INVESTIGATIONS OF MONOBACTAM BIOSYNTHESIS AND CARBAPENEM SYNTHASE ENGINEERING

Zinc metallo-β-lactamases, although long known, have only recently emerged in the clinic to confer potent resistance to penicillins, cephalosporins and even carbapenems, the β-lactam antibiotics of last resort in human medicine. The monobactams, however, having structurally distinct N-sulfonated monocyclic β-lactam rings are resistant to these enzymes and are the most effective class to combat e.g. carbapenem-resistant enterobacteria (CRE). Here we report unprecedented non-ribosomal peptide synthetase activities where an assembled tripeptide is N-sulfonated in trans prior to direct synthesis of the N-sulfonated β-lactam ring in a non-canonical, cysteine-containing thioesterase domain. This biosynthetic route is distinct from three other known tactics in Nature that generate β-lactam rings. These discoveries open the way to production of known and new monobactams by fermentation and semi-synthetic methods not previously available. In a similar vein, the comparatively well-studied biosynthesis of the simplest carbapenem affords the possibility, using the known biosynthetic enzymes, to produce more structurally complex and therapeutically useful carbapenems ie Meropenem by a fermentation process. Carbapenem synthase (CarC) performs the last step of the biosynthesis of the simplest carbapenem through a remarkable two-step reaction consisting of an epimerization followed by desaturation. Several attempts were made to expand the substrate scope of CarC to accommodate structures that represent intermediates in the production of commercially available carbapenems