Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3).
Membrane protein production is usually toxic to E. coli. However, using genetic screens strains can be isolated in which the toxicity of membrane protein production is reduced, thereby improving production yields. Best known examples are the C41(DE3) and C43(DE3) strains, which are both derived from the T7 RNA polymerase (P)-based BL21(DE3) protein production strain. In C41(DE3) and C43(DE3) mutations lowering t7rnap expression levels result in strongly reduced T7 RNAP accumulation levels. As a consequence membrane protein production stress is alleviated in the C41(DE3) and C43(DE3) strains, thereby increasing membrane protein yields. Here, we isolated Mutant56(DE3) from BL21(DE3) using a genetic screen designed to isolate BL21(DE3)-derived strains with mutations alleviating membrane protein production stress other than the ones in C41(DE3) and C43(DE3). The defining mutation of Mutant56(DE3) changes one amino acid in its T7 RNAP, which weakens the binding of the T7 RNAP to the T7 promoter governing target gene expression rather than lowering T7 RNAP levels. For most membrane proteins tested yields in Mutant56(DE3) were considerably higher than in C41(DE3) and C43(DE3). Thus, the isolation of Mutant56(DE3) shows that the evolution of BL21(DE3) can be promoted towards further enhanced membrane protein production.
Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, SE-106 91, Sweden.
Thomas
T
Baumgarten
Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, SE-106 91, Sweden.
Susan
S
Schlegel
Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, SE-106 91, Sweden.
Samuel
S
Wagner
Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, SE-106 91, Sweden.
Mirjam
M
Löw
Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, SE-106 91, Sweden.
Jonas
J
Eriksson
Technical University of Denmark, Novo Nordisk Foundation Center for Biosustainability, Kogle Alle 6, Hørsholm, 2970, Denmark.
Ida
I
Bonde
Technical University of Denmark, Novo Nordisk Foundation Center for Biosustainability, Kogle Alle 6, Hørsholm, 2970, Denmark.
Markus J
MJ
Herrgård
Helmholtz Centre for Environmental Research-UFZ, Department of Environmental Biotechnology, Permoserstrasse 15, Leipzig, 04318, Germany.
Hermann J
HJ
Heipieper
Technical University of Denmark, Novo Nordisk Foundation Center for Biosustainability, Kogle Alle 6, Hørsholm, 2970, Denmark.
Morten H H
MH
Nørholm
Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, Groningen, AG, 9747, The Netherlands.
Dirk Jan
DJ
Slotboom
Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, SE-106 91, Sweden.
Jan-Willem
JW
de Gier
D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011401:Promoter Regions, Genetic; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases