Optimization of Megalomicins Production in Escherichia coli

USEGLIO, M; PEIRÚ, S; RODRÍGUEZ, E; GRAMAJO, H

Puerto Madryn

Congreso; XLVI Reunión Anual de la Sociedad Argentina de Investigadores en Bioquímica y Biología Molecular (SAIB); 2010

SAIB - Sociedad Argentina de Investigación 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 the new megalomicin analogs in E. coli we carried out metabolic engineering of two endogenous pathways that consume the common deoxysugar intermediate, TDP-4-keto-6-deoxyglucose in E. coli BL21 strain. Higher intracellular levels of TKDG increase production of TDP-L-megosamine which result in improve of bioconversion experiment carried out by feeding erythromycin A and azithromycin. Expression of each protein from the megosamine operon by Western Blot allowed 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 y pTAC. Production of megalomicin analogs were evaluated by bioconversion experiment. In addition, bioconversion experiments using a fed-batch bioreactor allowed as to produce megalomicins analogous in minimal medium using different carbon sources The best amount of bioconversion was obtain using glycerol as carbon source.