RT Journal Article SR Electronic(1) A1 Wang, Peng A1 Gao, Xue A1 Chooi, Yit-Heng A1 Deng, Zixin A1 Tang, YiYR 2011 T1 Genetic characterization of enzymes involved in the priming steps of oxytetracycline biosynthesis in Streptomyces rimosus JF Microbiology, VO 157 IS 8 SP 2401 OP 2409 DO https://doi.org/10.1099/mic.0.048439-0 PB Microbiology Society, SN 1465-2080, AB 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., UL https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.048439-0