Combining Classical, Genetic, and Process Strategies for Improved Precursor-Directed Production of 6-Deoxyerythronolide B Analogues

A process for the production of erythromycin aglycone analogues has been developed by combining classical strain mutagenesis techniques with modern recombinant DNA methods and traditional process improvement strategies. A Streptomyces coelicolor strain expressing the heterologous 6‐deoxyerythronolide B (6‐dEB) synthase (DEBS) for the production of erythromycin aglycones was subjected to random mutagenesis and selection. Several strains exhibiting 2‐fold higher productivities and reaching >3 g/L total macrolide aglycones were developed. These mutagenized strains were cured of the plasmid carrying the DEBS genes and a KS1° mutant DEBS operon was introduced for the production of novel analogues when supplemented with a synthetic diketide precursor. The strains expressing the mutant DEBS were screened for improved 15‐methyl‐6‐dEB production, and the best clone, strain B9, was found to be 50% more productive as compared to the parent host strain used for 15‐methyl‐6‐dEB production. Strain B9 was evaluated in 5‐L fermenters to confirm productivity in a scalable process. Although peak titers of 0.85 g/L 15‐methyl‐6‐dEB by strain B9 confirmed improved productivity, it was hypothesized that the low solubility of 15‐methyl‐6‐dEB limited productivity. The solubility of 15‐methyl‐6‐dEB in water was determined to be 0.25–0.40 g/L, although higher titers are possible in fermentation medium. The incorporation of the hydrophobic resin XAD‐16HP resulted in both the in situ adsorption of the product and the slow release of the diketide precursor. The resin‐containing fermentation achieved 1.3 g/L 15‐methyl‐6‐dEB, 50% higher than the resin‐free process. By combining classical mutagenesis, recombinant DNA techniques, and process development, 15‐methyl‐6‐dEB productivity was increased by over 100% in a scalable fermentation process.