title: Combining classical, genetic, and process strategies for improved precursor-directed production of 6-deoxyerythronolide B analogues.
abstract: 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 degrees 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.
authors.affiliation: Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, California 94545, USA. desai@kosan.com
authors.forename: Ruchir P
authors.initials: RP
authors.lastname: Desai
authors.forename: Timothy
authors.initials: T
authors.lastname: Leaf
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Hu
authors.forename: C Richard
authors.initials: CR
authors.lastname: Hutchinson
authors.forename: Anderson
authors.initials: A
authors.lastname: Hong
authors.forename: Graham
authors.initials: G
authors.lastname: Byng
authors.forename: Jorge
authors.initials: J
authors.lastname: Galazzo
authors.forename: Peter
authors.initials: P
authors.lastname: Licari
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis; D019149:Bioreactors / Q000382:microbiology*; D018929:Cell Culture Techniques / Q000379:methods*; D004917:Erythromycin / Q000031:analogs & derivatives* / Q000096:biosynthesis*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods*; D016296:Mutagenesis / Q000502:physiology; D010865:Pilot Projects; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; C034277:6-deoxyerythronolide B; D004917:Erythromycin