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High-Throughput Analysis and Evolution of Stereoselective Enzymes using Flow Cytometry

使用流式细胞术对立体选择性酶进行高通量分析和进化

基本信息

批准号：
1904885
负责人：
Jennifer Heemstra
金额：
$ 39万
依托单位：
Emory University
依托单位国家：
美国
项目类别：
Continuing Grant
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2022-06-30
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1904885&HistoricalAwards=false
关键词：
Throughput Analysis Evolution Stereoselective Enzymes

项目摘要

Enzymes are among the most efficient catalysts known, and they are responsible for nearly all the reactions that make life possible. The capability of enzymes to catalyze chemical transformations can also be harnessed to provide environmentally benign routes for the synthesis of pharmaceuticals and other high-value chemicals. However, naturally occurring enzymes must be honed to function in these non-natural reactions. Key to augmenting enzyme capabilities is the development of fast, high-fidelity sensing tools that measure the function of enzymes in large numbers. With support from the Chemical Measurement and Imaging Program in the Division of Chemistry and partial co-funding from the Biosensing Program in the Division of Chemical, Bioengineering, Environmental and Transport Systems at NSF, Dr. Jennifer Heemstra at Emory University uses a high-speed sequential platform to measure the function of millions of different modified versions of naturally occurring enzymes generated by her team, thereby allowing for identification of those enzymes best suited to important non-natural reactions. This project is developing a user-friendly platform that can be widely applied to discover new enzymes for constructing these high-value chemicals. Integrated with this research, Dr. Heemstra develops and broadly disseminates multi-media resources that help to prepare the future workforce in science, technology, engineering, and mathematics. Specifically, these resources provide information and advice on professional skills, including oral and written communication, pioneering new research ideas, promoting diversity and inclusion, and professional networking. These resources are freely available to the general public and utilized by students and researchers nationwide.Dr. Jennifer Heemstra at Emory University is using enantiomeric DNA biosensors to enable high-throughput enantiopurity measurement as a broadly applicable approach to biocatalyst discovery. DNA biosensors are powerful in their ability to transduce the presence of a specific small-molecule target into a dose-dependent fluorescence output. Dr. Heemstra utilizes sensors comprised of each of the two enantiomers of DNA to quantify the concentrations of the two enantiomers of a target small molecule that are produced as a result of an enzymatic reaction. This analysis is integrated with droplet microfluidic technology to enable screening and sorting by flow cytometry. As a result, large libraries of enzyme variants can be rapidly screened, and those having the desired levels of stereoselectivity isolated, thereby accelerating the discovery of new biocatalysts for the synthesis of pharmaceuticals and other high-value chemicals. This research is integrated with an undergraduate curriculum in which students learn how to read and analyze the primary literature and craft original research proposals, as well as publicly available resources to promote the professional development of early-career researchers.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.

酶是已知的最有效的催化剂之一，它们负责几乎所有使生命成为可能的反应。酶催化化学转化的能力也可以用来为药物和其他高价值化学品的合成提供环境友好的途径。然而，天然存在的酶必须经过磨练才能在这些非天然反应中发挥作用。增强酶能力的关键是开发快速、高保真的传感工具，以大量测量酶的功能。在化学部化学测量和成像计划的支持下，以及NSF化学，生物工程，环境和运输系统部生物传感计划的部分共同资助下，埃默里大学的Jennifer Heemstra博士使用高速连续平台来测量她的团队产生的数百万种不同修饰版本的天然酶的功能，从而允许鉴定那些最适合于重要的非天然反应的酶。该项目正在开发一个用户友好的平台，可广泛应用于发现用于构建这些高价值化学品的新酶。与这项研究相结合，Heemstra博士开发并广泛传播多媒体资源，帮助培养未来的科学，技术，工程和数学人才。具体而言，这些资源提供专业技能方面的信息和建议，包括口头和书面沟通，开拓新的研究思路，促进多样性和包容性，以及专业网络。埃默里大学的Jennifer Heemstra博士正在使用对映体DNA生物传感器来实现高通量对映体纯度测量，作为一种广泛适用的生物催化剂发现方法。DNA生物传感器在其将特定小分子靶标的存在转化为剂量依赖性荧光输出的能力方面是强大的。Heemstra博士利用由DNA的两种对映异构体组成的传感器来定量酶促反应产生的靶小分子的两种对映异构体的浓度。该分析与液滴微流控技术相结合，以通过流式细胞术进行筛选和分选。因此，可以快速筛选酶变体的大型文库，并分离具有所需立体选择性水平的那些，从而加速发现用于合成药物和其他高价值化学品的新生物催化剂。该研究与本科课程相结合，学生学习如何阅读和分析主要文献，制作原创研究提案，以及公共资源，以促进早期职业研究人员的专业发展。该奖项反映了NSF的法定使命，并被认为值得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来支持。