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

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

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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.

项目摘要这项工作的总体目的是开发和应用液滴微流体系统，以促进快速酶的工程以合成药物分子。新药物的开发需要合成从初始铅化合物进行测试的复杂分子。一旦确定了药物，有效的合成是需要进行临床试验，并最终广泛产生。传统上，这样的合成利用了基于金属的合成催化剂。酶和基于细胞的系统具有许多潜在的优势，包括更高的选择性安装功能组，更绿色的反应，更有效的催化剂和低毒性。创建生物催化剂有了理想的选择性，需要酶工程。酶工程的潜力在授予2018年诺贝尔奖，并由制药商的吸收。酶工程需要创建和隔离数千种酶变体，与变体的底物孵育，筛选反应活性的变体以及鉴定用于进一步突变和进化的变体。当前方法用于使用机器人技术的工程，井板和液相色谱质量光谱法是时间和资源密集型，因此限制了医学化学中的使用。液滴微流体具有深刻的潜力通过更高的速度和大幅减少的材料需求来改善酶工程。在这些方法，单个酶变体被封装到液滴中，筛选以形成产品，并且根据信号进行分类。所需的低体积（<10 nl/反应）和高通量（超过1000 样本/s是对当前固定板方法的显着改进。但是，早期的演示液滴微流体的酶工程对于由于浮雕而对生物催化剂的发展不切实际筛选中的荧光检测。我们建议创建液滴微流体酶工程系统利用质谱（MS）的检测，提供了无标签和信息丰富的潜力屏幕高吞吐量。在初步工作中，我们开发了“大规模激活的液滴分类”（MADS）可以根据MS检测到的活性在体外表达的酶进行排序。我们将在此基础上建立成就创建具有高级分析测量酶工程的多功能系统。许多酶工程协议都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液滴生长单个微生物菌株，并通过MS分类。先前的工作通过MS对液滴的直接分析；但是，这排除了异构体的分离，可以是容易受到信号的矩阵影响。我们将通过将液滴连接到快速LC-MS来扩展分析选项在MS检测之前，可以提供异构体和矩阵的离子迁移率ms。该系统将用于工程师吡啶还毒素依赖性酶，用于多样化氨基酸底物和介导分子氧化物C – H/CH偶联反应的细胞色素P450s形成C – C键。