Engineering Bacillus subtilis for cell surface display

枯草芽孢杆菌具有许多吸引人的特性，可以作为微生物工厂生产用于生物技术应用的天然和工程蛋白。然而，它产生许多细胞外蛋白酶，可以降解感兴趣的蛋白质。虽然我的研究小组已经开发出一种缺乏8种蛋白酶的菌株，但我们仍然观察到存在一些残留的蛋白酶活性，可以降解某些细胞外蛋白质。这些活性可能来自两种膜蛋白酶，HtrA和HtrB。这两种蛋白质不仅具有蛋白酶活性，而且具有分子伴侣活性，可以帮助其他蛋白质正常折叠。在这个提议中，我们将通过选择性地在DNA水平上改变HtrA和HtrB的催化丝氨酸残基为丙氨酸来灭活它们的蛋白酶活性。通过这种方法，我们可以潜在地使蛋白酶活性失活，但保留分子伴侣活性。开发的新工程菌株将评估其产生完整重组蛋白的能力。本研究的下一个目标是进一步扩展枯草芽孢杆菌系统的应用，以在细胞表面显示有用的蛋白质。如果成功的话，这个系统可以展示复杂的酶复合物来降解纤维素、生物塑料和某些对环境有害的分子。它也可用于活疫苗的生产。为了使这个系统成功，我们必须设计一种链亲和素，它可以结合特定的肽标签，但不能结合生物素。在枯草芽孢杆菌细胞表面显示的工程链霉亲和素可以捕获携带该特异性肽标签的重组蛋白。最后，我们将描述枯草芽孢杆菌中一个新的分选酶系统。分选酶是一种将细胞壁蛋白与细胞表面共价连接的酶。这种分选酶的特性可以让我们通过共价键将蛋白质展示到细胞表面。在外部环境中，由于高剪切力，表面显示的巨大酶复合物脱落将不再是一个问题。分选酶很可能是细菌细胞壁生物合成酶复合物的一部分。将采用各种方法来研究这种关系。

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.