Erythromycin A biosynthesis in heterologous host

PEIRU, S; MENZELLA, H; KURTH, D; GRAMAJO, H

Bariloche

Congreso; XXXIX Annual Meeting of the Argentinean Society for Biochemistry and Molecular Biology (SAIB); 2003

SAIB

The increasing knowledge about the chemistry performed by polyketide synthetases (PKSs) has lead to the construction of polyketide libraries, generated by genetic modification of macrolide PKSs. Since these libraries were constructed in heterologous hosts lacking glycosylation pathways, only the corresponding aglycones were produced. We wished to expand the capabilities of the combinatorial biosynthesis strategies to incorporate post-PKS “tailoring” steps, in particular the adition of deoxysugar components. We decided to construct a strain capable to perform the tailoring steps that lead to the formation of Erythromycin A from the aglycon 6-dEB, wich is synthesized by the modular PKS 6-dEB synthetase (DEBS). These reactions include the attachment of two deoxysugar moieties, L-mycarose and D-desosamine, two hydroxilations and a methylation of the mycarose residue.One of the most widely used heterologous host for genetic manipulation of actinomycetes PKSs is Streptomyces coelicolor. We decided to genetically engineer the S coelicolor A3(2) strain transfering the methabolic pathways involved in the conversion of 6-dEB to Erythromycin A from Micromonospora megalomicea. This implied the construction of two operons including the genes involved in the biosynthetic pathways of the deoxysugars TDP-L-mycarose and TDP-D-desosamine and their corresponding glycosyltransferases, two hydroxylases and a methyltransferase. This strain could perform the bioconversion of the macrolide 6-dEB to the different erythromycin intermediates in batch cultures fed with this polyketide.