Enzyme evolution using in vitro compartmentalization of bacterial cell libraries

描述(申请人提供)：设计具有特制催化活性的人造蛋白质是分子科学的一个长期目标，具有潜在的改变化学、生物学和医学的意义。创造蛋白质催化剂的一个一般策略是通过计算将任意的催化活性设计成蛋白质支架，然后通过定向进化来优化这种人造酶的活性。现有的蛋白质定向进化方法对于酶的进化并不理想，因为它们不能对分子间底物结合施加直接选择压力，也不能为多个周转催化剂提供进化优势。这项建议通过将体外区隔、细菌细胞库和荧光激活细胞分类(FACS)整合到一个通用的选择系统中，用于定向进化成键反应的蛋白质催化剂，从而解决了这些限制。这项提议的第一个目标是设计一种能够分泌一种酶的大肠杆菌，同时在其表面显示一个合成的小分子底物。下一个目标是开发一种一般的键形成催化剂的选择方案，基于对细菌细胞的分区，这些细菌细胞被设计为分泌一种候选酶，该酶可以作用于显示在其细胞表面的底物分子。隔室内的键形成导致亲和手柄附着到细胞表面，这可以使用荧光抗体标记来检测。表现出最高水平荧光的细菌细胞，从而编码最活跃的多种周转酶，被FACS分离出来进行更多轮的选择。为了验证所提出的选择方案，将进行富含分泌活性磷酸铁氨酰转移酶的细菌细胞的模型选择。这项建议的最后一个目标是进化出用于Diels-Alder环加成反应的高效人造蛋白质催化剂，使用所提议的选择方案来优化David Baker教授的实验室产生的初始设计。将为2-吡喃酮和α-炔基酯之间的模型反应设计催化剂，并从选择的催化剂中确定最好的催化剂的催化效率、底物特性和区域选择性。与公众健康相关高效蛋白质催化剂的开发最终将使药品和其他重要化学品的生产更经济、更少浪费和更环保，从而有利于公众健康。作为环境修复的重要工具，酶还有望改善公众健康。

DESCRIPTION (provided by applicant): The design of artificial proteins with tailor-made catalytic activities represents a longstanding goal in the molecular sciences, with potentially transforming implications for chemistry, biology, and medicine. One general strategy for creating protein catalysts is to computationally design an arbitrary catalytic activity into a protein scaffold and then optimize the activity of this artificial enzyme through directed evolution. Existing methods for the directed evolution of proteins are not ideal for the evolution of enzymes because of their inabilities to apply direct selection pressure for intermolecular substrate binding and confer an evolutionary advantage for multiple turnover catalysts. This proposal addresses these limitations by integrating in vitro compartmentalization, bacterial cell libraries, and fluorescence-activated cell sorting (FACS) into a general selection system for the directed evolution of protein catalysts of bond-forming reactions. The first aim of this proposal is to engineer an Escherichia coli bacterium that is capable of secreting an enzyme while simultaneously displaying a synthetic small molecule substrate on its surface. The next aim is to develop a general selection scheme for catalysts of bond-formation, based on the compartmentalization of the bacterial cells that were engineered to secrete a candidate enzyme that can act upon substrate molecules displayed on their cell surfaces. Bond-formation within the compartments results in the attachment of an affinity handle to the cell surface, which can be detected using fluorescent antibody labeling. Bacterial cells exhibiting the highest levels of fluorescence, thereby encoding the most active multiple turnover enzymes, are isolated by FACS for more rounds of selection. To validate the proposed selection scheme, a model selection enriching for bacterial cells that secrete active phosphopantetheinyl transferase will be performed. The last aim of this proposal is to evolve highly efficient, artificial protein catalysts of a Diels-Alder cycloaddition reaction, using the proposed selection scheme to optimize initial designs generated by the laboratory of Professor David Baker. Catalysts will be designed for the model reaction between a 2-pyrone and an _-alkynyl ester, and the catalytic efficiencies, substrate specificities, and regioselectivites of the best catalysts isolated from the selections will be determined. PUBLIC HEALTH RELEVANCE The development of high-efficiency protein catalysts will benefit public health by eventually enabling a more economical, less wasteful, and more environmentally-friendly production of pharmaceuticals and other important chemicals. Enzymes also promise to improve public health as important tools for environmental remediation.