密旋链霉菌Act12中调控因子AdpA-s和LuxR-2306对寡霉素合成的影响

The effects of regulators AdpA-s and LuxR-2306 on oligomycin biosynthesis in Streptomyces pactum Act12

为揭示密旋链霉菌Act12(Streptomycespactum)中全局调控因子AdpA-s和途径特异调控因子LuxR-2306对寡霉素合成的影响,构建了AdpA-s和LuxR-2306的阻断突变株和高表达菌株,采用滤纸片法检验了发酵液对苹果腐烂菌(Valsamali)的抑生活性,利用高效液相色谱法(HPLC)分析了寡霉素的产量,通过定量PCR分析AdpA-s、LuxR-2306转录水平与寡霉素合成之间的关系.结果表明:高表达突变株AdpA-s HE和LuxR-2306 HE对苹果腐烂菌的抑生能力显著增强;而基因阻断突变株Spa LuxR-2306不再对苹果腐烂菌的生长具有抑制效果.AdpA-s HE和LuxR-2306 HE发酵液中寡霉素产量分别提高了1.8和1.9倍;Spa LuxR-2306中完全检测不到寡霉素的存在.实时定量PCR结果表明,LuxR-2306的转录水平与寡霉素生物合成核心酶的转录水平呈显著正相关.AdpA-s高表达突变株中LuxR-2306的转录水平也得到了显著提高.以上结果表明:LuxR-2306为寡霉素生物合成的途径特异性正调控因子,可以通过正调控寡霉素生物合成基因簇中核心酶的转录,进而促进寡霉素的合成.而全局性调控因子AdpA-s可能通过对LuxR-2306的转录调控,间接影响寡霉素的生物合成,后续实验将对此展开进一步的深入研究.此研究为后续对生防菌Act12中次级代谢的调控研究提供了一定的理论基础.

This study revealed the effects of AdpA-s and LuxR-2306 on oligomycin biosynthesis in Streptomyces pactum Act12.The deficient and overexpressed mutant strains strain of AdpA-s and LuxR-2306 were constructed by genetic engineering.Filter paper method was used to test the antimicrobial activity of extracted fermentation liquid on Valsa mali.High Performance liquid Chromatography(HPLC)was applied to analyze the yield of oligomycin.The relationship between the transcription of AdpA-s、LuxR-2306 and oligomycin biosynthesis was measured by qPCR.The antagonistic ability on Valsa mali was improved by the overexpression of AdpA-s and LuxR-2306,while LuxR-2306 dificient strain lost the antagonistic ability on Valsa mali.HPLC results showed that the yield of oligomycin increased 1.8 and 1.9 times than Act12 in AdpA-s HE and LuxR-2306 HE respectively but no oligomycin was detected in Spa LuxR-2306.LuxR-2306 could promote the transcription of oligomycin core synthetase gene,and that the transcribed level of oligomycin core synthetase gene decreased significantly when LuxR-2306 was knockout.The results suggested that the LuxR-2306 was accumulated rapidly by positive feedback between AdpA-s and LuxR-2306,which promotes the transcription of oligomycin core synthetase gene and speeds up the biosynthesis of oligomycin.The research provided a scientific basis for the regulation of the yield of oligomycin and its analogues.