Brasilicardin A生物合成相关二萜合酶的异源表达及产物鉴定

Heterologous expression and product identification of diterpene synthase involved in the biosynthesis of brasilicardin A

从致病性放线菌Nocardia brasiliensis IFM 0406中得到的二萜糖苷brasilicardin A,因免疫抑制活性显著、毒性较低且作用机制独特而成为新型免疫抑制剂的候选分子。由于原菌株中产率低,且结构复杂、化学合成困难,brasilicardin A及其类似物的获得成为该类新型免疫抑制剂的研究热点。根据目前报道的brasilicardin A可能的生物合成途径,通过生物信息学分析生物合成相关二萜合酶,定向从致病菌株N.brasiliensis IFM 0406中扩增可能合成brasilicardin A骨架的基因bra1~5,在白色链霉菌Streptomyces albus R1中成功实现异源表达。通过液体发酵培养,利用硅胶柱色谱及半制备液相色谱等方法进行化合物的分离纯化,共分离到6个新brasilicardins化合物,命名为brasilicardin H~M。利用脂多糖(LPS)活化的小鼠原代巨噬细胞炎症模型对分离鉴定的brasilicardins进行了体外活性筛选,结果显示,brasilicardin H~M具有较强抑制巨噬细胞释放一氧化氮(NO)的作用,IC_(50)分别为28.24±3.70、37.44±2.00、39.85±4.02、26.77±4.40、65.25±1.48、15.24±2.72μmol·L^(-1)(阳性对照吲哚美辛IC_(50)为34.28±4.10μmol·L^(-1)),表明6个化合物具有潜在的抗炎活性。本研究为brasilicardin A生物合成途径的彻底阐明提供了条件,也为研究该类化合物的构效关系、药物研发等奠定了基础。

Brasilicardin A,a diterpene glycoside isolated from pathogenic actinomycete Nocardia brasiliensis IFM 0406,has become a novel immunosuppressant candidate due to its significant immunosuppressive activity,low toxicity and unique mechanism of action.However,brasilicardin A and its analogues have become a research hotspot to the development of this promising immunosuppressant because of the low-yield production in the natural pathogenic producer and the synthetically challenging skeleton.According to the reported biosynthetic pathway of brasilicardin A,the function of involved diterpene synthase was analyzed by bioinformatics.Then the genes bra1−5 that synthesize the brasilicardin A skeleton were directionally amplified from the pathogenic strain N.brasiliensis IFM 0406,and heterologous expression was achieved successfully in Streptomyces albus R1.The compounds were isolated and purified by using various column chromatographies including silica gel column chromatography and semi-preparative HPLC.Six new brasilicardins were established and named brasilicardin H−M.The activity of brasilicardins was screened using lipopolysaccharide(LPS)-activated mouse primary macrophage inflammation model.Brasilicardin H−M exhibited good inhibitory activity on nitric oxide(NO)release with IC_(50) values of 28.24±3.70,37.44±2.00,39.85±4.02,26.77±4.40,65.25±1.48 and 15.24±2.72μmol·L^(-1),respectively(indomethacin as the positive control with IC_(50) value of 34.28±4.10μmol·L^(-1)).The results indicated that six compounds had potential anti-inflammatory activity.This study laid a foundation for the elucidation of the brasilicardin A biosynthetic pathway and evaluation of the structure-activity relationship as well as new drug developments.