Biosynthetic Pathways with Artificial Metalloenzymes

人工金属酶的生物合成途径

• 批准号: 2027943
• 负责人: John Hartwig
• 金额: $ 158.98万
• 依托单位: University of California-Berkeley
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-10-01 至 2024-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2027943&HistoricalAwards=false
• 关键词: Biosynthetic; Pathways; Artificial; Metalloenzymes

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.

微生物可以制造具有复杂结构的有机分子。许多这些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反应范围的限制。对细胞进行工程改造以生产人工金属酶（AME）将扩大可能的反应范围。通过用 AME 补充天然酶，将创建新的生物合成途径。研究生和博士后将在合成化学和合成生物学这一融合领域接受培训。 将向服务不足的高中生和教师以及 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总体概念之上，以及显示在细菌中创建这些途径的可行性的初步结果。我们将增加能够承载人工金属酶催化化学反应的微生物的数量和类型，以扩大与 AME 反应的天然产物的范围；扩大细胞内整合到 AME 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；并将非生物化学与自然生物合成以不同的顺序结合起来。具体来说，我们将1）将AME引入链霉菌菌株并测试异源产生的萜烯和聚酮化合物的活性； 2) 将新的辅因子整合到大肠杆菌和链霉菌中表达的AME中； 3) 扩大人工生物合成途径中 AME 催化的转化范围，以涵盖非生物 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的途径； 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智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Complete integration of carbene-transfer chemistry into biosynthesis

• DOI: 10.1038/s41586-023-06027-2
• 发表时间: 2023-05
• 期刊: Nature
• 影响因子: 64.8
• 作者: Jing Huang;A. Quest;Pablo Cruz-Morales;Kai Deng;J. H. Pereira;Devon Van Cura;Ramu Kakumanu;E. Baidoo;Q. Dan;Yan Chen;C. Petzold;T. Northen;Paul D. Adams;D. Clark;E. Balskus;J. Hartwig;A. Mukhopadhyay;J. Keasling