Directed Evolution of a Glycosynthase Via Chemical Complementation

通过化学互补进行糖合酶的定向进化

• 批准号: 0350183
• 负责人: Virginia Cornish
• 金额: $ 53.9万
• 依托单位: Columbia University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-15 至 2008-02-29
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0350183&HistoricalAwards=false
• 关键词: Directed; Evolution; Glycosynthase; Via; Chemical

With the support of the Organic Dynamics Program in the Chemistry Division, Professor Virginia W. Cornish of Columbia University will develop a general, high-throughput assay for enzyme catalysis that allows directed evolution to be applied to a broad range of chemical reactions. During the previous granting period, the Cornish laboratory developed a general, high throughput assay for enzyme catalysis based on the yeast three-hybrid assay (Chemical Complementation) that should allow directed evolution to be applied to a broad range of chemical reactions. This assay detects enzyme catalysis of bond formation or bond cleavage reactions based on covalent coupling of two small molecule ligands in vivo. The heterodimeric ligand reconstitutes a transcriptional activator, turning on transcription of a reporter gene. Bond formation is detected as activation of an essential reporter gene; bond cleavage, repression of a toxic reporter gene. The assay is high-throughput because it can be run as a growth selection where only the cells containing functional enzyme survive. The assay can be readily extended to new chemistry simply by synthesizing dimeric ligands with different substrates as chemical linkers. Here, it is proposed to apply this assay to the directed evolution of glycosynthases, enzymes that can be used for carbohydrate synthesis. Carbohydrates remain one of the few classes of natural products that are still difficult to synthesize with modern synthetic methods. Enzymes can provide an obvious alternative for carbohydrate synthesis because of their control of regio- and stereochemistry. In preliminary results, Professor Cornish has shown that Chemical Complementation can detect the glycosynthase activity of a known glycosidase variant using a LEU2 growth selection. The long-term goal of this research is to use directed evolution to generate glycosynthase variants with a range of substrate specificities for carbohydrate synthesis. The immediate objective of this proposal is to use the selection to improve the in vivo expression of the enzyme to allow for use of the glycosynthase on a preparative scale. The Organic and Macromolecular Chemistry Program supports Professor Virginia W. Cornish of Columbia University whose research using directed evolution has the potential to make it possible to routinely generate proteins with new functions for use as reagents for chemical synthesis and biomedical research, in chemical products, and even as therapeutics. This research also is ideal for training students to work at the interface of chemistry and biology. These projects involve synthetic chemistry, protein chemistry, and yeast genetics. In addition to training Ph.D. students, these projects are used to introduce undergraduates to laboratory research through an NSF-REU program and the Columbia University GSAS Summer Research Program for Minority Undergraduates.

在化学部的有机动力学计划的支持下，哥伦比亚大学的弗吉尼亚W. Cornish教授将开发一种用于酶催化的一般高通量测定法，允许将定向的进化应用于广泛的化学反应。在上一个授予期间，康沃尔实验室基于酵母的三杂化测定法（化学互补）开发了一种一般的高通量测定，用于酶催化，该测定法应允许将进化用于广泛的化学反应。该测定法检测基于体内两个小分子配体的共价偶联键形成或键裂解反应的酶催化。异二聚体配体重构转录激活剂，打开记者基因的转录。将键形成视为基本报告基因的激活。键裂解，抑制有毒的记者基因。该测定是高通量的，因为它可以作为生长选择运行，只有含有功能性酶的细胞才能存活。仅通过将不同底物作为化学接头的二聚体配体合成二聚体配体，可以轻松地扩展到新的化学。在这里，建议将该测定法应用于可用于碳水化合物合成的糖合成酶的定向演化。碳水化合物仍然是少数几类天然产品之一，它们仍然很难与现代合成方法合成。酶可以为碳水化合物合成提供明显的替代方法，因为它们对区域和立体化学的控制。在初步的结果中，康沃尔教授表明，化学互补可以检测使用LEU2生长选择的已知糖苷酶变体的糖合酶活性。这项研究的长期目标是使用定向进化来生成具有碳水化合物合成的一系列底物特异性的糖合酶变体。该提案的直接目的是使用选择来改善酶的体内表达，以便在制备量表上使用糖合酶。有机和大分子化学计划支持哥伦比亚大学的弗吉尼亚W. Cornish教授，其研究的研究有可能使具有新功能的蛋白质常规产生，以用作化学合成和生物医学研究的试剂，在化学产品中，甚至是治疗疗法。这项研究也是培训学生在化学和生物学界面工作的理想选择。这些项目涉及合成化学，蛋白质化学和酵母遗传学。除了培训博士学位学生，这些项目用于通过NSF-REU计划和哥伦比亚大学GSAS GSAS夏季研究计划为实验室研究介绍本科生。