1887

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 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 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.

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2011-08-01
2019-11-17
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vol. , part 8, pp. 2401 - 2409

[PDF](486 kb), including: PCR confirmation of the presence of relevant plasmids in strains WP3/pPW189, WP3/pDP33 and ATCC 13224/pDP12 Southern blot analysis of three mutants Genetic basis of ADOTC biosynthesis by (ATCC 13224) Overlay of the homology model structure of OxyD and the crystal structure of AsnB (PDB ID 1CT9) MS filter analysis of OTC and ADOTC production from organic extracts of different strains Primers for PCR confirmation of WP3/pPW189, WP3/pDP33 and ATCC 13224/pDP12



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