Construction of protein overproducer strains in Bacillus subtilis by an integrative approach

We evaluated the effect of several genetic factors reported as having a rol e in the induction of the expression of significant levels of recombinant p rotein in Bacillus subtilis. We utilized the beta -galactosidase reporter p rotein from Escherichia coli as our model for measuring the overproduction of heterologous proteins in B. subtilis. The lacZ gene was expressed in B. subtilis using the regulatory region of the subtilisin gene aprE. In this s tudy, we considered factors known to modulate the transcription and transla tion initiation rates and genetic and mRNA stability. We also consider the effects of different genetic backgrounds, such as degU32 and hpr2, that unt il now have been studied independently. By changing the native -35 promoter box to the consensus TTGACA sequence of the aprE promoter, a significant 1 00-fold increase in the beta -galactosidase activity was obtained. On the o ther hand, changes such as the GTG to ATG start codon, the construction of a consensus AAGGAGG ribosome binding site, and the addition of the cryIIIA transcription terminator at the 3' end of the lacZ gene, produced only marg inal effects on the final beta -galactosidase activity.