Development of a new gene expression vector for Thermus thermophilus using a silica-inducible promoter

Thermostable enzymes are commonly produced in mesophilic hosts for research and bioengineering purposes. However, these hosts do not overexpress the active forms of some biologically functional thermoenzymes. Therefore, an efficient thermophilic expression system is needed. Thermus thermophilus contains an easily manipulable genome and is therefore among the best candidate microbes for a "hot" expression system. We previously identified a strong and inducible promoter that was active in T. thermophilus under supersaturated silica conditions. Here, we report a new heterologous gene expression system based on a silica-inducible promoter in T. thermophilus.

A Thermus sp. A4 gene encoding thermostable β-galactosidase was cloned as a reporter gene into the expression vector pSix1, which contains a selection marker that confers thermostable resistance to hygromycin and a 600 bp DNA region containing a putative silica-inducible promoter. β-galactosidase activity was 11-fold higher in the presence than in the absence of 10 mM silicic acid. SDS-PAGE revealed a prominent band corresponding to 73 kDa of β-galactosidase, and this enzyme was expressed as an active and soluble protein (yield: 27 mg/L) in Thermus but as an inclusion body in Escherichia coli. Truncation of the putative silica-inducible promoter region in Thermus expression vector improved the yield of the target protein, possibly by avoiding plasmid instability due to homologous recombination. Finally, we developed an expression vector containing the pSix1 backbone and a 100 bp DNA region corresponding to the silica-inducible promoter. We used this vector to successfully express the active form of glutamate dehydrogenase from Pyrobaculum islandicum (PisGDH) without additional treatment (yield: 9.5 mg/L), whereas the expression of active PisGDH in E. coli required heat treatment.

We successfully expressed the thermostable β-galactosidase and PisGDH in T. thermophilus as active and soluble forms and achieved with our system the highest known protein expression levels in this species. These thermoenzymes were expressed in active and soluble forms. Our results validate the use of our silica-inducible expression system as a novel strategy for the intracellular overexpression of thermostable proteins.