Development of a high throughput platform for screening directed evolution libraries

开发用于筛选定向进化文库的高通量平台

• 批准号: 10574429
• 负责人: Laura Margaret Sanchez
• 金额: $ 16.28万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10574429
• 关键词: Acids; Acoustics; Agonist; Amino Acid Substitution; Analytical Chemistry; Automation; Biochemical; Biochemical Reaction; Biochemistry; Biological Assay; Biology; Biotechnology; Cells; Chemicals; Chemistry; Chromatography; Confounding Factors (Epidemiology); Constitution; Constitutional; Consumption; Coupled; Cyclization; Deposition; Detection; Development; Dioxygenases; Directed Molecular Evolution; Enzymes; Escherichia coli; Evolution; Family; Foundations; Future; Gas Chromatography; Generations; Genes; Glutamate Receptor; High Pressure Liquid Chromatography; Homologous Gene; Image; Individual; Industrialization; Industry; Infrastructure; Isomerism; Kainic Acid; Laboratories; Lactones; Lead; Libraries; Mass Fragmentography; Mass Spectrum Analysis; Measurement; Metabolic Biotransformation; Methodology; Molecular Weight; Mutate; Mutation; Natural Products; Nature; Neurotoxins; Nuclear Magnetic Resonance; Outcome; Polymerase Chain Reaction; Process; Production; Proteins; Pyrrolidines; Rapid screening; Reporter; Research; Research Personnel; Sampling; Screening Result; Site; Specificity; Spectrometry; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spottings; Statistical Data Interpretation; Synthesis Chemistry; System; Techniques; Technology; Therapeutic; Time; Variant; Work; alpha ketoglutarate; analog; antagonist; assay development; directed differentiation; experimental study; genomic locus; high dimensionality; imaging software; innovation; ion mobility; mass spectrometric imaging; member; microbial; microbial colonization; mutant; non-Native; novel; screening; small molecule; tandem mass spectrometry; tool; vector

The controlled evolution of proteins in the laboratory is a valuable biomedical tool for accessing biomolecules for industrial, therapeutic and research applications. This process, also known as directed evolution, allows one to employ the specificity and selectivity that Nature imbues within its privileged biomolecules to construct unnatural products that would otherwise be inefficient or laborious to generate chemosynthetically. While this process is incredibly powerful, an existing bottleneck is the subsequent screening of the resulting variants for these high value products. The directed evolution process typically generates hundreds to thousands of mutants or library members for biochemical analysis. In some cases, fluorescent reporter systems or bioactivity assays can be employed as a general biochemical readout, however, this does not inform on specific chemical transformations towards diverse small molecule targets. When high value chemical products are the subject of these directed evolution experiments, researchers employ multiple orthogonal analytical techniques, including: high performance liquid chromatography (HPLC); gas chromatography (GC); mass spectrometry (MS); and nuclear magnetic resonance (NMR). This becomes time and infrastructure intensive when thousands of variants need to be evaluated; even if variants are pooled in curated groups, considerable effort is needed for chromatographic assessment. Additionally, many of these methodologies may not be sensitive or specific enough to necessitate detection of low titer production of the desired product(s). Based on these shortcomings of the screening platforms, we are proposing to leverage our labs’ existing strengths to develop a high-throughput, specific, and sensitive mass spectrometry platform to screen directed evolution libraries for bioactive chemical products without chromatographic separation. The McKinnie lab has expertise in synthetic chemistry and biochemistry and has specifically worked on the α-ketoglutarate-dependent dioxygenase enzyme to construct neuroactive kainic acid on the gram scale. The Sanchez lab has expertise in natural product discovery and mass spectrometry techniques such as imaging mass spectrometry and tandem mass spectrometry. These respective strengths will allow us to develop an innovative pipeline for screening thousands of directed evolution library members to prioritize variants that direct the chemistry towards kainoid-ring glutamate receptor agonists and antagonists. Our pipeline will allow for unprecedented measurements in chemical specificity and be broadly applicable for any groups looking to conduct directed evolution. ● Current directed evolution screening platforms are time-consuming or low throughput ● The combined expertise of our team is highly interdisciplinary ● Mass spectrometry and trapped ion mobility spectrometry allow for high dimensionality measurements directly from mutant colonies without reliance on chromatography techniques

实验室中蛋白质的受控演化是一种有价值的生物医学工具，用于访问生物分子 工业，治疗和研究应用。这个过程，也称为导向的进化，允许一个人 利用自然在其特权生物分子中浸泡的特异性和选择性来构建不自然的 否则将无法效率或实验室产生化学合成的产品。虽然这个过程是 现有的瓶颈非常强大，是对这些高的变体的随后筛选 价值产品。定向的进化过程通常会产生数百个突变体或库 生化分析的成员。在某些情况下，荧光记者系统或生物活性测定可能是 但是，用作一般的生化读数，但这并不是特定化学转化的信息 朝向潜水员小分子靶标。当高价值化学产品是这些定向的主题时 进化实验，研究人员员工多种正交分析技术，包括：高 性能液相色谱（HPLC）；气相色谱（GC）；质谱法（MS）；和核 磁共振（NMR）。当成千上万的变体需要时，这将成为时间和基础设施密集型 要评估；即使将变体组合成策划的组，色谱也需要大量精力 评估。此外，这些方法中的许多方法可能对必要 检测所需产品的低滴度产生。基于筛选的这些缺点 平台，我们建议利用实验室的现有优势来发展高通量， 特定且敏感的质谱平台，用于筛选生物活性的定向演化库 没有色谱分离的化学产品。麦金尼实验室具有合成方面的专业知识 化学和生物化学，并专门用于α-酮戊二酸依赖性双氧酶 以克尺度构建神经活性海藻酸。桑切斯实验室在天然产品方面具有专业知识 发现和质谱技术，例如成像质谱和串联质量 光谱法。这些相对优势将使我们能够开发创新的管道来筛选数千个 定向演化库成员的优先级，将化学反应指向Kainoid-Chring 谷氨酸接收器激动剂和拮抗剂。我们的管道将允许在 化学特异性，并广泛适用于任何希望进行定向进化的群体。 ●当前的有向演进筛选平台是耗时或低吞吐量的 ●我们团队的组合专业知识是高度跨学科的 ●质谱和被困的离子迁移率光谱法可以提高高维度 直接从突变菌落中进行测量，而无需放松色谱技术