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

Rubradirin是由Streptomyces achromogenes var.红宝石。它与新生霉素、氯生霉素、香豆霉素Al和西莫环素酮一起属于氨基香豆素类抗生素。在所有氨基香豆素抗生素中，香豆素环3位的氨基通过酰胺键与酰基部分连接。德国申请人最近克隆并过表达了在新生霉素生物合成中形成该酰胺键的酶NovL（J.Biol.Chem.275，21754 [2000]），以及参与香豆霉素生物合成的相应酶CouL。这些酶主要不同于在其他抗生素生产者中详细研究的非核糖体肽合成酶。韩国申请人已经克隆并测序了红蔷薇苷生物合成基因簇。他们发现了三个与NovL和CouL具有高度同源性的基因。在本项目中，将通过基因失活实验和活性表达来功能性地鉴定rubradirin生物合成的酰胺合成酶。蛋白质将被过表达、纯化和生物化学表征，并且该酶的酰基底物将被鉴定。在与英国诺维奇的一个生物化学小组的合作中，将通过结晶和X射线晶体学研究蛋白质的结构。由此产生的信息将提供有关rubradirin生物合成的基础知识以及通过基因工程产生新抗生素的工具。

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.