title: Multiplexed integrating plasmids for engineering of the erythromycin gene cluster for expression in Streptomyces spp. and combinatorial biosynthesis.
abstract: Bacteria in the genus Streptomyces and its close relatives are prolific producers of secondary metabolites with antibiotic activity. Genome sequencing of these bacteria has revealed a rich source of potentially new antibiotic pathways, whose products have never been observed. Moreover, these new pathways can provide novel genes that could be used in combinatorial biosynthesis approaches to generate unnatural analogues of existing antibiotics. We explore here the use of multiple orthologous integrating plasmid systems, based on the int/attP loci from phages TG1, SV1, and ϕBT1, to express the polyketide synthase (PKS) for erythromycin in a heterologous Streptomyces host. Streptomyces strains containing the three polyketide synthase genes eryAI, eryAII, and eryAIII expressed from three different integrated plasmids produced the aglycone intermediate, 6-deoxyerythronolide B (6-dEB). A further pair of integrating plasmids, both derived from the ϕC31 int/attP locus, were constructed carrying a gene cassette for glycosylation of the aglycone intermediates, with or without the tailoring gene, eryF, required for the synthesis of erythronolide B (EB). Liquid chromatography-mass spectrometry of the metabolites indicated the production of angolosaminyl-6-dEB and angolosaminyl-EB. The advantages of using multiplexed integrating plasmids for engineering expression and for combinatorial biosynthesis were demonstrated.
authors.affiliation: Department of Biology, University of York, York, United Kingdom Chemistry of Natural and Microbial Products Department, National Research Centre, Cairo, Egypt.
authors.forename: Bahgat
authors.initials: B
authors.lastname: Fayed
authors.affiliation: Bioscience Technology Facility, Department of Biology, University of York, York, United Kingdom.
authors.forename: David A
authors.initials: DA
authors.lastname: Ashford
authors.affiliation: Chemistry of Natural and Microbial Products Department, National Research Centre, Cairo, Egypt.
authors.forename: Amal M
authors.initials: AM
authors.lastname: Hashem
authors.affiliation: Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo, Egypt.
authors.forename: Magdy A
authors.initials: MA
authors.lastname: Amin
authors.affiliation: Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt.
authors.forename: Omaima N
authors.initials: ON
authors.lastname: El Gazayerly
authors.affiliation: Isomerase Therapeutics, Science Village, Chesterford Research Park, Cambridge, United Kingdom.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Gregory
authors.affiliation: Department of Biology, University of York, York, United Kingdom maggie.smith@york.ac.uk.
authors.forename: Margaret C M
authors.identifier: http://orcid.org/0000-0002-4150-0496
authors.initials: MC
authors.lastname: Smith
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D002853:Chromatography, Liquid; D004917:Erythromycin / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000378:metabolism*; D005818:Genetic Engineering; D006031:Glycosylation; D013058:Mass Spectrometry; D009097:Multienzyme Complexes / Q000378:metabolism; D005810:Multigene Family / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D009097:Multienzyme Complexes; C034277:6-deoxyerythronolide B; C038315:erythronolide B; D004917:Erythromycin