Investigation of the amide synthetase reaction in the biosynthesis of rubradirin: Providing a new tool for combinatorial biosynthesis

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Rubradirin is an antibiotic produced by Streptomyces achromogenes var. rubradiris. It belongs to the aminocoumarin antibiotics, together with novobiocin, clorobiocin, coumermycin Al and the simocyclinones. In all aminocoumarin antibiotics, the amino group at position 3 of the coumarin ring is connected to an acyl component via an amide bond. The German applicants have recently cloned and overexpressed the enzyme NovL forming this amide bond in novobiocin biosynthesis (J. Biol. Chem. 275, 21754 [2000]), as well as the corresponding enzyme CouL involved in coumermycin biosynthesis. These enzymes are principally different from the non ribosomal peptide synthetases studied in detail in other antibiotic producers. The Korean applicants have cloned and sequenced the rubradirin biosynthetic gene cluster. They found three genes with high homology to NovL and CouL. In the present project, the amide synthetase of rubradirin biosynthesis shall be functionally identified by gene inactivation experiments and by active expression. The protein will be overexpressed, purified and biochemically characterized, and the acyl substrate of this enzyme will be identified. In cooperation with a biochemical group in Norwich, UK, the structure of the protein will be investigated by crystallization and X-ray crystallography. The resulting information will provide both basic knowledge about rubradirin biosynthesis as well as tools for the generation of new antibiotics by genetic engineering.