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Intermediates of rifamycin polyketide synthase produced by an Amycolatopsis mediterranei mutant with inactivated rifF gene

Novartis Pharma AG, Research, Core Technology Area, CH-4002 Basel , Switzerland¹

Author for correspondence: Thomas Schupp. Tel: +41 61 32 47903. Fax: +41 61 32 43279. e-mail: thomas.schupp{at}pharma.novartis.com

Rifamycin B biosynthesis in Amycolatopsis mediterranei N/813 was inactivated by introducing a small deletion in the rifF gene situated directly downstream of the rifamycin polyketide synthase (PKS) gene cluster. The corresponding mutant strain produced a series of linear intermediates of rifamycin B biosynthesis that are most probably generated by obstruction of the normal release of the end product of the rifamycin PKS. This result provides evidence that the rifF gene product catalyses the release of the completed linear polyketide from module 10 of the PKS and the intramolecular macrocyclic ring closure by formation of an amide bond, as indicated by sequence similarity of this protein to amide synthases. The chemical structures of the new rifamycin polyketide synthase intermediates released from modules 4 to 10 were determined by spectroscopic methods (UV, IR, NMR and MS) and gave insight into the reaction steps of rifamycin ansa chain biosynthesis and the timing of the formation of the naphthoquinone ring. The intermediates released from modules 6 and 8 were isolated as lactones formed by the terminal carboxyl group; proton NMR double resonance and ROESY(rotated frame nuclear Overhauser enhancement spectroscopy) experiments enabled the deduction of the relative configurations in the linear chain which correspond to the known absolute stereochemistry of rifamycin B.

Keywords: antibiotic biosynthesis, ansamycins, amide synthase, gene replacement, pathway engineering

Abbreviations: PKS, polyketide synthase; AHBA, 3-amino-5-hydroxybenzoic acid

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