We describe the development of an expression-secretion system in Bacillus subtilis to improve the quality and quantity of the secreted foreign proteins. This system consists of a strain (WB600) deficient in six extraceilular proteases and a set of sacB-based expression vectors. With the inactivation of all six chromosomal genes encoding neutral protease A, subtilisin, extracellular protease, metalloprotease, bacillopeptidase F, and neutral protease B, WB600 showed only 0.32% of the wild-type extracellular protease activity. No residual protease activity could be detected when WB600 was cultured in the presence of 2 mM phenylmethylsulfonyl fluoride.By using TEM P-lactamase as a model, we showed that WB600 can significantly improve the stability of the secreted enzyme. To further increase the production level we constructed an expression cassette carrying sacY, a sacB-specific regulatory gene. This gene was placed under the control of a strong, constitutively expressed promoter, P43. With this cassette in the expression vector, an 18-fold enhancement in ,-lactamase production was observed. An artificial operon, P43-sacY-degQ, was also constructed. However, only a partial additive enhancement effect (24-fold enhancement) was observed. Although degQ can stimulate the production of P-lactamase in the system, its ability to increase the residual extracellular protease activity from WB600 limits its application. The use of the P43-sacY cassette and WB600 would be a better combination for producing intact foreign proteins in high yield.With the capability of secreting extracellular enzymes directly into the culture medium, Bacillus subtilis potentially can serve as an efficient expression host (4,5,9,24,25). The secreted foreign proteins usually remain in biologically active forms (20,21), and the downstream purification is greatly simplified. However, at least two major limitations hinder the wide application of B. subtilis as a production microorganism. First, B. subtilis produces and secretes high levels of extracellular proteases which degrade the secreted foreign proteins (5, 41). It is well established that B. subtilis has six extracellular proteases: neutral protease A (45), subtilisin (also known as alkaline protease) (34, 42), extracellular protease (29,37), metalloprotease (30), bacillopeptidase F (31, 43), and neutral protease B (36). The construction of a strain deficient in five proteases GP263 (30,32), has eliminated over 99% of total extracellular protease activity. However, depending on the nature of the foreign proteins to be expressed in this strain, some of them remain unstable. Second, the lack of well-regulated inducible vectors also limits the wide application of the B. subtilis system. To overcome this problem, a few inducible vectors have been developed. Most of these vectors use either the E. coli lac system (16,46) or the temperature-sensitive repressor from lambda (3) or 4105 (23) to regulate the expression of foreign genes. Some others (10,39,47,48) are based on the regulatory region of a B. ...