Directed Evolution of a Glycosynthase Via Chemical Complementation

Columbia University, New York NY

Investigators

Abstract

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.