多杀菌素生物合成基因簇启动子探测和转录时序

Promoter detection and transcriptional analysis of the spinosad biosynthetic gene cluster

多杀菌素是对农业虫害防治及粮食仓储安全均具有重大意义的农用抗生素。为了深入揭示刺糖多孢菌合成多杀菌素的调控特点,首先通过建立基于报告基因的启动子探测技术,探测了多杀菌素生物合成基因簇的9个启动子活性。并进一步通过荧光定量PCR,分析了这9个基因和不在基因簇内的负责糖基前体供应和鼠李糖合成的4个基因的转录时序,结果表明多杀菌素生物合成基因簇内的9个基因在菌体生长进入稳定期时有较高的转录,这和发酵液中此时开始大量积累多杀菌素一致;同时还发现,簇外的4个与糖基供应相关的基因和基因簇内基因的转录时序不同,它们在菌体生长对数期有较高的转录活性,这暗示多杀菌素聚酮链的合成速率和参与后修饰的糖基底物供应的最优化匹配有可能是提高生物合成多杀菌素的前提和关键。

Spinosad represents a new class of insecticides produced by Saccharopolyspora spinosa.To understand the transcription of the spinosad biosynthetic gene cluster,two promoter detection plasmids based on different reporter genes were constructed and used to detect 9 promoters in the spinosad biosynthetic gene cluster.In addition,the temporal transcriptional profiles of the corresponding genes controlled by the 9 promoters,together with 4 genes outside of the spinosad cluster but are required for the synthesis of sugars in spinosad,were examined by real-time PCR.The results indicate that the 9 spinosad biosynthetic genes were highly expressed at the stationary phase,which coincides with the accumulation of spinosad in the fermentation broth.Of particular note is that the transcription of the 4 sugar synthetic genes showed higher level at the exponential phase,suggesting the expression of sugar synthetic genes is not correlated with the spinosad synthetic genes.The data suggest that spinosad biosynthesis could be improved by engineering the expression pattern of the sugar synthetic genes that lie outside the spinosad gene cluster.