title: A chromosomally encoded T7 RNA polymerase-dependent gene expression system for Corynebacterium glutamicum: construction and comparative evaluation at the single-cell level.
abstract: Corynebacterium glutamicum has become a favourite model organism in white biotechnology. Nevertheless, only few systems for the regulatable (over)expression of homologous and heterologous genes are currently available, all of which are based on the endogenous RNA polymerase. In this study, we developed an isopropyl-β-D-1-thiogalactopyranosid (IPTG)-inducible T7 expression system in the prophage-free strain C. glutamicum MB001. For this purpose, part of the DE3 region of Escherichia coli BL21(DE3) including the T7 RNA polymerase gene 1 under control of the lacUV5 promoter was integrated into the chromosome, resulting in strain MB001(DE3). Furthermore, the expression vector pMKEx2 was constructed allowing cloning of target genes under the control of the T7lac promoter. The properties of the system were evaluated using eyfp as heterologous target gene. Without induction, the system was tightly repressed, resulting in a very low specific eYFP fluorescence (= fluorescence per cell density). After maximal induction with IPTG, the specific fluorescence increased 450-fold compared with the uninduced state and was about 3.5 times higher than in control strains expressing eyfp under control of the IPTG-induced tac promoter with the endogenous RNA polymerase. Flow cytometry revealed that T7-based eyfp expression resulted in a highly uniform population, with 99% of all cells showing high fluorescence. Besides eyfp, the functionality of the corynebacterial T7 expression system was also successfully demonstrated by overexpression of the C. glutamicum pyk gene for pyruvate kinase, which led to an increase of the specific activity from 2.6 to 135 U mg(-1). It thus presents an efficient new tool for protein overproduction, metabolic engineering and synthetic biology approaches with C. glutamicum.
authors.affiliation: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, Jülich, D-52425, Germany.
authors.forename: Maike
authors.initials: M
authors.lastname: Kortmann
authors.forename: Vanessa
authors.initials: V
authors.lastname: Kuhl
authors.forename: Simon
authors.initials: S
authors.lastname: Klaffl
authors.forename: Michael
authors.initials: M
authors.lastname: Bott
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics; D002876:Chromosomes, Bacterial; D048230:Corynebacterium glutamicum / Q000201:enzymology* / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism*; D005434:Flow Cytometry; D005470:Fluorometry; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D017930:Genes, Reporter; D007544:Isopropyl Thiogalactoside / Q000378:metabolism*; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D011770:Pyruvate Kinase / Q000032:analysis / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C054562:yellow fluorescent protein, Bacteria; D007544:Isopropyl Thiogalactoside; D011770:Pyruvate Kinase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases