Dual Promoters Enhance Heterologous Enzyme Production from Bacterial Phage Based Recombinant Bacillus subtilis

The effect of dual promoters on recombinant protein production from bacterial phage based Bacillus subtilis expression system was investigated. Alpha amylase (from Bacillus amyloliquefaciens) and penicillin acylase (from Bacillus megaterium) were selected as the indicating enzymes. The prophage ϕ105 MU331, which contained the lacZ-cat insertion and had undergone cts-52 mutation, was a temperate phage of B. subtilis. Both the promoterless genes and the promoter-bearing genes were isolated through PCR amplification with properly designed primers, and were inserted into plasmid pSG703 that contained the lacZ-cat expression cartridge. The lysogenic B. subtilis (ϕ105 MU331) was transformed with the resultant recombinant plasmids, and the heterologous genes were thereby integrated into the chromosomal DNA of B. subtilis via homologous recombination. The transformants were designated as B. subtilis AMY1, B. subtilis AMY2, B. subtilis PA1, and B. subtilis PA2, respectively. In the recombinant B. subtilis strains, the inserted sequences were located downstream of a strong phage promoter that could be activated by thermal induction. In B. subtilis AMY1 and B. subtilis PA1, transcription of the heterologous genes was only initiated by the phage promoter after subjecting it to heat shock, whereas in B. subtilis AMY2 and B. subtilis PA2, transcription of the heterologous genes was initiated by dual promoters, namely, the phage promoter and the native promoter. The application of dual promoters increased the productivity of both enzymes, with 133 % enhancement for α-amylase production and 113 % enhancement for penicillin acylase production.