title: Bioassay-guided evolution of glycosylated macrolide antibiotics in Escherichia coli.
abstract: Macrolide antibiotics such as erythromycin are clinically important polyketide natural products. We have engineered a recombinant strain of Escherichia coli that produces small but measurable quantities of the bioactive macrolide 6-deoxyerythromycin D. Bioassay-guided evolution of this strain led to the identification of an antibiotic-overproducing mutation in the mycarose biosynthesis and transfer pathway that was detectable via a colony-based screening assay. This high-throughput assay was then used to evolve second-generation mutants capable of enhanced precursor-directed biosynthesis of macrolide antibiotics. The availability of a screen for macrolide biosynthesis in E. coli offers a fundamentally new approach in dissecting modular megasynthase mechanisms as well as engineering antibiotics with novel pharmacological properties.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, California, United States of America.
authors.forename: Ho Young
authors.initials: HY
authors.lastname: Lee
authors.forename: Chaitan
authors.initials: C
authors.lastname: Khosla
mesh_terms: D000606:Amino Sugars / Q000096:biosynthesis / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D001681:Biological Assay; D019020:Directed Molecular Evolution; D024901:Drug Resistance, Multiple, Bacterial / Q000502:physiology; D004917:Erythromycin / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D006031:Glycosylation; D006601:Hexoses / Q000096:biosynthesis / Q000235:genetics; D009154:Mutation; D009702:Nucleoside Diphosphate Sugars; D030781:Organisms, Genetically Modified; D013942:Thymine Nucleotides / Q000096:biosynthesis / Q000378:metabolism
chemical_list: C113834:6-deoxyerythromycin D; D000606:Amino Sugars; D000900:Anti-Bacterial Agents; D006601:Hexoses; D009702:Nucleoside Diphosphate Sugars; C513911:TDP-D-desosamine; D013942:Thymine Nucleotides; C045835:mycarose; D004917:Erythromycin