Transition Metal Catalysis and Metabolic Engineering using Artificial Metalloenzy

新合成分子用于改善人类健康的实际应用直接取决于 这些化合物的合成效率很高，但这常常受到反应产率低的限制 在整个长的反应序列中，中间体化合物必须被分离和纯化。 代谢工程师已经证明，新的生物合成途径可以组装，以 在体内产生化学物质，在含水需氧环境中没有分离中间体，但这些 序列仅限于由天然酶催化的转化。本提案描述了设计， 制备和应用的一类新的人工金属酶，结合化学的范围， 催化与生物合成的效率在一个前所未有的方式，以产生特殊的分子 生物学重要性。所提出的系统提供了一些显着的优势，比以前的人工 金属酶构建体，这使得其能够用于体内催化和代谢工程。这一雄心勃勃 项目将作为候选人的长期目标的一部分，提高有机的效率进行 合成，特别是用于生产生物活性分子。 在建议研究的指导阶段（K99），具有催化活性钯的氨基酸 侧链将被合成、表征并掺入合适的支架蛋白中。催化 所得金属酶的活性将使用各种C-C键形成反应来评价。的 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 它们掺入蛋白质将标志着UAA掺入领域的重大成就， 具有远远超出本申请范围的潜在应用的生物催化。这项研究将是 在蛋白质工程领域的领导者Frances Arnold教授的实验室中进行， 加州理工学院，世界知名的研究机构。阿诺德教授有着良好的记录 作为工业界和学术界成功成员的导师，她和候选人概述了一个 职业发展计划侧重于指导，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，教师和工作人员是完成拟议研究的理想选择， 对候选人作为独立科学家的全面发展有很大贡献。 独立（R 00）研究将集中在人工金属酶的定向进化， 钯催化的药学上重要的交叉偶联反应及其在有机化学中的潜在应用 合成和生物正交诊断。优化的金属酶也将表达额外的 E.大肠杆菌，以生物合成生物活性分子，包括吲哚并咔唑天然 产品衍生品。这一冒险的成功将大大扩大分子的范围， 代谢工程和简化新化合物的生产，以改善人类健康。这 工作将直接建立在候选人在阿诺德实验室的经验，并应促进发展， 一个令人兴奋的和合作的研究环境，在候选人的独立实验室，重点是 可持续有机合成酶的开发和应用。

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.