Transition Metal Catalysis and Metabolic Engineering using Artificial Metalloenzy

使用人工金属酶的过渡金属催化和代谢工程

• 批准号: 8214701
• 负责人: JARED C LEWIS
• 金额: $ 24.23万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8214701
• 关键词: Academia; Achievement; Aerobic; Alkenes; Amaze; Amino Acids; Amino Acyl-tRNA Synthetases; Anabolism; Binding; Biological; Biological Factors; Biology; Boronic Acids; California; Catalysis; Chemicals; Collection; Complex; Coupling; Development; Development Plans; Diagnostic; Discipline; Drug Industry; Engineering; Ensure; Environment; Enzymes; Escherichia coli; Faculty; Fostering; France; Goals; Health; Human; Individual; Industry; Institutes; Institution; Ions; Laboratories; Life; Medical; Mentors; Mentorship; Metabolic; Metabolic Pathway; Metals; Molecular Conformation; Nature; Organic Synthesis; Organic solvent product; Organism; Palladium; Pathway interactions; Peptides; Petroleum; Pharmacologic Substance; Phase; Physiological; Preparation; Procedures; Production; Protein Engineering; Proteins; Public Health; Reaction; Reagent; Research; Scaffolding Protein; Science; Scientist; Side; Site; Specificity; Speed; System; Technology; Transfer RNA; Transition Elements; Tryptophan; Work; Writing; aqueous; aryl halide; career development; catalyst; chemical reaction; chemical synthesis; design; directed evolution; experience; improved; in vivo; member; metalloenzyme; novel; practical application; professor; success; trend

Practical application of new synthetic molecules for the betterment of human health depends directly on the efficiency with which these compounds can be synthesized, but this is frequently limited by poor reaction yields throughout long reaction sequences in which intermediate compounds must be isolated and purified. Metabolic engineers have demonstrated that novel biosynthetic pathways can be assembled in order to produce chemicals in vivo with no isolation of intermediates in an aqueous aerobic environment, but these sequences are limited to transformations catalyzed by natural enzymes. This proposal describes the design, preparation, and application of a new class of artificial metalloenzymes that combines the scope of chemical catalysis with the efficiency of biosynthesis in an unprecedented manner to produce molecules of exceptional biological importance. The proposed system offers a number of significant advantages over previous artificial metalloenzyme constructs, which enable its use for in vivo catalysis and metabolic engineering. This ambitious project will be conducted as part of the candidate's long term goals of increasing the efficiency of organic synthesis, particularly for the production of biologically active molecules. In the mentored phase (K99) of the proposed research, amino acids with catalytically active palladacycle side chains will be synthesized, characterized, and incorporated into a suitable scaffold protein. The catalytic activity of the resulting metalloenzymes will be evaluated using a variety of C-C bond forming reactions. The proposed amino acids catalysts could prove highly useful for a variety of applications in their own right, and their incorporation into proteins would mark a significant achievement in the fields of UAA incorporation and biocatalysis with potential applications well beyond the scope of this application. This research will be conducted in the laboratory of Professor Frances Arnold, a leader in the field of protein engineering, at the California Institute of Technology, a world-renowned research institution. Professor Arnold has a strong record as a mentor of successful members of industry and academia, and she and the candidate have outlined a career development plan focusing on mentorship, writing, and research to ensure the candidate continues this trend. The facilities, faculty, and staff at Caltech are ideal for completion of the proposed research and will contribute greatly to the candidate's overall development as an independent scientist. Independent (R00) research will focus on directed evolution of artificial metalloenzymes for in vivo palladium catalysis of pharmaceutically important cross-coupling reactions with potential applications in organic synthesis and bio-orthogonal diagnostics. Optimized metalloenzymes will also be expressed with additional enzymes in E. coli in order to biosynthesize biologically active molecules, including indolocarbazole natural product derivatives. Success in this venture would greatly expand the scope of molecules available via metabolic engineering and simplify the production of new compounds for the betterment of human health. This work will build directly on the candidate's experiences in the Arnold lab, and should foster the development of an exciting and collaborative research environment in the candidate's independent laboratory focusing on the development and application of enzymes for sustainable organic synthesis.

新合成分子在改善人类健康方面的实际应用直接取决于 这些化合物可以合成的效率，但这通常受到反应产量不佳的限制 在长的反应序列中，必须分离和纯化中间化合物。 代谢工程师已经证明，可以组装新的生物合成途径 在体内产生化学物质，在水性有氧环境中没有分离中间体，但是 序列仅限于自然酶催化的转化。该建议描述了设计， 组合化学范围的新型人工金属酶的准备和应用 以前所未有的方式与生物合成的效率催化，以产生异常分子 生物学重要性。所提出的系统比以前的人工提供了许多重要的优势 金属酶构建体，可用于体内催化和代谢工程。这个雄心勃勃 项目将作为候选人提高有机效率的长期目标的一部分进行 合成，特别是用于生物活性分子的生产。 在拟议研究的指导阶段（K99）中，具有催化活性palladacycle的氨基酸 侧链将合成，表征并掺入合适的支架蛋白中。催化 将使用多种C-C键形成反应评估所得的金属酶的活性。这 拟议的氨基酸催化剂可能会自身对各种应用非常有用，并且 它们将其纳入蛋白质将标志着UAA成立领域的重大成就， 具有潜在应用的生物催化范围远远超出了本应用的范围。这项研究将是 在蛋白质工程领域的领导者弗朗西斯·阿诺德（Frances Arnold）教授的实验室中进行 加利福尼亚理工学院，一家世界知名的研究机构。阿诺德教授的记录很强 作为行业和学术界成功成员的导师，她和候选人概述了 职业发展计划着重于指导，写作和研究，以确保候选人继续这一点 趋势。加州理工学院的设施，教职员工和工作人员非常适合完成拟议的研究，并将 为候选人作为独立科学家的整体发展做出了巨大贡献。 独立（R00）研究将重点放在体内人工金属酶的定向演化上 药物上重要的交叉偶联反应的钯催化与有机中的潜在应用 合成和生物正交诊断。优化的金属酶也将以额外的方式表示 大肠杆菌中的酶以生物合成生物学活性分子，包括吲哚可自然 产品衍生物。该合资企业的成功将大大扩大通过 代谢工程并简化了新化合物的生产，以改善人类健康。这 工作将直接基于候选人在阿诺德实验室的经验，并应促进发展 候选人的独立实验室中令人兴奋的协作研究环境，重点是 酶为可持续有机合成的开发和应用。