title: Screening through the PLICable promoter toolbox enhances protein production in Escherichia coli.
abstract: Escherichia coli is a common host for recombinant protein production in which production titers are highly dependent on the employed expression system. Promoters are thereby a key element to control gene expression levels. In this study, a novel PLICable promoter toolbox was developed which enables in a single cloning step and after a screening experiment to identify out of ten IPTG-inducible promoters (T7, A3, lpp, tac, pac, Sp6, lac, npr, trc and syn) the most suitable one for high level protein production. The target gene is cloned under the control of different promoters in a single and efficient cloning step using the ligase-free cloning method PLICing (phosphorothioate-based ligase-independent gene cloning). The promoter toolbox was firstly validated using three well producible proteins (a cellulase from a metagenome library, a phytase from Yersinia mollaretii and an alcohol dehydrogenase from Pseudomonas putida) and then applied to two enzymes (3D1 DNA polymerase and glutamate dehydrogenase mutant) which are poorly produced in E. coli. By applying our PLICable pET-promoter toolbox, the authors were able to increase production by two-fold for 3D1 DNA polymerase (lac promoter) and 29-fold for glutamate dehydrogenase mutant H52Y (trc promoter).
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Aachen, Germany.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Yang
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Aachen, Germany.
authors.forename: Anna Joëlle
authors.initials: AJ
authors.lastname: Ruff
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Aachen, Germany.
authors.forename: Stefanie Nicole
authors.initials: SN
authors.lastname: Hamer
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Aachen, Germany.
authors.forename: Feng
authors.initials: F
authors.lastname: Cheng
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Aachen, Germany.|DWI-Leibniz Institut für Interaktive Materialien, Aachen, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: D010832:6-Phytase / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002480:Cellulase / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005969:Glutamate Dehydrogenase; D017853:Phosphotransferases (Alcohol Group Acceptor); D010832:6-Phytase; C106456:CELA2 protein, Streptomyces halstedii; D002480:Cellulase
keywords: Directed evolution; Escherichia coli; Phosphorothioate-based ligase-independent gene cloning; Promoter; Recombinant protein