Biosynthetic Pathways with Artificial Metalloenzymes

微生物可以制造具有复杂结构的有机分子。其中许多分子或其类似物是各种产品的核心成分，但这些分子的多样性受到天然酶催化的反应范围的限制。工程细胞生产人造金属酶(Ames)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和AMES来创建。研究生和博士后助理将接受合成化学和合成生物学这一融合领域的培训。将向服务不足的高中生和教师以及K-8年级的学生提供涵盖与人工生物合成有关的主题的外展活动。该项目将建立在创建包含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，该结果表明在细菌中创建这些途径的可行性。我们将增加能够承载人工金属酶催化化学的微生物的数量和类型，以扩大与Ames反应的天然产物的范围；扩大细胞内整合到Ames中的金属辅因子的类型，以增加这些途径中非自然反应的范围；并以不同的顺序将非生物化学与自然生物合成结合起来。具体地说，我们将1)将Ames引入链霉菌菌株，并测试异源产生的萜类和聚酮类化合物的活性；2)在大肠杆菌和链霉菌中表达的Ames中加入新的辅因子；3)扩大Ames在人工生物合成途径中催化转化的范围，包括非生物C-H键功能化；4)创建在人工生物合成过程中发生非自然化学作用的途径；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在整个微生物中创建人工生物合成途径的指导原则，这些途径将简单的碳源转化为有价值的非天然产品。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.