Enzyme evolution using in vitro compartmentalization of bacterial cell libraries

描述（由申请人提供）：具有定制催化活性的人工蛋白质的设计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变革意义。创造蛋白质催化剂的一个一般策略是通过计算将任意催化活性设计到蛋白质支架中，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，并赋予多个周转催化剂进化优势。该提案通过将体外区室化、细菌细胞库和荧光激活细胞分选（FACS）整合到用于键形成反应的蛋白质催化剂的定向进化的通用选择系统中来解决这些限制。该提案的第一个目的是工程化大肠杆菌细菌，其能够分泌酶，同时在其表面上展示合成的小分子底物。下一个目标是开发一个通用的选择方案的键形成的催化剂的基础上，划分的细菌细胞，工程分泌的候选酶，可以作用于底物分子显示在其细胞表面。隔室内的键形成导致亲和柄与细胞表面的附着，这可以使用荧光抗体标记来检测。通过FACS分离表现出最高水平的荧光的细菌细胞，从而编码最活跃的多重周转酶，用于更多轮的选择。为了验证所提出的选择方案，将进行富集分泌活性磷酸泛酰巯基乙胺基转移酶的细菌细胞的模型选择。该提案的最后一个目的是发展高效的，人工蛋白质催化剂的狄尔斯-阿尔德环加成反应，使用建议的选择方案，以优化由大卫贝克教授的实验室产生的初始设计。将针对2-吡喃酮和_-炔基酯之间的模型反应设计催化剂，并确定从选择中分离出的最佳催化剂的催化效率、底物特异性和区域选择性。公共卫生相关性高效蛋白质催化剂的开发将最终使药物和其他重要化学品的生产更经济、更少浪费和更环保，从而有益于公共卫生。酶还有望改善公众健康，成为环境修复的重要工具。

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.