OPTIMIZATION OF MACROLIDE GLYCOSYLATION IN E. COLI AND S. LIVIDANS

BÁRBARA A. BERCOVICH; HUGO GRAMAJO; EDUARDO RODRIGUEZ

Rosario

Congreso; SAIB 2014; 2014

SAIB

Glycosylation patterns of macrolides are important determinants of the bioactivities of the molecule. Previously we have demonstrated that substrate flexibility of the UTP-dependent glycosyltransferase pair MegDI-MegDVI from megalomycin gene cluster allowed production of two new antimalarial megosaminylated-macrolides in E. coli. However, the efficiency of the system only tolerates bioconversion of 20 mg/L of macrolide per fermentation. Analysis showed that the high G+C content megDI-megDVI genes from M. megalomycea were poorly expressed in E. coli. In order to optimize the production of megosaminylated-macrolides new TDP-megosamine operons were constructed using codon-optimazed megDI/DVI genes for E. coli. The new operons were introduced in E. coli and the protein expression will be analyzed by Western Blot and bioconversion experiment.In addition, we have developed a new glycosylation system using S. lividans. In this case we carried out metabolic engineering of endogenous pathways that consume the common glucose-1P intermediate in S. lividans. We perform single and double mutation in pgm, manB, glgC, glgA and galU genes. Here we expected that higher intracellular levels of glucose-1P, and hence of NTP-sugars, increase the biosynthesis of TDP-L-megosamine, which should improve glycosylation of macrolides. This will be tested by analyzing NDP-sugar pool and bioconversion experiment.