Engineering Bacillus subtilis for cell surface display

用于细胞表面展示的枯草芽孢杆菌工程

• 批准号: 36556-2012
• 负责人: Wong, SuiLam
• 金额: $ 1.89万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2012
• 资助国家: 加拿大
• 起止时间: 2012-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=505675
• 关键词: Engineering; Bacillus; subtilis; cell; surface

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.

枯草芽孢杆菌具有许多诱人的特性，可以作为微生物工厂来生产用于生物技术应用的天然和工程蛋白质。然而，它会产生许多胞外蛋白酶，可以降解感兴趣的蛋白质。虽然本课题组已经培育出一株八种蛋白水解酶缺陷型菌株，但我们仍观察到一些残留的水解酶活性可以降解某些胞外蛋白。这些活性可能来自两种膜蛋白，HtrA和HtrB。这两种蛋白质不仅具有蛋白酶活性，而且还具有分子伴侣活性，可以帮助其他蛋白质正确折叠。在这个方案中，我们将通过在DNA水平上选择性地将HtrA和HtrB的催化丝氨酸残基改变为丙氨酸来灭活HtrA和HtrB的蛋白酶活性。通过这种方式，我们可以潜在地灭活蛋白酶的活性，但保留分子伴侣的活性。新开发的工程菌株将被评估其生产完整重组蛋白的能力。这项研究的下一个目标是进一步扩大枯草杆菌系统的应用，将有用的蛋白质展示到细胞表面。如果成功，该系统可以展示复杂的酶复合体，以降解纤维素、生物塑料和某些对环境有害的分子。它也可以应用于活疫苗的生产。为了使这个系统成功，我们必须设计一种链霉亲和素，它可以结合特定的多肽标签，但不能结合生物素。携带这种特异性多肽标签的重组蛋白可以被显示在枯草杆菌细胞表面的工程化链霉亲和素捕获。最后，我们将对枯草芽孢杆菌中的一个新的分解酶系统进行表征。山梨酸酶是一种将细胞壁蛋白共价连接到细胞表面的酶。这种酸解酶的特性可以使我们通过共价键将蛋白质展示到细胞表面。由于外部环境的高剪切力，表面显示的巨型酶复合体的脱落将不再是一个令人担忧的问题。这很可能是细菌细胞壁生物合成酶复合体的一部分。将采用各种方法来研究这种关系。