In this study, we monitored the thiol–disulfide redox potential of different Escherichia coli strains using redox-sensitive variants of green fluorescent protein. The cells with extreme oxidizing cytoplasm were generated by introducing a highly efficient disulfide relay system. The developed cells have exceptionally efficient de novo disulfide bond formation and significantly improve the oxidative folding of the client multi-disulfide proteins. Superoxidizing E. coli strain provides an effective method for the high-level production of recombinant disulfide-containing proteins. Biotechnol. Bioeng. 2014;111: 2520–2527. © 2014 Wiley Periodicals, Inc.