title: Production of the polyketide 6-deoxyerythronolide B in the heterologous host Bacillus subtilis.
abstract: Polyketides, such as erythromycin, are complex natural products with diverse therapeutic applications. They are synthesized by multi-modular megaenzymes, so-called polyketide synthases (PKSs). The macrolide core of erythromycin, 6-deoxyerythronolide B (6dEB), is produced by the deoxyerythronolide B synthase (DEBS) that consists of three proteins each with a size of 330-370 kDa. We cloned and investigated the expression of the corresponding gene cluster from Saccharopolyspora erythraea, which comprises more than 30 kb, in Bacillus subtilis. It is shown that the DEBS genes are functionally expressed in B. subtilis when the native eryAI-III operon was separated into three individual expression cassettes with optimized ribosomal binding sites. A synthesis of 6dEB could be detected by using the acetoin-inducible acoA promoter and a fed-batch simulating EnBase-cultivation strategy. B. subtilis was capable of the secretion of 6dEB into the medium. In order to improve the 6dEB production, several genomic modifications of this production strain were tested. This included the knockout of the native secondary metabolite clusters of B. subtilis for the synthesis of surfactin (26 kb), bacillaene (76 kb), and plipastatin (38 kb). It is revealed that the deletion of the prpBD operon, responsible for propionyl-CoA utilization, resulted in a significant increase of the 6dEB product yield when exogenous propionate is provided. Although the presented B. subtilis 6dEB production strain is not competitive with established Escherichia coli 6dEB production strains, the results of this study indicate that B. subtilis is a suitable heterologous host for the secretory production of a complex polyketide.
authors.affiliation: Pharmaceutical Biotechnology, Institute of Pharmacy, Ernst-Moritz-Arndt-University, Felix-Hausdorff-Str. 3, 17489, Greifswald, Germany.|Present Address: Department of Biomolecular Chemistry, Leibniz Institute for Natural Product Research and Infection Biology, HKI, Beutenbergstr. 11a, 07745, Jena, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Kumpfmüller
authors.affiliation: Institute of Biochemistry, Ernst-Moritz-Arndt-University, Felix-Hausdorff-Str. 4, 17489, Greifswald, Germany.
authors.forename: Karen
authors.initials: K
authors.lastname: Methling
authors.affiliation: Department of Chemical and Biological Engineering, State University of New York at Buffalo, 904 Furnas Hall, Buffalo, NY, 14260-4200, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Fang
authors.affiliation: Department of Chemical and Biological Engineering, State University of New York at Buffalo, 904 Furnas Hall, Buffalo, NY, 14260-4200, USA.
authors.forename: Blaine A
authors.initials: BA
authors.lastname: Pfeifer
authors.affiliation: Institute of Biochemistry, Ernst-Moritz-Arndt-University, Felix-Hausdorff-Str. 4, 17489, Greifswald, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Lalk
authors.affiliation: Pharmaceutical Biotechnology, Institute of Pharmacy, Ernst-Moritz-Arndt-University, Felix-Hausdorff-Str. 3, 17489, Greifswald, Germany. schweder@uni-greifswald.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schweder
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004917:Erythromycin / Q000031:analogs & derivatives* / Q000096:biosynthesis; D060847:Metabolic Engineering; D061065:Polyketides / Q000378:metabolism*; D016930:Saccharopolyspora / Q000201:enzymology / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D061065:Polyketides; C034277:6-deoxyerythronolide B; D004917:Erythromycin
keywords: Bacillus subtilis; Deoxyerythronolide B synthase; Heterologous expression; Metabolic engineering; Polyketide; acoA promoter