细胞穿膜肽介导金色链霉菌胞内代谢产物特异性核酸探针入胞机制研究

微生物是人类重要的资源宝库。微生物单个活细胞内代谢产物检测技术在高产菌株高通量筛选方法建立中具有重要作用。分子信标生物传感器作为新型微生物胞内代谢物检测技术极具应用前景，但尚缺乏相应的分子信标载运手段。细胞穿膜肽（Cell-penetrating peptide，CPP）可载运分子信标进入动物活细胞并实现靶标的高灵敏检测，其优越的载运性能在微生物细胞中也展现了良好的应用前景。我们前期工作证实，可借助CPP将DNA分子信标载运至重要工业微生物菌种金色链霉菌细胞内，并初步实现了四环素类抗生素的单细胞水平活细胞检测。但相关载运机制不明，载运系统也尚待优化。本项目拟从核酸探针、CPP性质及二者互作角度综合解析CPP成功载运核酸探针进入金色链霉菌活细胞实现靶标检测的机制，为工业微生物高产菌株高通量筛选方法的建立奠定基础。本研究模型也有望用于高产四环素类抗生素菌株的高通量筛选，为产业的提升贡献力量。

Microorganisms produce a large variety of important metabolites products for multi-purpose use. There is a great demand for the imaging and detection of metabolites in single living microbial cell. Single living cell based metabolites detection may contribute to the establishment of high-throughput screen method for selecting high yield strains in the future. DNA biosensor has been widely studied and implemented in the field of live cell detection. And, the use of cell-penetrating peptide (CPP) for the development of new cellular imaging tools, DNA biosensors, has been highlighted in mammalian cell recent years. However, compared to mammalian cells, microorganisms have more complex cellular barriers. Thus, due to the lack of suitable probe delivery system, the method for DNA biosensors delivery into microbial live cells is still challenging. Our previous studies have shown that，the initial established tetracyclines (TCs) sensitive molecular beacon (MB) biosensor delivery platform comprising CPP could internalized into Streptomyces aureofaciens for live imaging and detection of intracellular TCs. However, mechanisms of how CPP mediated MB delivery into S. aureofaciens, and important factors which limiting the delivery efficiency and causing cytotoxicity should be evaluated. We thus intend to study mechanisms of CPP based nucleotide probe delivery into microorgnisms. First, the effect mechanism of MB-target interaction will be elucidated. Second, the mechanism of CPP character determined balance point of delivery efficiency and cytotoxicity will be explored. Third, based on the above results, mechanism of the effect of MB and CPP interaction on successful delivery and detection need be further explored and elucidated. This project would provide a suitable platform for imaging microbial metabolites with molecular beacon biosensors in live cells, which may not only help to further understand the dynamic microbial metabolism and metabolites under in vivo condition but also promote the development of high-throughput screen method establishment for selecting high-yield strains in the future. And the research model, CPP based MB delivery system for tetracyclines detection is supposed to be used in high tetracyclines producing strains’ high-throughput screening.