双组分转导系统CNX_RS34865/CNX_RS34870对安丝菌素生物合成的调控机制研究

Characterization of the two-component transduction system CNX_RS34865/CNX_RS34870 in regulating the biosynthesis of ansamitocin

目的探究珍贵束丝放线菌(Actinosynnema pretiosum)中双组分转导系统CNX_RS34865/CNX_RS34870对A.pretiosum中安丝菌素P-3(AP-3)生物合成的调控机制。方法本研究以A.pretiosum X47菌株为出发菌株,构建双组分转导系统CNX_RS34865/CNX_RS34870中调控基因CNX_RS34870的缺失突变菌株;利用HPLC分析及生物活性测定分析出发菌株X47与CNX_RS34870突变菌株(ΔRS34870)的AP-3产量差异;利用转录组测序分析菌株之间各基因的转录水平差异,分析CNX_RS34870对菌株AP-3生物合成的影响及作用机制。结果与出发菌株X47相比,ΔRS34870菌株在液体发酵条件下AP-3生物合成量显著下降,在第6天时,ΔRS34870菌株AP-3产量降低了36.94%。转录水平分析显示ΔRS34870菌株中1524个基因的转录较X47明显不同,其中AP-3生物合成基因簇中的asm1、asm2、asm30、asm32、asm33、asm34、asm35和asm37基因以及与初级代谢中与AP-3前体合成相关的pgk基因出现了显著下调。结论CNX_RS34870是菌株AP-3生物合成的正调控基因,可通过调控AP-3生物合成基因簇的转录以及影响初级代谢从而影响AP-3的产生。本研究将为了解AP-3生物合成的调控网络以及工业菌株的遗传改造提供理论指导。

Objective This study aims to explore the regulatory mechanism of the two-component transduction system CNX_RS34865/CNX_RS34870 on ansamitocin P-3(AP-3)biosynthesis in Actinosynnema pretiosum.Methods CNX_RS34870(the regulatory protein encoding gene of CNX_RS34865/CNX_RS34870)deletion mutant strain was constructed based on the original strain Actinosynnema pretiosum X47;The AP-3 yield in X47 andΔRS34870(CNX_RS34870 deletion mutant strain)was analyzed by HPLC and biological activity assays;The effect of CNX_RS34870 on AP-3 biosynthesis was analyzed by transcriptomic sequencing.Results The yield of AP-3 was significantly reduced in ΔRS34870 compared to X47 under liquid fermentation conditions.The yield of AP-3 was reduced by 36.94% in ΔRS34870 compared to X47 on the 6th day.Transcriptome analysis revealed that there were 1,524 differentially expressed genes in ΔRS34870 compared to X47;Among them,the transcriptional levels of asm1,asm2,asm30,asm32,asm33,asm34,asm35,and asm37 in the AP-3 biosynthetic gene cluster,and the pgk gene,which is related to the biosynthesis of AP-3 precursor,were significantly down-regulated inΔRS34870 compared to X47.Conclusion CNX_RS34870 positively regulates AP-3 biosynthesis through regulating AP-3 biosynthetic gene cluster and primary metabolism.This study will provide theoretical guidance for further understanding the regulatory network of AP-3 biosynthesis and the genetic modification of industrial Actinosynnema pretiosum strains.