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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.

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