Biosynthetic Pathways with Artificial Metalloenzymes

微生物可以制造具有复杂结构的有机分子。这些分子或其类似物中的许多是各种产品中的核心成分，但这些分子的多样性受到天然酶催化的反应范围的限制。工程细胞生产人工金属酶（艾姆斯）将扩大可能的反应范围。新的生物合成途径将通过补充天然酶与艾姆斯。研究生和博士后助理将在合成化学和合成生物学的这个融合领域进行培训。 将向服务不足的高中学生和教师以及K-8学生提供涵盖人工生物合成相关主题的外联活动。该项目将建立在创建含有人工金属酶的人工生物合成途径的一般概念和初步结果的基础上，这些结果显示了在细菌中创建这些途径的可行性。我们将增加微生物的数量和类型，这些微生物可以承载人工金属酶催化的化学反应，以扩大与艾姆斯反应的天然产物的范围;扩大细胞内掺入艾姆斯的金属辅因子的类型，以增加这些途径中的非天然反应的范围;并以不同的顺序将非生物化学与天然生物合成联合收割机结合起来。具体而言，我们将1）将艾姆斯引入链霉菌菌株中并测试对异源产生的萜烯和聚酮化合物的活性; 2）将新的辅因子引入在E.大肠杆菌和链霉菌; 3）拓宽人工生物合成途径中由艾姆斯催化的转化的范围以涵盖非生物C-H键官能化; 4）创建其中非天然化学发生在人工生物合成中间的途径;以及5）阐明含重氮小分子的途径。通过这样做，我们将产生基础知识，并展示指导原则，以创建人工生物合成途径，将简单的碳源转化为整个微生物中有价值的非天然产物。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Microbes can make organic molecules with complex structures. Many of these molecules or their analogs are central ingredients in a variety of products, but the diversity of these molecules is limited by the range of the reactions catalyzed by natural enzymes. Engineering cells to produce artificial metalloenzymes (AMEs) will expand the range of possible reactions. Novel biosynthetic pathways will be created by complementing natural enzymes with AMEs. Graduate students and postdoctoral associates will be trained in this convergent field of synthetic chemistry and synthetic biology. Outreach activities to encompass topics related to artificial biosynthesis will be offered to underserved high school students and teachers, as well as to K-8 students. This project will build upon the general concept of creating artificial biosynthetic pathways containing artificial metalloenzymes and preliminary results showing the feasibility of creating these pathways in bacteria. We will increase the numbers and types of microorganisms that can host the chemistry catalyzed by artificial metalloenzymes to expand the range of natural products that react with AMEs; expand the types of metallo-cofactors that are incorporated intracellularly into AMEs to increase the scope of unnatural reactions in these pathways; and combine the abiotic chemistry with natural biosynthesis in varying sequences. Specifically, we will 1) introduce AMEs into Streptomyces strains and test activity on heterologously produced terpenes and polyketides; 2) incorporate new cofactors into AMEs expressed in E. coli and Streptomyces; 3) broaden the scope of transformations catalyzed by AMEs in the artificial biosynthetic pathways to encompass abiotic C-H bond functionalizations; 4) create pathways in which the unnatural chemistry occurs in the middle of the artificial biosynthesis; and 5) elucidate the pathways for diazo-containing small molecules. By doing so, we will generate the fundamental knowledge and demonstrate guiding principles to create artificial biosynthetic pathways that convert simple carbon sources to valuable unnatural products in whole microorganisms.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.