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 工业、治疗和研究应用。这个过程，也被称为定向进化，允许一个人 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 用化学合成方法生产效率低或费力的产品。虽然这个过程是 令人难以置信的强大，现有的瓶颈是随后对这些高表达的结果变体进行筛选 超值产品。定向进化过程通常会产生成百上千个突变体或文库 用于生化分析的成员。在某些情况下，荧光报告系统或生物活性分析可以 然而，作为一般的生化读数，这并不能反映特定的化学变化。 朝向不同的小分子目标。当高价值的化学产品是这些指令的主题时 在进化实验中，研究人员使用了多种正交分析技术，包括：High 高效液相色谱(HPLC)；气相色谱(GC)；质谱学(MS)；以及核 磁共振(核磁共振)。当数以千计的变种需要时，这将变得时间和基础设施密集 待评估；即使变异体被集中在精心策划的组中，层析也需要相当大的努力 评估。此外，这些方法中的许多可能不够敏感或不够具体，不足以 检测所需产品的低效价生产(S)。基于这些缺点的筛选 平台，我们建议利用我们实验室的现有优势开发一种高通量、 筛选生物活性定向进化文库的特异、灵敏的质谱学平台 未经层析分离的化工产品。麦金尼实验室在合成方面拥有专业知识 化学和生物化学，专门研究依赖于α-酮戊二酸的双加氧酶 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 发现和质谱学技术，如成像质谱学和串联质谱仪 光谱分析。这些各自的优势将使我们能够开发一种创新的渠道，对数千人进行筛查 对定向进化文库成员进行优先排序，以确定将化学作用引向海人藻类环的变体 谷氨酸受体激动剂和拮抗剂。我们的管道将允许史无前例的 具有化学特异性，广泛适用于任何希望进行定向进化的群体。 目前的定向进化筛选平台存在耗时或吞吐量低的问题 我们团队的专业知识是高度跨学科的 质谱学和俘获离子迁移率光谱允许高维 直接从突变菌落进行测量，而不依赖于层析技术