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 获取用于工业、治疗和研究应用的生物分子。这一过程，也称为 定向进化，允许一个人利用大自然赋予其特权的专一性和选择性 构建非天然产物的生物分子，否则将产生效率低下或费力的产品 化学合成的。虽然这一过程非常强大，但现有的瓶颈是后续 筛选这些高价值产品的最终变种。定向进化过程通常 产生数十万个用于生化分析的突变体或文库成员。在某些情况下， 荧光报告系统或生物活性分析可以用作一般的生化读数， 然而，这并不能说明针对不同小分子靶标的特定化学转化。 当高价值的化学产品是这些定向进化实验的对象时，研究人员 采用多种正交分析技术，包括：高效液相色谱法； 气相色谱(GC)、质谱仪(MS)和核磁共振(核磁共振)。这就变成了 需要评估数千种变体时的时间和基础设施密集型；即使将变体汇集在一起也是如此 在策划组中，需要付出相当大的努力来进行层析评估。此外，其中许多 方法学可能不够灵敏或特异，不需要检测低滴度生产的 理想产品(S)。基于筛查平台的这些缺点，我们建议利用我们的 Labs的现有优势，开发高通量、特异和灵敏的质谱学平台 无需层析分离筛选生物活性化工产品定向进化文库。这个 麦金尼实验室在合成化学和生物化学方面拥有专业知识，并专门致力于α- 酮戊二酸依赖的双加氧酶在克尺度上构建神经活性红藻氨酸。这个 桑切斯实验室在天然产物发现和成像等质谱学技术方面拥有专业知识 质谱学和串联质谱学。这些各自的优势将使我们能够开发出一种 用于筛选数千个定向进化库成员的创新流水线，以确定 将化学作用引向海人藻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性测量，并广泛适用于任何希望 进行定向进化。 ·目前的定向进化筛选平台耗时或吞吐量低 ·我们团队的综合专业知识是高度跨学科的 ·质谱学和俘获离子迁移率光谱分析允许进行高维测量 直接来自突变菌落，而不依赖于层析技术

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