粤蓝链霉菌榴菌素生物合成基因orf20的功能

Function of the granaticin biosynthetic gene orf20 from Streptomyces vietnamensis

【目的】在大肠杆菌中,转录因子SoxR作为胞内氧化还原感应器,参与抗氧化胁迫的全局性调控。粤蓝链霉菌榴菌素生物合成基因簇内存在一个类soxR基因orf20,但其生理功能仍不清楚。【方法】将orf20基因在大肠杆菌中进行表达,分析携带重组质粒的大肠杆菌对百草枯抗性的变化。同时通过修改后的PCR-targeting方法构建粤蓝链霉菌orf20删除的突变株,分析突变株的表型变化和对百草枯抗性水平的变化。【结果】重组ORF20在羧基端含有组氨酸标签,并在大肠杆菌中获得可溶性表达,携带重组质粒pET28b-orf20的大肠杆菌对百草枯的抗性水平显著提高。粤蓝链霉菌orf20删除突变株仍具有产孢能力,生长特性没有改变,对百草枯的抗性水平也没有变化,但榴菌素的产量大幅提高,是野生株的3.3倍。【结论】在大肠杆菌中,orf20基因的编码产物能够被百草枯激活,替代SoxR参与抗氧化胁迫的调控。在粤蓝链霉菌中,orf20基因不参与抗氧化胁迫,而对榴菌素的产生有负调控效应。

[Objective]The transcription factor SoxR,which serves as a cellular redox sensor,is a global activator of anti-oxidative stress in Escherichia coli.The granaticin biosynthetic gene orf20 from Streptomyces vietnamensis was found to be a soxR-like gene.This study was carried out towards understanding the physiological function of this gene.[Methods]The orf20 gene was cloned and expressed in E.coli.An orf20 deletion mutant of S.vietnamensis was constructed by using a modified PCR-targeting disruption method.The growth curves of the recombinant E.coli strains and the mutant S.vietnamensis at various paraquat concentrations were assayed to resolve any changes of resistance levels.[Results]Soluble expression of orf20 gene in E.coli was achieved,and the recombinant ORF20 protein was tagged by seven histidines at the carboxylic end.An orf20 deletion mutant of S.vietnamensis DMR20 was successfully constructed.The DMR20 mutant showed no growth changes to the wild,and the ability of sporulation was retained,too.However,the production of granaticin was improved for more than three folds.Compared to the wild type,the DMR20 mutant had no visible changes of resistance to paraquat,however,E.coli carrying the recombinant plasmid pET28b-orf20 received an elevated resistance to paraquat.[Conclusion]The orf20 gene can complement the soxR gene in E.coli,but is not involved in the regulation of anti-oxidative stress response in S.vietnamensis.Instead,it imposes a negative effect on granaticin production.