醌那霉素生物合成基因簇中alp2F和alp2G基因的功能表征

Functional Characterization of alp2F and alp2G in the Kinamycin Biosynthetic Gene Cluster

醌那霉素是由Ⅱ型聚酮合酶系统产生的一类角蒽环类聚酮化合物。从结构上看,其具有三个显著的特征:苯并芴核、高度氧化的A环以及芴环上的重氮基团。醌那霉素因其特殊的苯并芴结构以及良好的生物活性,引起了科研人员广泛的研究兴趣。但是迄今醌那霉素的生物合成途径并没有得到完全的解析,尤其是对重氮基团的形成。前期研究因缺少醌那霉素完整的生物合成基因簇信息而受到阻碍。本课题组最近确证了醌那霉素的完整生物合成基因簇,共包含62个基因,其中有8个基因并没有被报道过。通过生物信息学分析,本研究发现其中alp2F和alp2G基因与cremeomycin生物合成中的creE和creD具有高度的同源性。creE和creD基因产物通过催化天冬氨酸生成亚硝酸,其后亚硝酸在酸性条件下自发加载到化合物的碳骨架上,实现重氮基团的加载。本研究通过体外酶催化反应证实了Alp2F和Alp2G同样可以催化天冬氨酸生成亚硝酸。亚硝酸钠喂养实验进一步确证了亚硝酸盐参与醌那霉素的合成。alp2F和alp2G基因功能体外和体内的确证,不仅是对醌那霉素生物合成基因簇中未知基因功能的表征,也对阐明醌那霉素家族天然产物中重氮基团的形成有启发意义。

Kinamycin is a class of angucyclinone produced by type II polyketide synthase system.Structurally,it has three distinctive features:benzofluorene core,highly oxidized A ring,diazo group on fluorene ring.Due to its benzfluorene unique structure and good bioactivity,kinamycin has attracted a wide range of research interests.However,up to now,the biosynthetic pathway of kinamycin has not been completely elucidated,especially for the formation of the diazo group.Previous studies have been hampered by lack of information regarding complete biosynthetic gene cluster of kinamycin.Our group has recently confirmed the complete biosynthetic gene cluster of kinamycin,which contains 62 genes.Among them,eight genes have not been reported.Through bioinformatics analysis,we found that the alp2F and alp2G is highly homologous to creE and creD in cremeomycin biosynthetic pathway.The gene products encoded by creE and creD catalyze the production of nitrous acid from aspartic acid.The nitrous acid is spontaneously loaded onto the carbon skeleton to form the diazo group under acidic conditions.In this study,we confirmed that Alp2F and Alp2G can also catalyze the formation of nitrous acid from aspartic acid by in vitro enzyme-catalyzed reaction.Furthermore,sodium nitrite feeding experiment confirmed the involvement of nitrite in the kinamycin biosynthesis.The in vitro and in vivo confirmation of the function of alp2F and alp2G is not only the characterization of the unknown gene function in the gene cluster of the clindamycin biosynthesis gene cluster,but also the significance of the formation of the diazonium group in the natural product of the clindamycin family.