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Abstract
Here we explore the use of multiple orthologous integrating plasmid systems, based on the int/attP loci from phages TG1, SV1 and zBT1, to express the 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. A further pair of integrating plasmids, both derived from the zC31 int/attP locus, were constructed encoding a gene cassette for glycosylation of the aglycone intermediates, with and without the tailoring gene, eryF, required for the synthesis of erythronolide B. Liquid chromatography {u2013} mass spectrometry of the metabolites indicated the production of angolosaminyl-6dEB and angolosaminyl-EB. Erythromycin was formulated into PLGA nanoparticles. Entrapment efficiency of erythromycin in PLGA nanoparticles was determined microbiologically. The effect of PLGA amount, PLGA type, organic solvent, sonication probe diameter, sonication time, organic phase: aqueous phase ratio as well as effect of PVA percent on microbiologically determined entrapment efficiency was assessed. The results showed that microbiologically determined entrapment efficiency was significantly enhanced after optimizing different parameters to reach 51.1±0.68 % for the optimum formula (F10). The size of the nanoparticles produced by optimal formulation observed from SEM micrographs was around 100 nm with narrow particle size distribution