title: Evaluation of novel inducible promoter/repressor systems for recombinant protein expression in Lactobacillus plantarum.
abstract: BACKGROUND
  Engineering lactic acid bacteria (LAB) is of growing importance for food and feed industry as well as for in vivo vaccination or the production of recombinant proteins in food grade organisms. Often, expression of a transgene is only desired at a certain time point or period, e.g. to minimize the metabolic burden for the host cell or to control the expression time span. For this purpose, inducible expression systems are preferred, though cost and availability of the inducing agent must be feasible. We selected the plasmid free strain Lactobacillus plantarum 3NSH for testing and characterization of novel inducible promoters/repressor systems. Their feasibility in recombinant protein production was evaluated. Expression of the reporter protein mCherry was monitored with the BioLector(®) micro-fermentation system.
  
  
  RESULTS
  Reporter gene mCherry expression was compared under the control of different promoter/repressor systems: PlacA (an endogenous promoter/repressor system derived from L. plantarum 3NSH), PxylA (a promoter/repressor system derived from Bacillus megaterium DSMZ 319) and PlacSynth (synthetic promoter and codon-optimized repressor gene based on the Escherichia coli lac operon). We observed that PlacA was inducible solely by lactose, but not by non-metabolizable allolactose analoga. PxylA was inducible by xylose, yet showed basal expression under non-induced conditions. Growth on galactose (as compared to exponential growth phase on glucose) reduced basal mCherry expression at non-induced conditions. PlacSynth was inducible with TMG (methyl β-D-thiogalactopyranoside) and IPTG (isopropyl β-D-1-thiogalactopyranoside), but also showed basal expression without inducer. The promoter PlacSynth was used for establishment of a dual plasmid expression system, based on T7 RNA polymerase driven expression in L. plantarum. Comparative Western blot supported BioLector(®) micro-fermentation measurements. Conclusively, overall expression levels were moderate (compared to a constitutive promoter).
  
  
  CONCLUSIONS
  We evaluated different inducible promoters, as well as an orthologous expression system, for controlled gene expression in L. plantarum. Furthermore, here we provide proof of concept for a T7 RNA polymerase based expression system for L. plantarum. Thereby we expanded the molecular toolbox for an industrial relevant and generally regarded as safe (GRAS) strain.
authors.affiliation: Christian Doppler Laboratory for Genetically Engineered Lactic Acid Bacteria, Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 11, 1190, Vienna, Austria.
authors.forename: Silvia
authors.initials: S
authors.lastname: Heiss
authors.affiliation: Christian Doppler Laboratory for Genetically Engineered Lactic Acid Bacteria, Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 11, 1190, Vienna, Austria.
authors.forename: Angelika
authors.initials: A
authors.lastname: Hörmann
authors.affiliation: Christian Doppler Laboratory for Genetically Engineered Lactic Acid Bacteria, Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 11, 1190, Vienna, Austria.
authors.forename: Christopher
authors.initials: C
authors.lastname: Tauer
authors.affiliation: Christian Doppler Laboratory for Genetically Engineered Lactic Acid Bacteria, Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 11, 1190, Vienna, Austria.
authors.forename: Margot
authors.initials: M
authors.lastname: Sonnleitner
authors.affiliation: Christian Doppler Laboratory for Genetically Engineered Lactic Acid Bacteria, Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 11, 1190, Vienna, Austria.
authors.forename: Esther
authors.initials: E
authors.lastname: Egger
authors.affiliation: Christian Doppler Laboratory for Genetically Engineered Lactic Acid Bacteria, Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 11, 1190, Vienna, Austria.
authors.forename: Reingard
authors.initials: R
authors.lastname: Grabherr
authors.affiliation: Christian Doppler Laboratory for Genetically Engineered Lactic Acid Bacteria, Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 11, 1190, Vienna, Austria. stefan.heinl@boku.ac.at.
authors.forename: Stefan
authors.initials: S
authors.lastname: Heinl
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D048191:Lactiplantibacillus plantarum / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D007785:Lactose / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism; D014994:Xylose / Q000494:pharmacology
chemical_list: D011994:Recombinant Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D007544:Isopropyl Thiogalactoside; D014994:Xylose; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D007785:Lactose
keywords: BioLector® micro-fermentation system; IPTG; Inducible expression; L. plantarum 3NSH; Orthologous expression system; T7 RNA polymerase