以DHN-黑色素合酶研究烟曲霉中次生代谢合成机器的亚细胞定位的分子与细胞机制

Aspergillus fumigatus, a human pathogenic fungi, is capable of synthesizing a large number of secondary metabolites and gains stronger competition. However, it is still enigmatic how the fungi limits self-toxification from secondary metabolites produced in itself. In summary of our previous results, we hypothesize that the secondary metabolites are produced and transported in specific subcellular space (endosomes or endosomes-related vesicles) and their biosynthetases are located in endosomes. In this proposal, we are going to explore the common molecular and cellular mechanisms for which the secondary metabolites biosynthetases and biosynthesis are locked in endosomes. Here, we take DHN-melanin and its synthases as a model to find the mechanism for their subcellular localization. First, we screen the key factors for melanin synthase subcellular localization through forward genetic screening. Second, we use protein-protein interactions and proteomics to identify melanin synthases interacting proteins and key proteins for melanin synthases subcellular localization. At last, we will systematically study the subcellular localization of secondary metabolites and their synthases in Aspergillus fumigatus, and we also test and verify the mechanisms for melanin synthase are also involved in other secondary metabolites and its synthase in subcellular localization.

人类致病真菌烟曲霉能够合成大量的次级代谢产物提高自身在环境中的竞争力。目前对于烟曲霉在产生次生代谢产物时是如何避免合成过程中代谢产物对自身造成的伤害仍然不清楚。根据已有结果，我们假设次生代谢合酶定位在内涵体相关的亚细胞空间中并合成次生代谢产物，密封的亚细胞空间能隔离代谢产物对合成宿主的毒性。本项目以烟曲霉中DHN-黑色素合酶的亚细胞空间定位的分子和细胞机制为研究方向，来寻找次生代谢产物合成过程中涉及到的合酶和产物的细胞定位的共同机制。首先，我们通过正向遗传学筛选与黑色素合酶定位相关的关键因子；其次，我们通过蛋白质相互作用以及蛋白质组学鉴定与黑色素合酶相互作用的蛋白，验证这些蛋白是否与黑色素合酶定位相关；最后，我们将系统性地研究次生代谢产物及其合酶在烟曲霉中的亚细胞定位，验证上述方法中鉴定出来的定位相关的关键蛋白在次生代谢合酶亚细胞定位的作用，寻找次生代谢合成机器的亚细胞定位的共同机制。

人类致病真菌烟曲霉能够合成大量的次级代谢产物提高自身在环境中的竞争力。本项目探索真菌次生代谢产物及其合酶胞内运输的通用机制，结合本项目申请人前期在黑色素合酶的细胞定位的研究基础，寻找次生代谢合酶的细胞定位的分子和细胞机制。通过正向遗传学筛选和蛋白组学的方法寻找控制次生代谢产物运输和分泌以及次生代谢合酶分选定位的重要蛋白元件，找到分子和细胞机制；系统性对次生代谢合酶的进行细胞定位。本项目发现，棕榈酰酰基化修饰在黑色素合酶定位至关重要，而且也参与黑色素合成。棕榈酰蛋白组学鉴定出与黑色素合酶被棕榈酰基化修饰有关。进一步系统性分析发现，烟曲霉菌其他几个与次生代谢合成相关的几个合酶的亚细胞定位也在分泌性颗粒中。发现分泌性蛋白Bmh1/2与黑色素合酶共定位在分泌性颗粒中。