Wang Lu, Du Deyao, Li Jin'e, Tian Yuqing, Liu Hao, Niu Guoqing, Tan Huarong
Wei Sheng Wu Xue Bao. 2015 Jun 4;55(6):707-18.
We expressed a nikkomycin biosynthetic gene cluster in the well-characterized surrogate Streptomyces coelicolor M1146.
By using PCR-targeting method, we replaced the promoters of sanG and sanF in pNIK, which contains nikkomycin biosynthetic gene cluster, with the hrdB 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.
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.
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.
我们在特征明确的替代宿主天蓝色链霉菌M1146中表达了多氧霉素生物合成基因簇。
采用PCR靶向法，用hrdB启动子替换含有多氧霉素生物合成基因簇的pNIK中sanG和sanF的启动子，构建pNIKm。通过属间接合将pNIK和pNIKm转入天蓝色链霉菌M1146，分别获得M1146-NIK和M1146-NIKm。然后通过RT-PCR评估该基因簇在异源宿主中的表达。此外，我们还通过对链格孢的生物测定和HPLC分析，比较了M1146-NIK和M1146-NIKm的抗真菌活性和多氧霉素产量。
M1146-NIK和M1146-NIKm均表现出抗真菌活性，且能产生微量的多氧霉素X、多氧霉素Z和假Z。在M1146-NIK中尿苷大量积累，而在M1146-NIKm中观察到尿苷、核糖呋喃糖基-4-甲酰基-4-咪唑酮和吡啶基高丝氨酸大量积累。
我们成功地在异源宿主中表达了多氧霉素生物合成基因簇，并鉴定出了多氧霉素及其一些关键的生物合成中间体。本研究将为两个多氧霉素部分之间的缩合酶促反应以及通过组合生物合成产生杂合抗生素提供基础。
