Thermostable enzymes, such as cellulases, xylanases, proteases, pectinases, etc. have wide biotechnological and industrial applications. Most of these proteins are currently produced in mesophilic bacteria using heterologous gene expression systems due to a lack of versatile expression system for thermophilic bacteria. This often leads to different post-translational modifications or incorrect folding and, thus, protein properties are affected. These obstacles could be overcome if thermostable proteins were produced by thermophilic bacteria. Until now most attempts were unsuccessful due to a lack of genetic tools necessary for thermophilic expression, including thermostable reporter proteins, selection markers, plasmid vectors and others. However, these genetic tools are being rapidly developed which allows further successful attempts to construct an expression system for thermophilic bacteria.
The aim of our work is to create a regulated gene expression system for (Para)Geobacillus spp. bacteria. (Para)Geobacillus are thermophiles that usually have higher reaction rates at elevated temperatures and often are susceptible to genetic modifications, which makes them a suitable host for thermophilic expression.
To achieve this, pMSN4-GFP vector was developed. It was constructed by inserting genes of geobacillin I two component induction regulation system (geoR, geoK), promoter (geoA), reporter protein (sfGFP) and terminator (PgeoT) into pNW33N plasmid. sfGFP was inserted between PgeoA promoter and geoT terminator. Its expression is regulated by adding subinhibitory amount of geobacillin I. Added geobacillin I binds to a histidine-protein kinase (GeoK) which is then autophosphorylated. Subsequently, phosphate group is transfered to a response regulator (GeoR) which in turn leads to an activation of geoA promoter. Finally, activated PgeoA induces the expression of sfGFP or another protein in its place. Geobacillin I two component induction regulation system and sfGFP were chosen because of their stability at higher temperatures (55 °C).
pMSN4-GFP vector was later successfully inserted into Parageobacillus thermoglucosidasius DSM 2542 electrocompetent cells by electroporation method. Geobacillin I produced by Geobacillus thermodenitrificans DSM 465 was used for pMSN4-GFP expression system induction. However, sfGFP fluorescence was not detected. Therefore, we will use recombinant geobacillin I which was synthesized in E. coli [1] for further induction efficiency assessment.