INVESTIGADORES
MAZZAFERRO Laura
congresos y reuniones científicas
Título:
Alteration of the route to menaquinone (vitamin K2) in Escherichia coli towards unnatural chorismate-derived metabolites
Autor/es:
ALEXANDER FRIES; LAURA S. MAZZAFERRO; MICHAEL MULLER
Reunión:
Simposio; III Simposio Latinoamericano de Biocatálisis y Biotransformaciones; 2018
Resumen:
Whereas metabolic engineering is generally devoted towards the production of a single target compound, the shikimate biosynthetic pathway results in a diverse set of products, comparable to a tree with many branches1,2. In this pathway, chorismate constitutes a branch- point intermediate and, as such, is the precursor in the biosynthesis of aromatic primary and secondary metabolites in microorganisms and plants1,2. To obtain functionalized artificial cyclic compounds starting from chorismate and its regioisomer isochorismate, we modified the route to menaquinone (vitamin K2) in Escherichia coli. In menaquinone biosynthesis, the thiamine diphosphate dependent MenD (SEPHCHC synthase) catalyzes the decarboxylative carboligation of α-ketoglutarate and isochorismate. We propose a model for the binding of isochorismate to the active site of E. coli MenD that explains the regio- and stereoselective outcome of the reaction. Based on this model, we have designed variants of MenD for the in vitro conversion of several isochorismate analogues. The double variant EcMenD Asn117Arg- Leu478Thr preferentially converts (5S,6S)-5,6-dihydroxycyclohexa-1,3-diene-1-carboxylate with a >70-fold higher ratio than that for the wild type. The single variant EcMenD Arg107Ile uses (5S,6S)-6-amino-5-hydroxycyclohexa-1,3-diene-1-carboxylate as substrate with >sixfold conversion compared to wild-type MenD. In both cases, the functionalized new compounds have been made accessible by microbial fermentation (up to 5.3 g/L), thus extending the already broad diversity of chorismate-derived metabolites.
rds']