形成之间的关联，与黑曲霉（Aspergillus niger）、产黄青霉（Penicillium chrysogenum）等模式微生物的已有研究结论一致。