Mutations in sigma 70 transcription factor improves expression of functional eukaryotic membrane proteins in Escherichia coli

Scientific Reports
Pablo Emiliano TomatisAndreas Plückthun

Abstract

Eukaryotic integral membrane proteins (IMPs) are difficult to study due to low functional expression levels. To investigate factors for efficient biogenesis of eukaryotic IMPs in the prokaryotic model organism Escherichia coli, important, e.g., for isotope-labeling for NMR, we selected for E. coli cells expressing high levels of functional G protein-coupled receptors (GPCRs) by FACS. Utilizing an E. coli strain library with all non-essential genes systematically deleted, we unexpectedly discovered upon whole-genome sequencing that the improved phenotype was not conferred by the deleted genes but by various subtle alterations in the "housekeeping" sigma 70 factor (RpoD). When analyzing effects of the rpoD mutations at the transcriptome level we found that toxic effects incurred on wild-type E. coli during receptor expression were diminished by two independent and synergistic effects: a slower but longer-lasting GPCR biosynthesis and an optimized transcriptional pattern, augmenting growth and expression at low temperature, setting the basis for further bacterial strain engineering.

References

Jun 1, 1992·Journal of Bacteriology·M LonettoC A Gross
Jan 1, 1993·Methods in Enzymology·R M HortonL R Pease
Aug 1, 1996·The Biochemical Journal·J Tucker, R Grisshammer
Jul 1, 1998·Annual Review of Biophysics and Biomolecular Structure·K H Gardner, L E Kay
Jun 1, 2000·Proceedings of the National Academy of Sciences of the United States of America·K A Datsenko, B L Wanner
May 23, 2002·Genes & Development·Miki JishageThomas Nyström
Feb 15, 2003·Biochimica Et Biophysica Acta·Da-Neng WangXiao-Dan Li
Oct 7, 2003·Annual Review of Microbiology·Tanja M Gruber, Carol A Gross
Jun 17, 2006·Current Opinion in Biotechnology·Reinhard Grisshammer
Jul 6, 2006·Trends in Biotechnology·Samuel WagnerJan-Willem de Gier
Apr 21, 2007·Molecular & Cellular Proteomics : MCP·Samuel WagnerJan-Willem de Gier
Jun 16, 2007·Applied Microbiology and Biotechnology·C Perry Chou
Feb 29, 2008·Protein Science : a Publication of the Protein Society·Daniel Martinez MolinaMarina Ignatushchenko Sabet
Jul 3, 2008·Protein Science : a Publication of the Protein Society·A James LinkGeorge Georgiou
Aug 5, 2008·Current Opinion in Structural Biology·Elisabeth P CarpenterSo Iwata
Sep 18, 2008·Proceedings of the National Academy of Sciences of the United States of America·Samuel WagnerJan-Willem de Gier
Sep 25, 2008·Proceedings of the National Academy of Sciences of the United States of America·Casim A SarkarAndreas Plückthun
Jan 24, 2009·Protein Science : a Publication of the Protein Society·Elizabeth Massey-GendelJames U Bowie
Jul 10, 2009·Biotechnology Letters·Lucy Y Xu, A James Link
Dec 31, 2009·Microbial Cell Factories·Maria FreigassnerAnton Glieder
Jan 25, 2011·Microbial Biotechnology·Susan SchlegelJan-Willem de Gier
Feb 15, 2011·Journal of Molecular Biology·Mirjam M KlepschJan-Willem L de Gier
Mar 8, 2011·Journal of Molecular Biology·Igor Dodevski, Andreas Plückthun
Dec 29, 2011·Biochimica Et Biophysica Acta·Joen LuirinkJan-Willem de Gier
Jun 6, 2012·Proceedings of the National Academy of Sciences of the United States of America·Karola M SchlinkmannAndreas Plückthun
Jun 12, 2012·Journal of Molecular Biology·Karola M SchlinkmannAndreas Plückthun
Aug 4, 2012·Journal of Molecular Biology·Susan SchlegelJan-Willem de Gier
Sep 20, 2012·Proceedings of the National Academy of Sciences of the United States of America·Heewook LeePatricia L Foster

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Citations

Sep 27, 2020·Metabolic Engineering·Elisa GayánAbram Aertsen

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Methods Mentioned

BETA
fluorescence-activated cell sorting
PCR
genetic modification
FACS
RNA-seq
NMR

Software Mentioned

Genevestigator
EnPresso

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