title: A T7 RNA polymerase-based toolkit for the concerted expression of clustered genes.
abstract: Bacterial genes whose enzymes are either assembled into complex multi-domain proteins or form biosynthetic pathways are frequently organized within large chromosomal clusters. The functional expression of clustered genes, however, remains challenging since it generally requires an expression system that facilitates the coordinated transcription of numerous genes irrespective of their natural promoters and terminators. Here, we report on the development of a novel expression system that is particularly suitable for the homologous expression of multiple genes organized in a contiguous cluster. The new expression toolkit consists of an Ω interposon cassette carrying a T7 RNA polymerase specific promoter which is designed for promoter tagging of clustered genes and a small set of broad-host-range plasmids providing the respective polymerase in different bacteria. The uptake hydrogenase gene locus of the photosynthetic non-sulfur purple bacterium Rhodobacter capsulatus which consists of 16 genes was used as an example to demonstrate functional expression only by T7 RNA polymerase but not by bacterial RNA polymerase. Our findings clearly indicate that due to its unique properties T7 RNA polymerase can be applied for overexpression of large and complex bacterial gene regions.
authors.affiliation: Institut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum Jülich, D-52426 Jülich, Germany.
authors.forename: Solmaz
authors.initials: S
authors.lastname: Arvani
authors.forename: Annette
authors.initials: A
authors.lastname: Markert
authors.forename: Anita
authors.initials: A
authors.lastname: Loeschcke
authors.forename: Karl-Erich
authors.initials: KE
authors.lastname: Jaeger
authors.forename: Thomas
authors.initials: T
authors.lastname: Drepper
mesh_terms: D017123:Bacteriophage T7 / Q000201:enzymology; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005818:Genetic Engineering / Q000379:methods*; D006864:Hydrogenase / Q000235:genetics; D008957:Models, Genetic; D005810:Multigene Family; D011401:Promoter Regions, Genetic; D016958:Pseudomonas putida / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000201:enzymology / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014764:Viral Proteins; D006864:Hydrogenase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases