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。这些分子或类似物中的许多是各种产物中的中心成分，但是这些分子的多样性受到天然酶催化的反应范围的限制。产生人工冶金酶（AME）的工程细胞将扩大可能的反应范围。新型的生物合成途径将通过与AMES补充天然酶来创建。研究生和博士后同伴将在这个合成化学和合成生物学的融合领域接受培训。 外展活动涵盖了与人工生物合成有关的主题，将向服务不足的高中生和老师以及K-8学生提供。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和初步结果的一般概念上，显示在细菌中创建这些途径的可行性。我们将增加可以托管人工金属酶催化的化学物质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；扩展细胞内掺入AME的金属构件的类型，以增加这些途径中不自然反应的范围；并将非生物化学与自然生物合成结合在不同的序列中。具体而言，我们将1）将AME引入链霉菌菌株中，并在异源产生的萜烯和聚酮化合物上进行测试。 2）将新的辅助因子纳入大肠杆菌和链霉菌表达的AME； 3）扩大了AME在人工生物合成途径中催化的转化范围，以包含非生物C-H键功能化； 4）创建在人工生物合成中间发生不自然化学的途径； 5）阐明了含有重氮的小分子的途径。通过这样做，我们将产生基本的知识并展示指导原则，以创建人工生物合成途径，这些途径将简单的碳源转换为全体微生物中有价值的不自然产品。该奖项反映了NSF的法定任务，并被认为是通过该基金会的知识分子优点和广泛影响的评估来评估的。

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