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进化是获取生物分子的宝贵生物医学工具 这一过程也称为定向进化，可用于工业、治疗和研究。 利用大自然赋予其特权生物分子的特异性和选择性来构建非自然的 否则，通过化学合成方法生成的产品将效率低下或费力。 令人难以置信的强大，现有的瓶颈是随后筛选这些高的结果变体 定向进化过程通常会产生数百到数千个突变体或文库。 在某些情况下，可以使用荧光报告系统或生物活性测定。 然而，用作一般生化读数，这并不能提供特定化学转化的信息 当高价值化学产品是这些定向的主题时。 在进化实验中，研究人员采用了多种正交分析技术，包括： 高效液相色谱法（HPLC）；气相色谱法（MS）； 当需要数千个变体时，这会变得非常耗时和基础设施密集。 进行评估；即使将变体汇集到策划组中，也需要付出相当大的努力来进行色谱分析 此外，许多这些方法可能不够敏感或具体，不足以进行评估。 基于筛选的这些缺点来检测所需产物的低滴度生产。 平台，我们建议利用我们实验室的现有优势来开发高通量、 特异性、灵敏的质谱平台，用于筛选生物活性的定向进化文库 无需色谱分离的化学产品 McKinnie 实验室拥有合成方面的专业知识。 化学和生物化学，特别研究α-酮戊二酸依赖性双加氧酶 构建克级神经活性红藻氨酸 桑切斯实验室拥有天然产品方面的专业知识。 发现和质谱技术，例如成像质谱和串联质量 这些各自的优势将使我们能够开发出用于筛选数千种的创新管道。 定向进化库成员的优先顺序，将化学导向凯诺德环 我们的管道将允许在谷氨酸受体激动剂和拮抗剂方面进行前所未有的测量。 化学特异性并广泛适用于任何寻求进行定向进化的群体。 目前的定向进化筛选平台耗时或通量低 ● 我们团队的综合专业知识具有高度跨学科性 ● 质谱分析和俘获离子迁移谱分析可实现高维度 直接从突变菌落进行测量，无需依赖色谱技术