The desosaminyl transferase, DesVII, from methymycin/pikromycin biosynthetic pathway of Streptomyces venezuelae, is proposed for use in in vitro glycosylation of polyketide libraries generated by combinatorial biosynthesis. Preliminary genetic studies have demonstrated that DesVII has unusually flexible specificity toward both polyketide aglycone and deoxy sugar substrates. The overall goal of this project is to develop an in vitro glycosylation technology using DesVII. The recombinant DesVII enzyme will be purified and characterized. Its specificity toward polyketide aglycones of different ring sizes and structures as well as five deoxy sugars will be studied in detail. Its ability to glycosylate a ketolide library will be tested. This project, marking the first purification and study of polyketide glycosyltransferase, will provide valuable information about the substrate specificity of glycosyltransferases. DesVII will be further modified to improve its glycosylation capability by mutagenesis. This project will provide an efficient glycosylation technique that is much needed yet unavailable in the field of combinatorial biosynthesis. PROPOSED COMMERCIAL APPLICATION: The need for glycosyltransferase is immense and so ther is a large market potential for the proposed in vitro glycosylation technique. The target enzyme, DesVII, is a glycosyltransferase with wide ranging substrate specificity. It is expected to glycosylate polyketide compounds with a number of TDP-deoxy sugars, thus providing a much needed tool for glycosylation.
