CONICET | Buscador de Institutos y Recursos Humanos

Glycosyltransferases from polyketide gene cluster determinate the glycosylation patterns of macrolides which define the bioactivities of these molecules. Previously we have demonstrated substrate flexibility of the UTP-dependent glycosyltransferase pair MegDI-MegDVI from megalomycin gene cluster toward both the TDP-zugar and macrolide substrates. Thus, a new megosaminyl-azitromycin derivative with improved antimalaria and antibiotic activity were produced by bioconversion experiments in E. coli. In order to study structural contribution for antibacterial and antimalarial activity of this compound, new derivatives were produced. Modifications into desosamine residue were introduced by synthetic chemistry and megosamine residue was introduced by bioconversion experiments generating two new megosaminil-azitromycin derivatives. The structures of the compounds were confirmed by mass spectrometry. Scaling up of this process will allow validating its structure and the biological activity will be analyzed in order to test the effect of structural modifications on activities. In addition, due to low efficiency of bioconversion experiments in E. coli, we have developed a new glycosylation system using S. lividans. For this end, a S. lividans Δmgt strain was developed toghether with an integrative plasmid carrying the megosamine operon. This pathway include five enzimes that convert glu-1-P into TDP-megosamine which is further transfere to the macrolide by glycosyltransferase pair MegDI-MegDVI. To optimize the system, we carried out metabolic engineering of endogenous pathways that consume the common glu-1-P intermediate in S. lividans. Single and double mutations in pgm and manB genes were performed and test improving carbon flux distribution toward TDP-megosamine when using galactose as carbon source. This hypothesis is being tested by analyzing NDP-sugar pool and bioconversion efficiency.