A host-vector system for analysis and manipulation of rifamycin polyketidebiosynthesis in Amycolatopsis mediterranei

Modular polyketide synthases (PKSs) are a large family of multifunctional e nzymes responsible for the biosynthesis of numerous bacterial natural produ cts such as erythromycin and rifamycin. Advanced genetic analysis of these remarkable systems is often seriously hampered by the large size (>40 kb) o f PKS gene clusters, and, notwithstanding their considerable fundamental an d biotechnological significance, by the lack of suitable methods for engine ering non-selectable modifications in chromosomally encoded PKS genes. The development of a facile host-vector strategy for genetic engineering of the rifamycin PKS in the producing organism. Amycolatopsis mediterranei S699, is described here. The genes encoding all 10 modules of the rifamycin PKS w ere replaced with a hygromycin-resistance marker gene. In a similar constru ction, only the first six modules of the PKS were replaced. The deletion ho sts retained the ability to synthesize the primer unit 3-amino-5-hydroxyben zoic acid (AHBA), as judged by co-synthesis experiments with a mutant strai n lacking AHBA synthase activity. Suicide plasmids carrying a short fragmen t from the 5' flanking end of the engineered deletion, an apramycin-resista nce marker gene, and suitably engineered PKS genes could be introduced via electroporation into the deletion hosts, resulting in the integration of PK S genes and biosynthesis of a reporter polyketide in quantities comparable to those produced by the wild-type organism. Since this strategy for engine ering recombinant PKSs in A. mediterranei requires only a selectable single crossover and eliminates the need for tedious non-selectable double-crosso ver experiments, it makes rifamycin PKS an attractive target for extensive genetic manipulation.