Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases.
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 extracellular 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 beta-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 beta-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 beta-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.
Wu
X C
XC
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Lee
W
W
Tran
L
L
Wong
S L
SL
D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D010450:Endopeptidases / Q000378:metabolism*; D015870:Gene Expression / Q000235:genetics; D005809:Genes, Regulator / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics
D011994:Recombinant Proteins; D010450:Endopeptidases; D001618:beta-Lactamases