Deng Mingrong, Guo Jun, Huang Yongheng, Zhu Honghui
Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, Guangdong Institute of Microbiology, Guangzhou 510070, China.
Wei Sheng Wu Xue Bao. 2011 Mar;51(3):402-9.
The transcription factor SoxR, which serves as a cellular redox sensor, is a global activator of antioxidative 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.
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.
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 orJ20 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.
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.
转录因子SoxR作为细胞氧化还原传感器，是大肠杆菌抗氧化应激的全局激活剂。发现来自越南链霉菌的granaticin生物合成基因orf20是一个类soxR基因。本研究旨在了解该基因的生理功能。
将orf20基因克隆并在大肠杆菌中表达。采用改良的PCR靶向破坏方法构建了越南链霉菌的orf20缺失突变体。测定了重组大肠杆菌菌株和突变型越南链霉菌在不同百草枯浓度下的生长曲线，以确定抗性水平的任何变化。
实现了orf20基因在大肠杆菌中的可溶性表达，重组ORF20蛋白在羧基末端带有七个组氨酸标签。成功构建了越南链霉菌DMR20的orf20缺失突变体。DMR20突变体与野生型相比生长无变化，且仍保留产孢能力。然而，granaticin的产量提高了三倍多。与野生型相比，DMR20突变体对百草枯的抗性没有明显变化，但是携带重组质粒pET28b-orf20的大肠杆菌对百草枯的抗性增强。
orf20基因可互补大肠杆菌中的soxR基因，但不参与越南链霉菌抗氧化应激反应的调控。相反，它对granaticin的产生有负面影响。
