Application of a high throughput platform for screening directed evolution libraries

家长奖摘要。实验室中蛋白质的受控进化是一种有价值的生物医学工具， 获取生物分子用于工业、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的特异性和选择性， 生物分子来构建非天然产物，否则产生非天然产物是低效或费力的 化学合成虽然这个过程非常强大，但现有的瓶颈是随后的 筛选这些高价值产品的所得变体。定向进化过程通常 产生数百至数千个突变体或文库成员用于生化分析。在某些情况下， 荧光报告系统或生物活性测定可用作一般的生物化学读出， 然而，这并不能说明向不同小分子靶的特定化学转化。 当高价值的化学产品成为这些定向进化实验的对象时， 采用多种正交分析技术，包括：高效液相色谱法（HPLC）; 气相色谱法（GC）;质谱法（MS）;和核磁共振（NMR）。这成为 当需要评估数千个变体时，需要大量的时间和基础设施;即使变体被合并， 在精选组中，色谱评估需要相当大的努力。此外，其中许多 方法学可能不够灵敏或特异，不需要检测低滴度的 所需产品（S）。基于筛查平台的这些缺点，我们建议利用我们的 利用实验室现有的优势，开发高通量、特异性和灵敏度的质谱分析平台， 筛选定向进化文库中的生物活性化学产品，而无需色谱分离。的 McKinnie实验室在合成化学和生物化学方面拥有专业知识，并专门研究α- 酮戊二酸依赖性双加氧酶来构建克级的神经活性红藻氨酸。的 桑切斯实验室拥有天然产物发现和质谱技术的专业知识， 质谱法和串联质谱法。这些各自的优势将使我们能够发展一个 用于筛选数千个定向进化文库成员的创新管道， 将化学导向类海人酸环谷氨酸受体激动剂和拮抗剂。我们的管道将允许 用于前所未有的化学特异性测量，并广泛适用于任何寻求 进行定向进化。 ·目前的定向进化筛选平台耗时或低通量 ·我们团队的综合专业知识是高度跨学科的 ·质谱和捕获离子迁移谱允许高维测量 直接从突变菌落中分离，而不依赖色谱技术

Parent award abstract. 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