Construction of a highly efficient host-vector system for secretion of heterologous protein in Bacillus subtilis

Abstract

An efficient secretion host-vector system in Bacillus subtilis was constructed using two Bacillus amyloliquefaciens genes, sacQ (BamF) responsible for the increase of extracellular levansucrase and protease levels and npr for extracellular neutral protease. The former was introduced into the genomic DNA of the extracellular alkaline and neutral protease deficient Bacillus subtilis and the latter was used to construct a secretion vector utilizing the promoter and head portion of the prepropeptide coding region. This introduction of sacQ(BamF) permitted the increase of levansucrase level of the recipient strain. In contrast, the residual proteolytic activity of the double mutant was further reduced by sacQ(BamF). The expression level of chloramphenicol acetyltransferase gene located in a multicopy plasmid in the double mutant, of which mRNA synthesis was directed by the npr promoter, was elevated 1.7-fold by the introduction of sacQ(BamF). The extracellular production level of human growth hormone (hGH) in the double mutant, whose gene was inserted into the multicopy secretion vector based on npr, was increased 4- to 5-fold by the introduction of sacQ(BamF). This increase seemed to be mainly dependent upon the continuity of the hGH accumulation even in long cultivation rather than stimulation of the production rate. Using this host-vector system, we demonstrated the highly efficient secretion of hGH (200 mg 1 −1) in B. subtilis.