title: Tailoring Escherichia coli for the l-Rhamnose PBADPromoter-Based Production of Membrane and Secretory Proteins.
abstract: Membrane and secretory protein production in Escherichia coli requires precisely controlled production rates to avoid the deleterious saturation of their biogenesis pathways. On the basis of this requirement, the E. coli l-rhamnose PBADpromoter (PrhaBAD) is often used for membrane and secretory protein production since PrhaBAD is thought to regulate protein production rates in an l-rhamnose concentration-dependent manner. By monitoring protein production in real-time in E. coli wild-type and an l-rhamnose catabolism deficient mutant, we demonstrate that the l-rhamnose concentration-dependent tunability of PrhaBAD-mediated protein production is actually due to l-rhamnose consumption rather than regulating production rates. Using this information, a RhaT-mediated l-rhamnose transport and l-rhamnose catabolism deficient double mutant was constructed. We show that this mutant enables the regulation of PrhaBAD-based protein production rates in an l-rhamnose concentration-dependent manner and that this is critical to optimize membrane and secretory protein production yields. The high precision of protein production rates provided by the PrhaBAD promoter in an l-rhamnose transport and catabolism deficient background could also benefit other applications in synthetic biology.
authors.affiliation: Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University , SE-106 91 Stockholm, Sweden.
authors.forename: Anna
authors.initials: A
authors.lastname: Hjelm
authors.affiliation: Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University , SE-106 91 Stockholm, Sweden.
authors.forename: Alexandros
authors.initials: A
authors.lastname: Karyolaimos
authors.affiliation: Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University , SE-106 91 Stockholm, Sweden.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University , SE-106 91 Stockholm, Sweden.
authors.forename: Edurne
authors.initials: E
authors.lastname: Rujas
authors.affiliation: Xbrane Biopharma AB , SE-111 45 Stockholm, Sweden.
authors.forename: David
authors.initials: D
authors.lastname: Vikström
authors.affiliation: Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen , 9712 CP Groningen, The Netherlands.
authors.forename: Dirk Jan
authors.initials: DJ
authors.lastname: Slotboom
authors.affiliation: Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University , SE-106 91 Stockholm, Sweden.
authors.forename: Jan-Willem
authors.identifier: 0000-0001-5537-4358
authors.initials: JW
authors.lastname: de Gier
mesh_terms: D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D012210:Rhamnose / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D011994:Recombinant Proteins; D012210:Rhamnose
keywords: E. coli; l-rhamnose metabolism; l-rhamnose promoter; membrane protein; protein production; secretory protein