Biosynthesis of Erythromycin in E. coli: improvement of the mycarosylation step

S. PEIRÚ, H. GRAMAJO AND E. RODRIGUEZ

Pinamar, BA, Argentina

Congreso; XL Reunión Anual de la Sociedad Argentina de Investigadores en Bioquímica y Biología Molecular (SAIB; 2005

Biosynthesis of erythromycin C involves the synthesis of the polyketide 6-deoxyerythronolide B through the condensation of one proprionate and six methylmalonyl-CoA units. Further modifications of the macrolactone ring are sequentially introduced, which comprise a number of enzymatic activities encoded by 20 genes. These “tailoring” steps involve a hydroxylation at C-6, the sequential addition of the deoxysugars mycarose and desosamine, and a second hydroxylation at C-12. Recently, we developed an E. coli strain that functionally expressed two operons for the biosynthesis and transfer of mycarose and desosamine deoxysugars from Micromonospora megalomicea, together with the 6-dEB synthetase genes from Saccharopolyspora erythraea. This allowed us to obtain erythromycin C from batch cultures, together with other intermediate compounds. The characterization by NMR and LC/MS of one of these products indicated the absence of a methyl group in the mycarose moiety. In the present work, we were able to identify the biosynthetic step responsible for the accumulation of this intermediate product. Surprisingly, this was not due to a failure in the methyl transferase step but the previous enzyme in the pathway of dTDP-L-mycarose biosynthesis, the ketoreductase MegBII. The addition of megDVII gene from megalomicin cluster to the mycarose operon allowed improving the mycarosylation efficiency. Structural analysis suggests that MegBII and MegDVII interact to form a functional complex.