Glycosylation reactions in secondary metabolism : glycosylation events in C-mannosylation and the biosynthesis of kijanimicin

White-Phillip, Jessica Ann
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In this work, we examine two disparate aspects of glycosylation. The first project involves the elucidation of the glycosylation of the novel tetronolide natural product, kijanimicin. The biosynthesis of the deoxysugar TDP-L-digitoxose from the kijanimicin natural product pathway was achieved in vitro. The genes were identified from the cluster, cloned, expressed and the products were purified. Activity was demonstrated for the novel enzymes and the pathway was reconstructed in vivo using Streptomyces lividans. These strains of S. lividans were used to examine kijanimicin glycosyltransferase activity. We were able to demonstrate activity for 3 of 4 digitoxosyltransferases in the biosynthetic pathway and propose a biosynthetic scheme by which the tetrasaccharide chain is formed. We identified two putative glycosidases with novel folds, and one glycosyltransferase that appears to have unprecedented activity, attaching 2 if not 3 sugars in sequence. In the second portion of this work, we attempted to identify the eukaryotic C-mannosyltransferase enzyme and demonstrate its activity in vitro and in vivo. Here, we describe our efforts to identify the CMT. Through in silico analysis, putative C-mannosyltransferase genes were identified. These genes were expressed in E. coli and S. cerevisiae, however gene expression was apparently toxic to E. coli. S. cerevisiae expression was acceptable, but extraction proved to be somewhat problematic. We describe our efforts to develop a CMT assay for use in vitro by expressing the putative CMT in insect cells, which was much more promising. We also attempted to knock down the putative CMT genes using shRNA, which demonstrated that the genes of unknown function that were identified were essential for cellular viability. This work has contributed to the fields of both C-mannosylation and natural product glycosylation. We have elucidated the biosynthetic pathway of a novel deoxysugar, and identified potentially valuable tools for glycoengineering including a glycosyltransferase that appears to exhibit novel polymeric activity, as well as identifying two glycosyltransferase proteins that are apparent glycosidases. Our attempts to identify the CMT provided valuable insight into the future development of a C-mannosylation assay, and we have identified several promising protein candidates that are apparently essential for H. sapiens cellular viability.