Bioassay-guided evolution of glycosylated macrolide antibiotics in Escherichia coli.
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.
Lee
Ho Young
HY
Department of Chemistry, Stanford University, Stanford, California, United States of America.
Khosla
Chaitan
C
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
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