De-convoluting the Genetic Adaptations of E. coli C41(DE3) in Real Time Reveals How Alleviating Protein Production Stress Improves Yields

Cell Reports
Susan SchlegelJan-Willem de Gier

Abstract

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.

References

Aug 15, 1991·Proceedings of the National Academy of Sciences of the United States of America·J W Drake
Jul 1, 1985·Proceedings of the National Academy of Sciences of the United States of America·V M WattW J Rutter
May 5, 1986·Journal of Molecular Biology·F W Studier, B A Moffatt
Apr 24, 1987·Cell·B A Moffatt, F W Studier
Jul 1, 1970·Proceedings of the National Academy of Sciences of the United States of America·A E SilverstoneB Magasanik
Jan 1, 1995·Annual Review of Genetics·R D Camerini-Otero, P Hsieh
Jun 1, 2000·Proceedings of the National Academy of Sciences of the United States of America·K A Datsenko, B L Wanner
Apr 3, 2004·The Journal of Biological Chemistry·Kyoung-Seok RyuChankyu Park
Aug 6, 2004·Protein Expression and Purification·Laurence Dumon-SeignovertLaurent Vuillard
May 26, 2005·Protein Expression and Purification·F William Studier
Mar 24, 2006·Nature Methods·David DrewJan-Willem de Gier
Apr 21, 2007·Molecular & Cellular Proteomics : MCP·Samuel WagnerJan-Willem de Gier
Sep 18, 2008·Proceedings of the National Academy of Sciences of the United States of America·Samuel WagnerJan-Willem de Gier
Jan 24, 2009·Protein Science : a Publication of the Protein Society·Elizabeth Massey-GendelJames U Bowie
Sep 30, 2009·Journal of Molecular Biology·Haeyoung JeongJihyun F Kim
Dec 17, 2009·BMC Bioinformatics·Christiam CamachoThomas L Madden
Dec 7, 2010·Metabolic Engineering·Tomohiro MakinoGeorge Georgiou
Dec 28, 2010·Cell·Robert J NicholsCarol A Gross
Jun 12, 2012·Journal of Bacteriology·Tobias KraxenbergerKirsten Jung
Sep 20, 2012·Proceedings of the National Academy of Sciences of the United States of America·Heewook LeePatricia L Foster
Nov 13, 2014·Molecular Biology and Evolution·Ram Maharjan, Thomas Ferenci

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Citations

Oct 13, 2016·Cellular and Molecular Life Sciences : CMLS·Susan SchlegelJan-Willem de Gier
Jan 25, 2017·Proceedings of the National Academy of Sciences of the United States of America·Yorke ZhangFloyd E Romesberg
Jan 26, 2017·Biotechnology Journal·Wieland N ReicheltChristoph Herwig
Aug 8, 2018·Proceedings of the National Academy of Sciences of the United States of America·Pragya MittalGrzegorz Kudla
Jun 21, 2019·Protein Science : a Publication of the Protein Society·Germán L RosanoEduardo A Ceccarelli
Jul 10, 2019·Nature Biotechnology·Peter Rugbjerg, Morten O A Sommer
Mar 25, 2017·Scientific Reports·Thomas BaumgartenJan-Willem de Gier
Oct 14, 2017·Scientific Reports·Heather M JensenAindrila Mukhopadhyay
Aug 3, 2017·Microbial Cell Factories·Ida LauritsenMorten H H Nørholm
Feb 18, 2020·Frontiers in Bioengineering and Biotechnology·Alexandros KaryolaimosJan-Willem de Gier
Feb 7, 2020·International Journal of Molecular Sciences·Kangsan KimByung-Kwan Cho
Feb 26, 2016·Biotechnology and Applied Biochemistry·Simone Brixius-AnderkoRita Bernhardt
Jul 15, 2018·FEMS Microbiology Letters·Alexandros KaryolaimosJan-Willem de Gier
Apr 15, 2018·Applied and Environmental Microbiology·Thomas BaumgartenJan-Willem de Gier
Oct 6, 2020·Chemical Society Reviews·Michiko Kimoto, Ichiro Hirao
Feb 1, 2021·Bioresource Technology·Adriano GennariClaucia Fernanda Volken de Souza
Aug 14, 2021·Communications Biology·Sophia A H Heyde, Morten H H Nørholm
Jul 3, 2019·ACS Synthetic Biology·Nico J ClaassensJohn van der Oost

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