Construction of an efficient Bacillus subtilis system for extracellular production of heterologous proteins.

An efficient expression/secretion vector, designated pM2Veg, was constructed for extracellular production of heterologous proteins in Bacillus subtilis. To construct pM2Veg, a synthetic cassette, the Veg cassette carrying: (1) the strong vegetative vegI promoter from B. subtilis, (2) the Escherichia coli lac operator, (3) the B. subtilis consensus ribosome-binding site, (4) the Staphylococcal protein A leader sequence, (5) a cloning region for insertion of foreign genes, (6) translational stop codons in all three reading frames, and (7) the gnt transcriptional terminator, was cloned into a derivative of the stable pRB373 B. subtilis/E. coli shuttle plasmid, the pM2 vector. The application of pM2Veg to effect secretory production of heterologous proteins was illustrated using two widely different proteins: the endoglucanase (Eng) encoded by the cenA gene of Cellulomonas fimi and human epidermal growth factor (hEGF). Levels of Eng and hEGF measured in culture supernatant samples of B. subtilis transformants harboring recombinant constructs formed between pM2Veg and the cenA and hEGF genes were 8.3 U ml-1 and 7.0 mg l-1, respectively. The Eng activity is more than four times higher than the yield from the best cenA recombinant construct previously reported, and the hEGF data represents the first successful expression of the factor in B. subtilis.