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

In this study, we developed a robust expression system based on optimized P tetR 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, P tetA and P tetR . Both promoters were inserted upstream of eGFP on a pUB110-derivative with high copy number. Reduction of the 18bp spacer region of both P tetA and P tetR to 17bp significantly increased their strength in B. subtilis. Nevertheless, only the optimized P tetR with 17bp spacer region (P tetR2) directed high level of eGFP expression. In the second step, regulation of the system was optimized by testing of tetR using well-known promoters, such as P mtlA , P mtlR , P ptsG and P penP . Expression of tetR by P ptsG resulted in a tight regulation of P tetR2-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.

Source: Plasmid - October 10, 2015 Category: Biotechnology Source Type: research