Evaluation of novel inducible promoter/repressor systems for recombinant protein expression in Lactobacillus plantarum

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 (R) micro-fermentation system. Results: Reporter gene mCherry expression was compared under the control of different promoter/repressor systems: P-lacA (an endogenous promoter/repressor system derived from L. plantarum 3NSH), P-xylA (a promoter/repressor system derived from Bacillus megaterium DSMZ 319) and P-lacSynth (synthetic promoter and codon-optimized repressor gene based on the Escherichia coli lac operon). We observed that P-lacA was inducible solely by lactose, but not by non-metabolizable allolactose analoga. P-xylA 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. P-lacSynth was inducible with TMG (methyl beta-D-thiogalactopyranoside) and IPTG (isopropyl beta-D-1-thiogalactopyranoside), but also showed basal expression without inducer. The promoter P-lacSynth 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 (R) 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.