中国生物工程杂志

A bacterial phage f105 based recombinant Bacillus subtilis for secretive production of penicillin G acylase (PGA) was constructed and expression of the cloned gene was optimized. Both the promoterless penicillin G acylase gene (pga) and the native promoter bearing penicillin G acylase gene (pgaPB) were isolated by PCR amplification from the chromosomal DNA of Bacillus megaterium with properly designed primers. The isolated genes were then inserted into the chromosomal DNA of a lysogenic B. subtilis bearing prophage f105MU331 through homologous recombination, with the aid of the integrating vector pSG703 that contained the homologous fragments of the prophage f105MU331. The resultant recombinant strains were denoted as B. subtilis PA1 and B. subtilis PA2, respectively, in which the recombinant genes were covalently integrated into the chromosomal DNA, and was located downstream of a strong thermal inducible phage promoter. The recombinant strains showed high stability and effective recombinant PGA production. The productivity of recombinant PGA from B. subtilis PA2, in which transcription of the heterologous gene was initiated from the phage promoter and the native pga promoter, was higher than B. subtilis PA1 where transcription of the pga gene was initiated from the phage promoter only. Inducing the recombinant strain at 50 ℃ for 3 min in the late exponential phase of growth led to best PGA production. Addition of phenylacetic acid failed to stimulate PGA production, and glucose exhibited inhibitory effect on PGA production. The pH stat fed-batch fermentation with strain B. subtilis PA2 was carried out on a 20 L fermentor, and a high productivity of 18.28 U/ml was achieved.