Development of an anhydrotetracycline-inducible expression system for expression of a neopullulanase in B. subtilis.

Bacillus subtilis is a widely used bacterium for production of heterologous and homologous proteins. The primary challenge in the production of proteins in B. subtilis is choosing a relevant expression system. In this study, we developed a robust expression system based on optimized PtetR of transposon Tn1721, which is repressible by its specific repressor, TetR. The first step of this work was focused on the optimization of structure and core elements of Tn1721 anhydrotetracycline-inducible promoters, PtetA and PtetR. Both promoters were inserted upstream of eGFP on a pUB110-derivative with high copy number. Reduction of the 18 bp spacer region of both PtetA and PtetR to 17 bp significantly increased their strength in B. subtilis. Nevertheless, only the optimized PtetR with 17 bp spacer region (PtetR2) directed high level of eGFP expression. In the second step, regulation of the system was optimized by testing the expression of tetR using well-known promoters, such as PmtlA, PmtlR, PptsG and PpenP. Expression of tetR by PptsG resulted in a tight regulation of PtetR2-eGFP showing 44-fold induction. By using the final expression plasmid in B. subtilis, neopullulanase was produced up to 15% of the total soluble protein.