糖基化诺西肽衍生物的定向生物合成及活性评价

Nosiheptide, a thiopeptide antibiotic, possesses a potent activity against multidrug-resistant bacterial strains in vitro. However, the poor water solubility and structural stability of nosiheptide caused a poor plasma concentration and a negligible in vivo antibacterial activity. Given the availability of glycosylation modification in improving the water solubility of drugs, nosiheptide derivative with dimethylamino glucose group (NDDG) was biosynthesized and its activity was evaluated, As a result, glycosylation can effectively improve the in vivo antibacterial activity of nosiheptide. On the base of biosynthetic pathway for synthesis of NDDG, the present study will biosynthesize a series of glycosylated nosiheptide derivatives by specific modification of structural peptide and redesign of biosynthetic pathway. After screening, selected glycosylated nosiheptide derivative(s) with excellent solubility, improved structural stability and better in vivo antibacterial activity than vancomycin will be obtained, which could provide a foundation for the development of nosiheptide. Thus, this study is not only an example for the development of new antibiotic against resistant bacteria, but also provides ideas for targeted structural modification of complex microbial secondary metabolites.

诺西肽是一种体外抗多重耐药菌活性超强的硫肽类抗菌化合物，但体内抗菌活性微弱，至今未能成药，主要原因在于其极低的水溶性和较差的结构稳定性导致无法达到有效的血药浓度。鉴于糖基化修饰是改善药物水溶性的重要手段，本课题组前期定点生物合成了二甲氨基葡萄糖诺西肽，并完成了活性研究，初步证明糖基化修饰能够有效提高诺西肽的体内抗菌活性。本课题将在二甲氨基葡萄糖诺西肽生物合成途径的基础上通过结构肽和基因簇的理性改造，进而定向生物合成一系列糖基化诺西肽衍生物，筛选获得水溶性改善、结构稳定性提高和体内抗菌活性优于万古霉素的新型抗菌化合物，为诺西肽的成药性开发提供物质基础。本课题不仅可为开发新型抗耐药菌抗生素奠定了一定基础，也可为复杂微生物次生代谢产物的结构改造提供思路。

针对硫肽类抗生素诺西肽体内抗菌活性微弱的关键问题，本课题在前期实验的基础上，通过结构肽改造或生物合成途径再设计，对诺西肽进行针对性的糖基化修饰或结构改造，以期获得体内活性显著改善的新型糖基化诺西肽衍生物。具体研究内容如下：1）对二甲氨基葡萄糖诺西肽产生菌S. actuosus L1009G的发酵条件和培养基进行优化，以期提高生物合成效价。2）阐明前导肽对诺西肽生物合成途径的影响和调控蛋白NosP对诺西肽生物合成的影响，尝试了前导肽和调控系统改造以提高合成效价。3）在无法提高二甲氨基葡萄糖合成效价的情况下，构建了不同来源的糖基转移酶库，并筛选获得能够催化诺西肽糖基化修饰的糖基转移酶GT-9。4）将GT-9整合至诺西肽产生菌的基因簇，改造结构肽，发酵后检测到多个糖基化衍生物，分离纯化获得葡萄糖基化产物Nos-G1和Nos-G2，这两个化合物更加稳定，而且体内活性明显优于万古霉素。该研究方法不仅实现了利用合成途径改造改善诺西肽体内活性的目的，也为其他体外抗菌活性强，而体内无活性的硫肽类抗生素开发提供了新的方法和思路。