防御假单胞菌H78中sRNA基因crcY/Z正调控藤黄绿菌素合成

Positve Regulation of sRNA Genes crcY/Z on Pyoluteorin Biosynthesis in Pseudomonas protegens H78

防御假单胞菌(Pseudomonas protegens)H78能产生藤黄绿菌素(pyoluteorin,Plt)等多种广谱抗生素,这些抗生素的合成存在复杂的调控机制与网络。本研究旨在研究H78菌株碳分解代谢阻遏调控系统(Cbr/Crc)中两个同源小RNA(small RNA,sRNA)基因crcY和crcZ对Plt合成的调控机制。首先,利用无痕敲除方法构建crcY/Z双基因敲除突变体,发酵分析结果显示crcY/Z双敲除使H78菌株几乎完全丧失Plt的合成;其次,基因互补实验证实crcY或crcZ基因的外源互补能恢复crcY/Z双敲除突变体的Plt合成;最后,利用lacZ报告基因分析技术进一步证实crcY/Z双突变使Plt合成操纵子pltLABCDEFG的表达显著降低。结果表明:防御假单胞菌sRNA基因crcY/Z对Plt合成及基因表达存在显著的正调控作用。本研究将为进一步阐明Plt合成的调控网络及通过代谢工程提高Plt产量奠定基础。

Pseudomonas protegens H78 can produce multiple broad-spectrum antibiotics such as pyoluteorin(Plt).The biosynthesis of these antibiotics is controlled by the complex regulatory mechanism and network.This study aims to ascertain the regulatory mechanism of Plt biosynthesis by two homologous small RNA(sRNA)genes crcY and crcZ in the carbon catabolism repression regulatory system(Cbr/Crc)of strain H78.Firstly,the crcY/Z dual mutant was constructed by the non-scar deletion technology.The fermentation analysis result showed that Plt biosynthesis was almost completely inhibited by the crcY/Z dual deletion in H78.Secondly,the gene complementary experiment showed the complementation of crcY or crcZ gene could restore Plt biosynthesis of the crcY/Z dual mutant.Finally,the lacZ reporter analysis result further confirmed that the crcY/Z deletion significantly down regulated the expression of Plt biosynthetic operon pltLABCDEFG.These results demonstrated that the sRNA genes crcY/Z had a significant positive regulatory effect on the Plt biosynthesis and its gene expression in P.protegens.This study will lay the foundation for further elucidating the regulatory network of Plt biosynthesis and improving Plt production through metabolic engineering.