Microfluidic Systems to Enable Enzyme Engineering for Chemical Synthesis

项目总结 这项工作的总体目标是开发和应用一种液滴微流控系统，以促进快速 合成药物分子的酶工程学。新药的开发需要合成 从最初的先导化合物中提取复杂的分子进行测试。一旦一种药物被鉴定出来，有效的合成就是 临床试验和最终广泛生产所需的。传统上，这类合成使用的是基于金属的 催化剂。基于酶和细胞的系统提供了许多潜在的优势，包括更好的选择性 安装官能团，反应更绿色，催化剂更高效，毒性更低。创造生物催化剂 具有所需的选择性需要酶工程。酶工程的潜力体现在 2018年诺贝尔奖的颁发和制药商的吸收。酶工程需要 创造和分离数千种酶变异体，用变异体培养底物，筛选 反应活性的变异，以及进一步突变和进化的变异的鉴定。当前的方法 对于使用机器人技术的工程来说，井板和液-质联用是时间和 资源密集型，限制了其在药物化学中的应用。液滴微流体学具有深刻的 通过更快的速度和大幅减少的材料需求来改进酶工程。在……里面 这些方法，单个酶变体被包裹在液滴中，筛选产物形成，以及 根据信号进行排序。所需的低容量(&lt；10 nL/反应)和高吞吐量(超过1000 样本/S)是对当前井板方法的重大改进。然而，早期的演示 基于液滴微流体的酶工程对于生物催化剂的发展是不切实际的 关于荧光检测在筛查中的应用。我们建议创建液滴微流控酶工程系统 利用基于质谱仪(MS)的检测，提供了无标签和信息丰富的潜力 以高吞吐量进行筛分。在前期工作中，我们开发了“质量激活液滴分类”(MADS)。 它可以根据女士检测到的酶活性对体外表达的酶进行分类。我们将在此基础上 成就创造了一个多功能系统，具有先进的酶工程分析测量方法。 许多酶工程协议要求在微生物中表达这些变体，因此我们将 开发工具，允许单个微生物菌株在单个液滴中生长并由女士进行分类 依赖于MS对液滴的直接分析；然而，这排除了异构体的分离，并且可以 易受信号上的矩阵影响。我们将通过将液滴连接到快速LC-MS来扩展分析选择 和离子迁移率-MS，在MS检测之前提供异构体和基质的分离。该系统将用于 设计依赖于磷酸吡哆醛的酶，用于氨基酸底物的多样化和 介导分子间氧化C-H/C-H偶联反应形成C-C键的细胞色素P450。

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.