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）：新合成分子在改善人类健康方面的实际应用直接取决于可以合成这些化合物的效率，但这通常受到在长期反应序列中必须隔离中间化合物必须隔离并纯化并纯化的长反应序列的较差的反应产量的限制。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，而在水性有氧环境中没有分离中间体，但是这些序列仅限于由天然酶催化的转化。该提案描述了一种新型的人工金属酶的设计，制备和应用，这些人工金属酶将化学催化的范围与生物合成的效率相结合，以前所未有的方式产生具有非凡生物学重要性的分子。拟议的系统比以前的人造金属酶构建体具有许多重要的优势，这使其可以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，特别是用于生物活性分子的生产。在拟议研究的指导阶段（K99）中，将合成，表征和掺入具有催化活性的palladacycle侧链的氨基酸中，并将其掺入合适的脚手架蛋白中。将使用多种C-C键形成反应评估所得金属酶的催化活性。所提出的氨基酸催化剂可能会自身对各种应用非常有用，并且将其掺入蛋白质将标志着UAA掺入和生物催化领域的重大成就，并具有远远超出本应用范围的潜在应用。这项研究将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，加利福尼亚州理工学院是一家世界知名的研究机构。阿诺德教授作为工业和学术界成功成员的指导者有着很强的记录，她和候选人概述了一项职业发展计划，重点是指导，写作和研究，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巨大贡献。独立（R00）的研究将集中于人工金属酶的定向演化，用于在体内钯催化，对有机合成和生物正交诊断中的潜在应用，药物上重要的交叉偶联反应。优化的金属酶还将用大肠杆菌中的其他酶表达，以使生物合成生物学活性分子，包括吲哚可核酸甲氮烷基天然产物衍生物。在这项合资企业中的成功将大大扩大通过代谢工程可用的分子范围，并简化新化合物的生产，以改善人类健康。这项工作将直接基于候选人在阿诺德实验室的经验，并应促进候选人独立实验室的激动人心和协作研究环境的发展，重点是开发和应用酶以用于可持续的有机合成。公共卫生相关性：该提案中概述的研究有可能通过为合成具有生物活性分子的新型人工金属酶来大大改善公共卫生。该平台将以前所未有的方式将强大的过渡金属催化剂包含在代谢途径中，以便在体内有效生产化学物质。