title: Genetic characterization of enzymes involved in the priming steps of oxytetracycline biosynthesis in Streptomyces rimosus.
abstract: Tetracyclines are clinically important aromatic polyketides whose biosynthesis is catalysed by bacterial type II polyketide synthases (PKSs). Tetracyclines are biosynthesized starting with an amide-containing malonamate starter unit and the resulting C-2 carboxyamide is critical for the antibiotic activities. In this work, we genetically verified that an amidotransferase, OxyD, and a thiolase, OxyP, are involved in the biosynthesis and incorporation of the starter unit. First, two mutations, R248T and D268N, were found to be present in OxyD* encoded in Streptomyces rimosus ATCC 13224, a strain that produces the acetate-primed 2-acetyl-2-decarboxyamido-oxytetracycline (ADOTC) instead of the malonamate-primed oxytetracycline (OTC). Homology modelling suggested that in particular D268N may inactivate OxyD. Complementation of S. rimosus ATCC 13224 with wild-type OxyD restored OTC biosynthesis, thereby confirming the essential role of OxyD in the synthesis of the amide starter unit. Second, using a series of knockout and complementation approaches, we demonstrated that OxyP is most likely involved in maintaining fidelity of the amide-priming process via hydrolysis of the competing acetate priming starter units. While the inactivation of OxyP does not eliminate OTC biosynthesis, the ratio of acetate-primed ADOTC to malonamate-primed OTC is significantly increased. This suggests that OxyP plays an ancillary role in OTC biosynthesis and is important for minimizing the levels of ADOTC, a shunt product that has much weaker antibiotic activities than OTC.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, USA.|Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiaotong University, Shanghai 200030, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, USA.
authors.forename: Xue
authors.initials: X
authors.lastname: Gao
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, USA.
authors.forename: Yit-Heng
authors.initials: YH
authors.lastname: Chooi
authors.affiliation: Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiaotong University, Shanghai 200030, China.
authors.forename: Zixin
authors.initials: Z
authors.lastname: Deng
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, USA.|Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Tang
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D053898:Biosynthetic Pathways; D004798:Enzymes / Q000737:chemistry / Q000235:genetics; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D008954:Models, Biological; D008958:Models, Molecular; D020125:Mutation, Missense; D010118:Oxytetracycline / Q000096:biosynthesis*; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004798:Enzymes; D010118:Oxytetracycline