AN EFFICIENT EXPRESSION AND SECRETION SYSTEM BASED ON BACILLUS-SUBTILIS PHAGE PHI-105 AND ITS USE FOR THE PRODUCTION OF BACILLUS-CEREUS BETA-LACTAMASE-I

A novel expression system based on the Bacillus subtilis bacteriophage phi105 has been developed to permit the high-level synthesis and secr etion of beta-lactamase I (BlaI) from Bacillus cereus. Shotgun inserti on of a promoterless lacZ gene into the phage genome permitted the ide ntification of a clone producing large amounts of beta-galactosidase ( betaGal), indicating the transcription of the reporter gene from a str ong 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 prophage s contain two additional mutations: an ind mutation, which greatly enh ances the normally poor transformability of phi105 lysogens, and a cts mutation, which allows thermo-induction of phage development and prot ein production. Induction of a derivative prophage containing the blaI gene from B. cereus resulted in the production of up to 500 mug of se creted BlaI per ml of culture supernatant.