Directed Evolution of a Glycosynthase Via Chemical Complementation

在化学系有机动力学项目的支持下，弗吉尼亚W。哥伦比亚大学的康沃尔语将开发一种通用的、高通量的酶催化分析方法，使定向进化应用于广泛的化学反应。在上一个授予期间，康沃尔语实验室开发了一个通用的，高通量的酶催化分析的基础上，酵母三杂交测定（化学互补），应允许定向进化应用于广泛的化学反应。该测定基于体内两个小分子配体的共价偶联检测键形成或键裂解反应的酶催化。异二聚体配体重建转录激活因子，开启报告基因的转录。键形成被检测为必需报告基因的激活;键断裂，有毒报告基因的抑制。该测定是高通量的，因为它可以作为生长选择运行，其中仅含有功能酶的细胞存活。该测定可以容易地扩展到新的化学方法，简单地通过合成具有不同底物作为化学接头的二聚体配体。在这里，它提出了应用这种测定法的定向进化的糖苷酶，酶，可用于碳水化合物的合成。碳水化合物仍然是少数几类仍然难以用现代合成方法合成的天然产物之一。酶可以提供一个明显的替代碳水化合物的合成，因为它们的控制区域和立体化学。在初步结果中，康沃尔语教授已经表明，化学互补可以使用LEU 2生长选择检测已知糖苷酶变体的糖苷合酶活性。本研究的长期目标是利用定向进化产生具有一系列碳水化合物合成底物特异性的糖合酶变体。该提议的直接目的是使用选择来改善酶的体内表达，以允许以制备规模使用糖合酶。有机和高分子化学计划支持教授弗吉尼亚W。哥伦比亚大学的康沃尔语的研究利用定向进化有可能使常规产生具有新功能的蛋白质成为可能，这些蛋白质可用作化学合成和生物医学研究的试剂，用于化学产品，甚至用作治疗剂。这项研究也是理想的培养学生在化学和生物学的接口工作。这些项目涉及合成化学、蛋白质化学和酵母遗传学。除了培养PhD学生，这些项目是用来介绍本科生通过NSF-REU计划和哥伦比亚大学GSAS少数民族本科生暑期研究计划的实验室研究。

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.