Erythromycin biosynthesis in E. coli

S. PEIRÚ, HG. MENZELLA, E. RODRIGUEZ AND H. GRAMAJO

Iguazu, Argentina

Congreso; XL Reunión Anual de la Sociedad Argentina de Investigadores en Bioquímica y Biología Molecular (SAIB); 2004

Many polyketide synthetases (PKSs) has been genetically and biochemically characterized during the last years, leading to the construction of combinatorial polyketide libraries, generated by genetic modification of macrolide PKSs. However, these libraries were constructed in heterologous hosts which lack the post-PKS “tailoring” steps (glicosylations, hydroxylations, methylations, etc) so only the corresponding aglycones were produced. We wished to expand the capabilities of the combinatorial biosynthesis strategies to incorporate these post-PKS steps, in particular the addition of deoxysugar moieties, essential for the macrolides activity. Since E. coli is the most widely used host for heterologous gene expression, we decided to genetically engineer this strain to perform both the synthesis of the polyketide 6-dEB and the tailoring steps that lead to the formation of the antibiotic erythromycin C. This implied the construction of two operons including the genes from Micromonospora megalomicea involved in the biosynthesis of the deoxysugars TDP-L-mycarose and TDP-D-desosamine and their corresponding glycosyltransferases, two hydroxylases and an erythromycin resistance gene. The expression of this two operons in E. coli strain K207-3 together with the 6-dEB synthetase genes from Saccharopolyspora erythraea allowed us to obtain erythromycin C, and other intermediate compounds, from batch cultures fed with propionate.