基于高通量CRISPR/Cas9基因组编辑的黑曲霉葡萄糖转运系统协同调控机制研究

黑曲霉是有机酸与酶制剂的重要工业生产菌株。研究其葡萄糖转运系统对理解黑曲霉的生长发育特性与改造优化高产菌株具有重要意义。丝状真菌的葡萄糖转运系统往往具有组分多且调控复杂，但由于黑曲霉的基因编辑效率低，遗传操作通量低，对黑曲霉葡萄糖转运系统的分子鉴定和协同调控机制研究十分有限。针对此问题，本项目将利用基因组挖掘与比较转录组分析，结合前期已建立的高效CRISPR/Cas9系统，系统鉴定黑曲霉葡萄糖转运系统的关键组分，并定量表征各组分对细胞生长和柠檬酸生产的影响；借助黑曲霉高通量基因操作平台与Tet-on诱导表达系统，构建各关键组分元件间的组合突变体，通过关键组合的比较转录组与胞内代谢组分析，阐明葡萄糖转运系统的协同调控机制，实现葡萄糖高效转运，促进柠檬酸生产。本项目不仅将加深对黑曲霉葡萄糖转运系统协同调控网络的认识，也将为人工理性设计优化黑曲霉细胞工厂提供坚实理论基础。

Aspergillus niger is the main industrial strain used for producing organic acids and industrial enzymes, owing to its unique inherent physiological characters and better fitness for industrial fermentation. As the most important carbon substrate, glucose transport system plays an essential role in cell growth and industrial application. A. niger has low- and high-affinity dual glucose transporter system, which is composed of various glucose transporters with complicated molecular regulation. Nevertheless, until recently, owing to the lack of highly efficient high-throughput gene editing system, the essential genes and regulation mechanism of glucose transport of A. niger still limit. To break this bottleneck, we will systematically examine the 9 potential glucose transporters predicted by genomic mining and dynamic transcriptomic analysis, using the highly efficient CRISPR/Cas9 gene editing system, with special focus on mechanistic insights into the link between, cell growth, citric acid production and glucose transport of A. niger. We will exploit systems and synthetic biology tools established in our labs for A. niger including transcriptomics and metabolomics, high throughput CRISPR/Cas9 genome editing system and Tet-on system to comprehensively discover the coordinate regulation of glucose transport in A. niger. Understanding and unveiling the glucose transport coordinated network will facilitate targeted gene editing in A. niger to rationally optimize the carbon utilization and thus improve the performance of this cell factory during industrial production.