Transition Metal Catalysis and Metabolic Engineering using Artificial Metalloenzy

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合成这些化合物的效率，但这经常受到必须分离和纯化中间体化合物的整个长反应序列中的不良反应产率的限制。代谢工程师已经证明，可以组装新的生物合成途径，以在体内产生化学品，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。该提案描述了一类新的人工金属酶的设计、制备和应用，其以前所未有的方式将化学催化的范围与生物合成的效率相结合，以产生具有特殊生物重要性的分子。所提出的系统提供了一些显着的优势，比以前的人工金属酶的结构，使其在体内催化和代谢工程的使用。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进行，特别是用于生产生物活性分子。 在拟议研究的指导阶段（K99），将合成具有催化活性的palladaclide侧链的氨基酸，表征并将其纳入合适的支架蛋白中。将使用各种C-C键形成反应来评价所得金属酶的催化活性。所提出的氨基酸催化剂可以证明其本身对于各种应用是非常有用的，并且它们掺入蛋白质中将标志着UAA掺入和生物催化领域中的重大成就，其潜在应用远远超出本申请的范围。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的领军人物Frances Arnold教授的实验室进行。阿诺德教授作为工业界和学术界成功成员的导师有着良好的记录，她和候选人概述了一个职业发展计划，重点是导师，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，教师和工作人员是完成拟议研究的理想选择，并将大大有助于候选人作为独立科学家的整体发展。 独立（R00）研究将集中在人工金属酶的定向进化，用于体内钯催化药学上重要的交叉偶联反应，在有机合成和生物正交诊断中具有潜在的应用。优化的金属酶也将与另外的酶一起在E.大肠杆菌，以便生物合成生物活性分子，包括吲哚并咔唑天然产物衍生物。这项事业的成功将大大扩大通过代谢工程获得的分子的范围，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，并应促进在候选人的独立实验室的一个令人兴奋的和协作的研究环境的发展，重点是酶的开发和应用可持续有机合成。 公共卫生相关性：该提案中概述的研究有可能通过创造一类新的人工金属酶来合成生物活性分子，从而大大改善公众健康。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，以便在体内有效地产生化学物质。

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.