Objective: 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.
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 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.
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.