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

基本信息

批准号：
7787792
负责人：
JARED C LEWIS
金额：
$ 9万
依托单位：
CALIFORNIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-02-01 至 2010-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7787792
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 Development Development Plans Diagnostic Discipline Drug Industry Engineering Ensure Environment Enzymes Escherichia coli Faculty Fostering 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 public health relevance success trend

项目摘要

DESCRIPTION (provided by applicant): 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. Public Health Relevance: The research outlined in this proposal has the potential to greatly improve public health by creating a new class of artificial metalloenzymes for the synthesis biologically active molecules. This platform will enable inclusion of powerful transition metal catalysts in metabolic pathways in unprecedented fashion in order to efficiently produce chemicals in vivo.

描述（由申请人提供）：用于改善人类健康的新合成分子的实际应用直接取决于这些化合物的合成效率，但这通常受到整个长反应序列中反应收率差的限制，其中中间化合物必须进行分离和纯化。代谢工程师已经证明，可以组装新的生物合成途径，以便在体内产生化学物质，而无需在水性有氧环境中分离中间体，但这些序列仅限于天然酶催化的转化。该提案描述了新型人造金属酶的设计、制备和应用，该酶以前所未有的方式将化学催化范围与生物合成效率结合起来，产生具有特殊生物学重要性的分子。所提出的系统比以前的人工金属酶结构具有许多显着的优点，使其能够用于体内催化和代谢工程。这个雄心勃勃的项目将作为候选人提高有机合成效率的长期目标的一部分，特别是生物活性分子的生产。在拟议研究的指导阶段（K99），将合成、表征具有催化活性环环侧链的氨基酸，并将其整合到合适的支架蛋白中。所得金属酶的催化活性将使用各种 C-C 键形成反应进行评估。所提出的氨基酸催化剂本身可以被证明对各种应用非常有用，并且它们与蛋白质的结合将标志着 UAA 结合和生物催化领域的重大成就，其潜在应用远远超出了本应用的范围。这项研究将在世界著名研究机构加州理工学院蛋白质工程领域领军人物 Frances Arnold 教授的实验室中进行。阿诺德教授作为工业界和学术界成功人士的导师有着良好的记录，她和候选人制定了一份侧重于指导、写作和研究的职业发展计划，以确保候选人继续这一趋势。加州理工学院的设施、教职员工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巨大贡献。独立（R00）研究将重点关注人工金属酶的定向进化，用于药学上重要的交叉偶联反应的体内钯催化，并在有机合成和生物正交诊断方面具有潜在的应用。优化的金属酶也将在大肠杆菌中用其他酶表达，以生物合成生物活性分子，包括吲哚并咔唑天然产物衍生物。这项事业的成功将极大地扩大代谢工程可用分子的范围，并简化新化合物的生产，以改善人类健康。这项工作将直接建立在候选人在阿诺德实验室的经验基础上，并应促进候选人独立实验室中令人兴奋的协作研究环境的发展，重点关注可持续有机合成酶的开发和应用。公共健康相关性：该提案中概述的研究有可能通过创建一类用于合成生物活性分子的新型人造金属酶来极大地改善公共健康。该平台将以前所未有的方式将强大的过渡金属催化剂纳入代谢途径，以便在体内有效地生产化学物质。