De-convoluting the Genetic Adaptations of E-coli C41(DE3) in Real Time Reveals How Alleviating Protein Production Stress Improves Yields
2015 (English)In: Cell reports, ISSN 2211-1247, Vol. 10, no 10, 1758-1766 p.Article in journal (Refereed) Published
The well-established E. coli protein production strain C41(DE3) was isolated from the T7 RNA polymerase-based BL21(DE3) strain for its ability to produce difficult recombinant proteins, and it acquired multiple mutations during its isolation. Standard allelic replacement and competition experiments were insufficient to de-convolute these mutations. By reconstructing the evolution of C41(DE3) in real time, we identified the time frames when the different mutations occurred, enabling us to link them to particular stress events. Starvation stress imposed by the isolation procedure selected for mutations enhancing nutrient uptake, and protein production stress for mutations weakening the lacUV5 promoter, which governs t7rnap expression. Moreover, recapitulating protein production stress in BL21(DE3) showed that mutations weakening the lacUV5 promoter occur through RecA-dependent recombination with the wild-type lac-promoter and are selected for upon the production of any protein. Thus, the instability of the lacUV5 promoter in BL21(DE3) alleviates protein production stress and can be harnessed to enhance production.
Place, publisher, year, edition, pages
2015. Vol. 10, no 10, 1758-1766 p.
IdentifiersURN: urn:nbn:se:su:diva-116610DOI: 10.1016/j.celrep.2015.02.029ISI: 000351316700012OAI: oai:DiVA.org:su-116610DiVA: diva2:809117