An efficient expression and secretion system based on Bacillus subtilis phage phi 105 and its use for the production of B. cereus beta-lactamase I.

Abstract

A novel expression system based on the Bacillus subtilis bacteriophage phi 105 has been developed to permit the high-level synthesis and secretion of beta-lactamase I (BlaI) from Bacillus cereus. Shotgun insertion of a promoterless lacZ gene into the phage genome permitted the identification of a clone producing large amounts of beta-galactosidase (beta Gal), indicating the transcription of the reporter gene from a strong phage promoter. The insertion also blocked lysis of the host cell. Although the insertion in the original prophage was complex, plasmid vectors and prophage derivatives have been developed to facilitate the replacement of lacZ with other genes for expression. The new prophages contain two additional mutations: an ind mutation, which greatly enhances the normally poor transformability of phi 105 lysogens, and a cts mutation, which allows thermo-induction of phage development and protein production. Induction of a derivative prophage containing the blaI gene from B. cereus resulted in the production of up to 500 micrograms of secreted BlaI per ml of culture supernatant.