Engineering Bacillus subtilis for cell surface display

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Bacillus subtilis has many attractive features to serve as a microbial factory to produce natural and engineered proteins for biotechnological applications. However, it produces many extracellular proteases that can degrade the proteins of interest. Although my group has developed an eight-protease deficient strains, we still observe the presence of some residual protease activities that can degrade certain extracellular proteins. These activities are likely from two membrane proteases, HtrA and HtrB. These two proteins have not only protease activities but also the molecular chaperone activities which can help other proteins to fold properly. In this proposal, we will inactivate the protease activities of HtrA and HtrB by selectively changing their catalytic serine residues to alanine at the DNA level. In this manner, we can potentially inactivate the protease activities but preserve the molecular chaperone activity. New engineered strains developed will be evaluated for its ability to produce intact recombinant proteins. The next objective for this study is to further extend the application of the B. subtilis system to display useful proteins to the cell surface. If it is successful, this system can display sophisticated enzyme complexes to degrade cellulose, bioplastics and certain environmentally harmful molecules. It can also be applied for live vaccine production. To make this system successful, we have to engineer a streptavidin which can bind a specific peptide tag but cannot bind biotin. Recombinant proteins carrying this specific peptide tag can be captured by the engineered streptavidin displayed on the B. subtilis cell surface. Finally, we will characterize a new sortase system in B. subtilis. Sortase is an enzyme to covalently link the cell wall proteins to the cell surface. Characterization of this sortase can allow us to display proteins to the cell surface via covalent linkages. Shedding off of the surface displayed giant enzyme complexes by the high shearing force in the external environment will no longer be a concern. It is likely that sortases are part of the bacterial cell wall biosynthetic enzyme complexes. Various approaches will be applied to study this relationship.