Transition Metal Catalysis and Metabolic Engineering using Artificial Metalloenzy

描述(申请人提供)：新的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的合成效率，但这往往受到整个长反应序列中反应产率较低的限制，在这些反应序列中，中间化合物必须被分离和提纯。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，而不需要在水中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。这项提案描述了一种新型人工金属酶的设计、制备和应用，这种酶以前所未有的方式结合了化学催化的范围和生物合成的效率，以产生具有特殊生物学意义的分子。与以前的人工金属酶构建相比，该系统提供了许多显著的优势，使其能够用于体内催化和代谢工程。这一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，特别是在生产生物活性分子方面。在拟议研究的指导阶段(K99)，将合成具有催化活性的软环侧链的氨基酸，对其进行表征，并将其并入合适的支架蛋白中。将使用各种C-C键形成反应来评估所生成的金属酶的催化活性。所提出的氨基酸催化剂本身可能被证明是非常有用的各种应用，它们被整合到蛋白质中将标志着UAA结合和生物催化领域的重大成就，潜在的应用远远超出了这一应用的范围。这项研究将在世界知名研究机构加州理工学院蛋白质工程领域的领军人物弗朗西斯·阿诺德教授的实验室进行。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，她和这位候选人概述了一项专注于导师、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、教师和工作人员是完成拟议研究的理想选择，并将为候选人作为一名独立科学家的整体发展做出巨大贡献。独立(R00)研究将集中在人工金属酶的定向进化，用于体内钯催化具有重要药学意义的交叉偶联反应，并可能在有机合成和生物正交诊断中应用。优化的金属酶还将在大肠杆菌中用额外的酶进行表达，以便生物合成生物活性分子，包括吲哚咔唑天然产物衍生物。这一项目的成功将极大地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基础上，并应促进候选人独立实验室专注于可持续有机合成酶的开发和应用的令人兴奋和合作的研究环境的发展。 公共健康相关性：这项提案中概述的研究有可能通过创造一种新的人造金属酶来合成生物活性分子，从而极大地改善公众健康。这一平台将使强大的过渡金属催化剂以前所未有的方式包含在代谢途径中，以便在体内高效地生产化学品。

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.