Genetic characterization of enzymes involved in the priming steps of oxytetracycline biosynthesis in streptomyces rimosus

P. Wang, X. Gao, Heng Chooi, Z. Deng, Y. Tang

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Tetracyclines are clinically important aromatic polyketides whose biosynthesis is catalysed by bacterial type 2 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. © 2011 SGM Printed in Great Britain.
Original languageEnglish
Pages (from-to)2401-2409
JournalMicrobiology
Volume157
Issue number8
DOIs
Publication statusPublished - 2011

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Streptomyces rimosus
Oxytetracycline
Enzymes
Amides
Tetracyclines
Acetates
Polyketide Synthases
Anti-Bacterial Agents
Polyketides
Hydrolysis

Cite this

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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 2 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. {\circledC} 2011 SGM Printed in Great Britain.",
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Genetic characterization of enzymes involved in the priming steps of oxytetracycline biosynthesis in streptomyces rimosus. / Wang, P.; Gao, X.; Chooi, Heng; Deng, Z.; Tang, Y.

In: Microbiology, Vol. 157, No. 8, 2011, p. 2401-2409.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Genetic characterization of enzymes involved in the priming steps of oxytetracycline biosynthesis in streptomyces rimosus

AU - Wang, P.

AU - Gao, X.

AU - Chooi, Heng

AU - Deng, Z.

AU - Tang, Y.

PY - 2011

Y1 - 2011

N2 - Tetracyclines are clinically important aromatic polyketides whose biosynthesis is catalysed by bacterial type 2 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. © 2011 SGM Printed in Great Britain.

AB - Tetracyclines are clinically important aromatic polyketides whose biosynthesis is catalysed by bacterial type 2 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. © 2011 SGM Printed in Great Britain.

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DO - 10.1099/mic.0.048439-0

M3 - Article

VL - 157

SP - 2401

EP - 2409

JO - MICROBIOLOGY-SGM

JF - MICROBIOLOGY-SGM

SN - 1350-0872

IS - 8

ER -