chlorothricin and 6-methylsalicylic-acid

The enzymes 6-methylsalicylic acid (6-MSA) synthases (6-MSASs) are involved in the building of an aryl moiety in many bioactive secondary metabolites produced by fungi and bacteria. Using the bacterial 6-MSAS ChlB1 in the biosynthesis of spirotetronate antibiotic chlorothricin (CHL) as a model, functional analysis of its dehydratase (DH) and ketoreductase (KR) domains by site-specific mutagenesis revealed that selective ketoreduction is not essential for polyketide chain extension. Promiscuity of the ketoacylsynthase domain in beta functionality recognition allows for engineering ChlB1 to an orsellinic acid (OSA) synthase (OSAS) by inactivating KR at the active site. The engineered ChlB1 is compatible with the enzymes for late-stage tailoring in CHL biosynthesis, featuring specific protein recognitions that facilitate variable aryl group incorporation. The resulting spirotetronates, which bear an OSA-derived aryl group, exhibited antibacterial activities comparable to those of the parent products.

Topics: Acyltransferases; Aminoglycosides; Anti-Bacterial Agents; Bacterial Proteins; Ligases; Multienzyme Complexes; Oxidation-Reduction; Oxidoreductases; Protein Engineering; Protein Structure, Tertiary; Resorcinols; Salicylates; Streptomyces

The biosynthetic gene cluster for chlorothricin (CHL) was localized to a 122 kb contiguous DNA from Streptomyces antibioticus DSM 40725, and its involvement in CHL biosynthesis was confirmed by gene inactivation and complementation. Bioinformatic analysis of the sequenced 111.989 kb DNA region revealed 42 open reading frames, 35 of which were defined to constitute the CHL gene cluster. An assembly model for CHL biosynthesis from D-olivose, 2-methoxy-5-chloro-6-methylsalicyclic acid, and chlorothricolide building blocks was proposed. This work represents cloning of a gene cluster for spirotetronate antibiotic biosynthesis and sets the stage to investigate the unusual macrolide biosynthesis including tandem Diels-Alder cyclizations, Baeyer-Villiger oxidation, and incorporation of an enoylpyruvate unit.

Topics: Amino Acid Sequence; Aminoglycosides; Anti-Bacterial Agents; Carbohydrate Metabolism; Chlorine; Chromatography, High Pressure Liquid; Cloning, Molecular; Conserved Sequence; Drug Resistance, Bacterial; Gene Expression Regulation, Bacterial; Models, Genetic; Molecular Sequence Data; Molecular Structure; Multigene Family; Oxidation-Reduction; Salicylates; Sequence Alignment; Streptomyces antibioticus