新型免疫抑制剂Brasilicardin A类似物的发现及其生物合成机制研究

Brasilicardin A (BraA) is a natural diterpene glycoside isolated from the pathogenic antinomycete N. brasiliensis IFM 0406 with highly potent immunosuppressive activity (IC50=0.057μg/mL). However, the development of this promising immunosuppressant as drug for medical use was so far hindered as BraA has the unusual and synthetically challenging skeleton and the low yield production in the natural pathogenic producer. Many non-pathogenic bacteria with BraA gene cluster homologs have been identified from published genomes and it has been predicted that BraA analogs can be produced in these stains by preliminary genome mining and LC-MS analysis. On the basis of that, these metabolites will be searched for by directed LC-MS and characterized. Combined with the results of immunosuppressive activity evaluation, the best active molecules were selected to study the biosynthetic mechanism. The function of each module in the biosynthetic gene cluster of the target molecule was elucidated by gene knockout or heterologous recombination. The ability to extend nature's chemistry through combinatorial biosynthesis—deleting, altering or inserting functional modules from these strains through the manipulation of biosynthetic enzymes—will be exploited and offers unique opportunities to create a library of BraA analogs that would be impractical to produce by chemical methods. Therefore, the project is expected to discover active molecules with novel structures and promising immunosuppressive activity. Furthermore, the elucidation of biosynthesis mechanism lays a foundation for the construction of engineering non-pathogenic bacteria which can produce large amount of active BraA-like molecules. It has important scientific significance and application prospect.

Brasilicardin A是由致病性放线菌N. brasiliensis产生的新型二萜糖苷类强免疫抑制剂（IC50=0.057μg/mL）。因其结构复杂、合成困难，原菌种产率低且具有致病性，BraA及其类似物的获得性已成为此类新型免疫抑制剂研究的瓶颈。本项目利用基因组挖掘和LC-MS等技术，在前期已经发现多株具有合成BraA类似物潜能的非致病菌基础上，以LC-MS为导向，分离鉴定系列BraA类似物，并进行免疫抑制活性评价；优选最佳活性分子进行生物合成机制研究，利用基因敲除或异源重组表达技术阐明目标分子生物合成基因簇中各模块的功能；通过模块敲除、置换及插入，尝试组合合成系列BraA类似物，并进行免疫抑制活性评价。项目研究，不仅有望发现结构新颖的强免疫抑制分子，为创新药物的发现提供先导结构；而且阐明其生物合成机制，为工程菌构建和规模化制备奠定基础。具有重要的科学意义和应用前景。

Brasilicardin A（BraA）是由致病性放线菌N. brasiliensis产生的新型二萜糖苷类强免疫抑制剂（IC50=0.057μg/mL）。因其结构复杂、原菌种具有致病性，产BraA非致病菌株的发现及BraA类似物的合成已成为此类新型免疫抑制剂研究的焦点。本研究利用基因组挖掘技术共筛选到7株含有BraA相似基因簇的候选菌，对其基因簇进行表达分析后，发现3株菌具有合成BraA的潜力。重点对S. erythraea NRRL 2338进行比较代谢组、GNPS聚类、底物喂饲、基因整合等分析，结合LC-MS导向技术找到发酵产物中的目标成分并进行化学分离、结构鉴定，获得多个BraA类似物。对分离鉴定的BraA类似物进行细胞毒实验测定，在确定化合物无细胞毒作用后进行免疫抑制活性评价，获得结构新颖的活性分子。对BraA类似物生物合成基因簇中相关生物合成模块进行克隆和生物信息学分析，系统研究其催化功能，利用基因敲除鉴定基因簇中Se_Br3基因的功能；利用异源重组表达技术获得了C-16位羟化酶Bra0、C-16位氧甲基转移酶Se_Br11、C-2位羟化酶Bra6，阐明生物合成基因簇中各模块的功能，并开展对BraA类似物进行酶催化组合合成的结构修饰，合成相应的BraA类似物产物。项目研究克服了从致病菌N. brasiliensis中分离发现BraA的限制，为从非致病菌中筛选免疫抑制活性BraA类似物提供新途径，BraA生物合成基因簇中相关基因的表达及鉴定，也为工程菌构建和规模化制备奠定基础。