Enzyme evolution using in vitro compartmentalization of bacterial cell libraries

描述（由申请人提供）：设计具有定制催化活性的人造蛋白质代表了分子科学的长期目标，对化学，生物学和医学具有潜在的转变意义。制造蛋白质催化剂的一般策略是通过计算设计任意催化活性的蛋白质支架，然后通过定向进化优化这种人工酶的活性。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，也不能赋予多种转化催化剂进化优势。该提案通过将体外区隔化、细菌细胞文库和荧光激活细胞分选（FACS）整合到一个用于定向进化成键反应的蛋白质催化剂的一般选择系统中来解决这些局限性。这项提议的第一个目标是设计一种大肠杆菌，使其能够分泌一种酶，同时在其表面显示合成的小分子底物。下一个目标是根据细菌细胞的区隔化，开发出一种可以作用于细胞表面底物分子的候选酶，从而形成一种通用的键形成催化剂选择方案。隔室内的键形成导致亲和柄附着在细胞表面，这可以使用荧光抗体标记检测到。细菌细胞表现出最高水平的荧光，从而编码最活跃的多重转换酶，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，将对分泌活性磷酸甲磷酰基转移酶的细菌细胞进行模型选择。本提案的最后一个目标是发展高效的Diels-Alder环加成反应的人工蛋白质催化剂，使用建议的选择方案来优化由David Baker教授实验室生成的初始设计。催化剂将被设计用于2-吡啶酮和-炔基酯之间的模型反应，并从选择中分离出最佳催化剂的催化效率、底物特异性和区域选择性将被确定。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，从而有利于公共卫生。作为环境修复的重要工具，酶也有望改善公众健康。

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.