Optimization of megalomicin production in E. coli

MARIANA USEGLIO; PEIRÚ, SALVADOR; EDUARDO RODRÍGUEZ; GRAMAJO, HUGO

Puerto Madryn

Congreso; XLVI Annual Meeting SAIB; 2010

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular

Previously we have demonstrated that substrate flexibility of the sugar transferases MegDI/DVI allowed production of two new megalomicin analogs, megosaminyl-erythromycin A and megosaminyl-azithromycin. Antibacterial activity of these new macrolides did not change notably compared to original molecules. However, megosaminyl-erythromycin A showed a significant increase in potency against P falsiparum. In order to optimize the production of megalomicin analogs in E. coli we carried out metabolic engineering of two endogenous pathways that consume the common deoxysugar intermediate, TKDG in E. coli BL21 strain. Higher intracellular levels of TKDG increase production of TDP-L-megosamine, this improved bioconversion experiment carried out by feeding erythromycin A and azithromycin. Each protein of megosamine operon was expressed and analyzed by Western Blot determining which of them is limiting the efficiency of the bioconversion experiments. New megosamine operons were constructed with alternative gene order and different promoters: pT7, pBAD, pT5, pLacUV5, pC24 and pTAC. Production of megalomicin analogs were evaluated by bioconversion experiment. In addition, bioconversion experiments using a fed-batch bioreactor allowed as to produce megalomicins analogs in minimal medium using different carbon sources. The best amount of bioconversion was obtain using glycerol as carbon source