尼可霉素生物合成基因簇的改造及其异源表达

Engineering and heterologous expression of a nikkomycin biosynthetic gene cluster

【目的】异源表达尼可霉素生物合成基因簇,为尼可霉素核苷和肽基缩合机制研究以及尼可霉素与其它核苷类抗生素的组合生物合成奠定基础。【方法】以含有尼可霉素生物合成基因簇的pNIK为出发质粒,通过PCR-targeting的方法,将基因簇中san G和san F的启动子替换为组成型hrd B启动子,构建重组质粒pNIKm。通过接合转移的方法分别将pNIK和pNIKm导入天蓝色链霉菌M1146中,获得异源表达菌株M1146-NIK和M1146-NIKm,并通过RT-PCR检测基因簇的表达情况。最后通过抗菌活性实验和产物的分离鉴定,比较M1146-NIK和M1146-NIKm的抗菌活性和尼可霉素的产生情况。【结果】pNIK和pNIKm在异源宿主天蓝色链霉菌M1146成功表达;M1146-NIK和M1146-NIKm均有明显的抗菌活性;M1146-NIK和M1146-NIKm均能产生少量的尼可霉素X、Z和假尼可霉素Z;M1146-NIK大量积累尿苷,而M1146-NIKm大量积累尿苷、核糖基-4-甲酰-4-咪唑-2-酮和吡啶同型苏氨酸。【结论】尼可霉素生物合成基因簇成功异源表达,并分离鉴定了尼可霉素产物及其生物合成中间体。本研究将为尼可霉素核苷和肽基缩合的酶学机制研究以及尼可霉素与其它核苷类抗生素组合合成新型杂合抗生素提供理论依据和指导。

[Objective] We expressed a nikkomycin biosynthetic gene cluster in the well-characterized surrogate Streptomyces coelicolor M1146.[Methods] By using PCR-targeting method,we replaced the promoters of san G and san F in pNIK,which contains nikkomycin biosynthetic gene cluster,with the hrd B promoter to generate pNIKm.We transferred pNIK and pNIKm into S.coelicolor M1146 by intergeneric conjugation and obtained M1146-NIK and M1146-NIKm,respectively.We then evaluated expression of the gene cluster in the heterologous host by RT-PCR.Furthermore,we also compared the antifugal activity and nikkomycin production of M1146-NIK and M1146-NIKm by bioassay against Alternaria longipes and HPLC analysis.[Results] M1146-NIK and M1146-NIKm exhibited antifungal activity,and they can produce a trace amount of nikkomycin X,nikkomycin Z and pseudo-Z.There was a substantial accumulation of uridine in M1146-NIK,whereas substantial accumulations of uridine,ribofuranosyl-4-formyl-4-imidazolone and pyridylhomothreonine were observed in M1146-NIKm.[Conclusion]We successfully expressed the nikkomycin biosynthetic gene cluster in the heterologous host and identified nikkomycins and some of its key biosynthetic intermediates.This study will provide the basis for enzymatic reaction of the condensation between the two nikkomycin moieties and for the generation of hybrid antibiotics by combinatorial biosynthesis.