Expression and Purification of Glycosyltransferase DnmS from <i>Streptomyces peucetius</i> ATCC 27952 and Study on Catalytic Characterization of Its Reverse Glycosyltransferase Reaction

Abstract

Anthracyclines are an important class of natural antitumor drugs. They have a conservative aromatic tetracycline backbone that is substituted with different deoxyglucoses. The deoxyglucoses are crucial for the biological activity of many bacterial natural products after the proper modification from glycosyltransferases (GTs). The difficulty in obtaining highly purified active GTs has prevented biochemical studies on natural product GTs. In this paper, a new Escherichia coli fusion plasmid pGro7′, which introduces the Streptomyces coelicolor chaperone genes groEL1, groES and groEL2, was constructed. The glycosyltransferase DnmS from Streptomyces peucetius ATCC 27952 was co-expressed with the plasmid pGro7′, and unprecedented high-efficiency and soluble expression of DnmS in the E. coli expression system was realized. Subsequently, the reverse glycosylation reaction characteristics of DnmS and DnmQ were verified. We found that DnmS and DnmQ had the highest enzyme activity when they participated in the reaction at the same time. These studies provide a strategy for the soluble expression of GTs in Streptomyces and confirm the reversibility of the catalytic reaction of GTs. This provides a powerful method for the production of active anthracyclines and to enhance the diversity of natural products.

Keywords

• glycosyltransferase
• fusion protein
• DnmS
• DnmQ
• chaperone plasmids
• co-expression