CONSTRUCTION OF INDUCIBLE SECRETION VECTORS AND THEIR APPLICATION FOR THE SECRETION OF FOREIGN EXTRACELLULAR AND INTRACELLULAR PROTEINS IN BACILLUS-SUBTILIS

We have constructed inducible secretion vector (pISA412) which has (i) a unique BamHI site as a cloning site between the signal sequence and transcriptional terminator of Bacillus subtilis alpha-amylase gene (amyE), and (ii) penI-penJ operon and promoter-operator regions of penP (penicillinase gene) from Bacillus licheniformis as a regulation system upstream to the amyE signal sequence. Using pISA412, we examined the expression in B. subtilis of several cloned secretory protein genes such as the penicillinase gene from B. licheniformis (penP), alpha-amylase genes from Bacillus stearothermophilus (amyT) and human salivary (hsa), and the thermostable endoglucanase gene from Clostridium thermocellum (engD). In each case, gene expression was induced by the addition of 2-(2'-carboxyphenyl) benzoyl-6-aminopenicillanic acid (CBAP). Significant levels of extracellular activity of these gene products could be detected. Furthermore, when an intracellular enzyme gene (beta-galactosidase gene of Escherichia coli; lacZ) was subcloned in the secretion vector, beta-galactosidase activity could be detected in the culture supernatant. We also constructed a secretion vector, pISA(ts)412, with the temperature-sensitive repressor (P70L) in order to induce gene expression simply by shifting temperature of the culture from 30-degrees-C to 48-degrees-C. Expression of the cloned gene into the pISA(ts)412 could be controlled by the temperature shift. These results indicate that pISA412 and pISA(ts)412 vectors are useful inducible secretion vectors in B. subtilis.