Enzyme evolution using in vitro compartmentalization of bacterial cell libraries

利用细菌细胞库的体外区室化进行酶进化

• 批准号: 7783797
• 负责人: Irwin Chen
• 金额: $ 4.14万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2011-01-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7783797
• 关键词: Address; Affinity; Alder plant; Bacteria; Binding; Biology; Cell surface; Cells; Chemicals; Chemistry; Development; Engineering; Enzymes; Escherichia coli; Esters; Evolution; Exhibits; Fluorescence; Fluorescence-Activated Cell Sorting; Fluorescent Antibody Technique; Goals; In Vitro; Label; Laboratories; Libraries; Medicine; Methods; Modeling; Molecular; Pharmacologic Substance; Production; Proteins; Public Health; Pyrones; Reaction; Scaffolding Protein; Scheme; Science; Substrate Specificity; Surface; System; base; catalyst; cycloaddition; design; directed evolution; improved; model design; phosphopantetheinyl transferase; pressure; professor; public health relevance; remediation; small molecule; tool

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.

描述（由申请人提供）：具有量身定制的催化活性的人造蛋白质的设计代表了分子科学中的长期目标，并有可能改变对化学，生物学和医学的影响。创建蛋白质催化剂的一种一般策略是在计算中将任意催化活性设计成蛋白质支架，然后通过定向进化来优化该人造酶的活性。现有的蛋白质导向演化的方法对于酶的演化不是理想的选择，因为它们的无能为力，无法为分子间底物结合施加直接选择压力并赋予多种失误催化剂的进化优势。该建议通过整合体外隔室化，细菌细胞库以及荧光激活的细胞分选（FACS）来解决这些局限性，以纳入一个通用选择系统，以定向键形形成反应的蛋白质催化剂。该提案的第一个目的是设计一种能够分泌酶的大肠杆菌细菌，同时同时在其表面显示合成的小分子底物。下一个目的是基于对细菌细胞的分室化开发键构催化剂的一般选择方案，该细菌细胞的分隔是为分泌可以对其细胞表面上显示的底物分子作用的候选酶的。隔室内的键形成导致亲和力将其附着在细胞表面上，可以使用荧光抗体标记检测。 FACS分离出表现出最高水平的荧光水平的细菌细胞，从而编码最活跃的多重周转酶，以进行更多的选择。为了验证所提出的选择方案，将对细菌细胞的模型选择富含分泌活性磷酸乙基转移酶的细菌细胞。该提案的最后一个目的是使用拟议的选择方案来进化高效的人工蛋白催化剂，以优化由戴维·贝克（David Baker）教授实验室生成的初始设计。催化剂将设计用于2-吡喃和_-碱基酯之间的模型反应，以及将确定从选择中分离出的最佳催化剂的催化效率，底物特异性和区域性电位。公共卫生相关性高效蛋白催化剂的发展将通过最终实现更经济，浪费且对环境友好的药物和其他重要化学物质的生产而受益。酶还有望改善公共卫生作为环境修复的重要工具。