Microfluidic Systems to Enable Enzyme Engineering for Chemical Synthesis

微流体系统使酶工程能够用于化学合成

基本信息

批准号：
10715356
负责人：
ROBERT T KENNEDY
金额：
$ 47.32万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10715356
关键词：
Acceleration Achievement Amino Acids Award Cells Chemical Engineering Chromatography Clinical Trials Complex Coupling Cytochrome P450 DNA Detection Development Encapsulated Engineering Enzymes Escherichia coli Evaluation Evolution Fluorescence Genetic Transcription Goals In Vitro Incubated Individual Industrialization Investigation Isomerism Label Lead Manufacturer Mass Spectrum Analysis Measurement Mediating Medical Medicine Metals Methods Microbe Microfluidics Miniaturization Mutation Nobel Prize Pharmaceutical Chemistry Pharmaceutical Preparations Pharmacologic Substance Preparation Process Production Protocols documentation Pyridoxal Phosphate Reaction Resources Rhodium Robotics Sampling Signal Transduction Sorting Spectrometry, Mass, Electrospray Ionization Speed Structure-Activity Relationship System Technology Testing Time Toxic effect Translations Variant Work Yeasts advanced analytics catalyst cellular engineering chemical synthesis cost drug synthesis experimental study functional group improved ion mobility ionization liquid chromatography mass spectrometry nanolitre novel screening tool uptake

项目摘要

PROJECT SUMMARY The overall objective of this work is to develop and apply a droplet microfluidic system to facilitate rapid engineering of enzymes to synthesize drug molecules. Development of new medicines requires synthesis of complex molecules from initial lead compounds for testing. Once a drug is identified, efficient synthesis is needed for clinical trials and ultimately widespread production. Traditionally such syntheses utilize metal-based catalysts. Enzyme and cell-based systems offer numerous potential advantages including greater selectivity in installing functional groups, greener reactions, more efficient catalysts, and low toxicity. Creating biocatalysts with the desired selectivity requires enzyme engineering. The potential of enzyme engineering is seen in the awarding of a Nobel Prize in 2018 and uptake by pharmaceutical manufacturers. Enzyme engineering requires creation and isolation of thousands of enzyme variants, incubation of substrates with variants, screening of the variants for reaction activity, and identification of variants for further mutation and evolution. Current methods for engineering that use robotics, well plates, and liquid chromatography-mass spectrometry are time and resource intensive thus limiting the use in medicinal chemistry. Droplet microfluidics has potential to profoundly improve enzyme engineering through greater speed and substantially reduced materials requirements. In these methods, individual enzyme variants are encapsulated into droplets, screened for product formation, and sorted based on signal. The low volumes required (< 10 nL/reaction) and high-throughput (over 1000 samples/s) are dramatic improvements over current well-plate methods. However, early demonstrations of enzyme engineering by droplet microfluidics are impractical for development of biocatalysts due to a reliance on fluorescence detection in screening. We propose to create droplet microfluidic enzyme engineering systems that utilize mass spectrometry (MS)-based detection, offering the potential for label-free and information-rich screens at high throughput. In preliminary work, we have developed “mass-activated droplet sorting” (MADS) which can sort enzymes expressed in vitro based on their activity detected by MS. We will build on this achievement to create a versatile system with advanced analytical measurements for enzyme engineering. Many enzyme engineering protocols call for expressing the variants in microbes, therefore we will develop tools to allow individual microbe strains to be grown in single droplets and sorted by MS. Prior work has relied on direct analysis of droplets by MS; however, this precludes separations of isomers and can be vulnerable to matrix effects on signal. We will expand analytical options by interfacing droplets to rapid LC-MS and ion mobility-MS to offer separations of isomers and matrix before MS detection. The system will be used to engineer pyridoxal phosphate-dependent enzymes for the diversification of amino acid substrates and cytochrome P450s that mediate intermolecular oxidative C–H/C–H coupling reactions to form C–C bonds.

项目概要这项工作的总体目标是开发和应用液滴微流控系统以促进快速酶工程合成药物分子的开发需要合成一旦确定了药物，就可以从最初的先导化合物中提取复杂的分子进行测试。传统上，此类合成利用金属基材料进行临床试验和最终广泛生产。酶和基于细胞的系统提供了许多潜在的优势，包括更高的选择性。安装官能团、更环保的反应、更高效的催化剂以及低毒性的生物催化剂。具有所需的选择性需要酶工程酶工程的潜力可见于。 2018 年诺贝尔奖的颁发和制药制造商的采用需要酶工程。创建和分离数千种酶变体、用变体孵育底物、筛选反应活性的变体以及进一步突变和进化的鉴定当前方法。对于使用机器人技术、孔板和液相色谱-质谱分析的工程来说，时间和资源密集，从而限制了液滴微流控技术的应用。通过提高速度和大幅减少材料需求来改进酶工程。这些方法将单个酶变体封装到液滴中，筛选产物形成，并根据信号进行排序。所需体积低（< 10 nL/反应）和高通量（超过 1000）。样本/秒）比当前的孔板方法有了显着的改进，但是，早期的演示。由于依赖，通过液滴微流体进行酶工程对于生物催化剂的开发是不切实际的我们建议创建液滴微流控酶工程系统。利用基于质谱 (MS) 的检测，提供无标记和信息丰富的潜力在前期工作中，我们开发了“质量激活液滴分选”（MADS）。它可以根据 MS 检测到的酶活性对体外表达的酶进行分类。成就为酶工程创建了具有先进分析测量的多功能系统。许多酶工程方案要求在微生物中表达变体，因此我们将开发工具以允许单个微生物菌株在单液滴中生长并通过 MS 进行分类。依赖于通过 MS 对液滴进行直接分析；然而，这排除了异构体的分离，并且可以我们将通过将液滴与快速 LC-MS 连接来扩展分析选项。和离子淌度 MS，在 MS 检测之前提供异构体和基质的分离。设计吡哆醛磷酸盐依赖性酶以实现氨基酸底物的多样化和细胞色素 P450 介导分子间氧化 C-H/C-H 偶联反应以形成 C-C 键。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Mass-Activated Droplet Sorting for the Selection of Lysine-Producing Escherichia coli.

用于选择产生赖氨酸的大肠杆菌的质量激活液滴分选。

DOI：
10.1021/acs.analchem.3c03080
发表时间：
2023-10-11
期刊：
Analytical chemistry
影响因子：
7.4
作者：
Emory M. Payne;Bridget E Murray;Laura I Penabad;Eric Abbate;R. T. Kennedy
通讯作者：
R. T. Kennedy

High-Throughput Liquid Chromatographic Analysis Using a Segmented Flow Injector with a 1 s Cycle Time.

使用循环时间为 1 秒的分段流动注射器进行高通量液相色谱分析。

DOI：
发表时间：
2023-11-21
期刊：
影响因子：
7.4
作者：
Makey, Devin M;Diehl, Roger C;Xin, Yue;Murray, Bridget E;Stoll, Dwight R;Ruotolo, Brandon T;Grinias, James P;Narayan, Alison R H;Lopez;Stark, Michaela;Johnen, Philipp;Kennedy, Robert T
通讯作者：
Kennedy, Robert T

High-Throughput Capillary Liquid Chromatography Using a Droplet Injection and Application to Reaction Screening.

使用液滴注射的高通量毛细管液相色谱及其在反应筛选中的应用。

DOI：
发表时间：
2024-03-19
期刊：
影响因子：
7.4
作者：
Xin, Yue;Foster, Samuel W;Makey, Devin M;Parker, Deklin;Bradow, James;Wang, Xiaochun;Berritt, Simon;Mongillo, Robert;Grinias, James P;Kennedy, Robert T
通讯作者：
Kennedy, Robert T