Defining the genes for the final steps in biosynthesis of the complex polyketide antibiotic mupirocin by Pseudomonas fluorescens NCIMB10586

Jack Connolly; Amber Wilson; Malgorzata Macioszek; Zhongshu Song; Luoyi Wang; Hadi Mohammad; Mukul Yadav; Maura di Martino; Claire Litchfield; Joanne Hothersall; Anthony Haines; Elton Stephens; Matthew P. Crump; Christine L. Willis; Thomas J. Simpson; Peter Winn; Christopher Thomas

doi:10.1038/s41598-018-38038-9

Defining the genes for the final steps in biosynthesis of the complex polyketide antibiotic mupirocin by Pseudomonas fluorescens NCIMB10586

Jack Connolly, Amber Wilson, Malgorzata Macioszek, Zhongshu Song, Luoyi Wang, Hadi Mohammad, Mukul Yadav, Maura di Martino, Claire Litchfield, Joanne Hothersall, Anthony Haines, Elton Stephens, Matthew P. Crump, Christine L. Willis, Thomas J. Simpson, Peter Winn, Christopher Thomas

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Abstract

The mupirocin trans-AT polyketide synthase pathway, provides a model system for manipulation of antibiotic biosynthesis. Its final phase involves removal of the tertiary hydroxyl group from pseudomonic acid B, PA-B, producing the fully active PA-A in a complex series of steps. To further clarify requirements for this conversion, we fed extracts containing PA-B to mutants of the producer strain singly deficient in each mup gene. This additionally identified mupM and mupN as required plus the sequence but not enzymic activity of mupL and ruled out need for other mup genes. A plasmid expressing mupLMNOPVCFU+macpE together with a derivative of the producer P. fluorescens strain NCIMB10586 lacking the mup cluster allowed conversion of PA-B to PA-A . MupN converts apo-mAcpE to holo-form while MupM is a mupirocin-resistant isoleucyl tRNA synthase, preventing self-poisoning. Surprisingly, the expression plasmid failed to allow the closely related P. fluorescens strain SBW25 to convert PA-B to PA-A.

Original language	English
Article number	1542
Journal	Scientific Reports
Volume	9
Issue number	1
Early online date	7 Feb 2019
DOIs	https://doi.org/10.1038/s41598-018-38038-9
Publication status	Published - May 2019

Keywords

antibiotic biosynthesis
polyketide synthase
pseudomonas fluorescens
mupirocin
pseudomonic acid
synthetic biology

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Connolly_et_al_Defining_the_genes_for_the_final_Scientific_Reports_2019
Checked for eligibility 07/02/2019 Published in Scientific Reports https://doi.org/10.1038/s41598-018-38038-9
Final published version, 1.6 MBLicence: Creative Commons: Attribution (CC BY)

https://www.nature.com/articles/s41598-018-38038-9Licence: Creative Commons: Attribution (CC BY)

Novel hybrid anti-MRSA antibiotics from manipulation of the mupirocin and thiomarinol biosynthetic pathways
Thomas, C. & Winn, P.
Biotechnology & Biological Sciences Research Council
1/10/11 → 31/12/14
Project: Research Councils

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Connolly, J., Wilson, A., Macioszek, M., Song, Z., Wang, L., Mohammad, H., Yadav, M., di Martino, M., Litchfield, C., Hothersall, J., Haines, A., Stephens, E., Crump, M. P., Willis, C. L., Simpson, T. J., Winn, P., & Thomas, C. (2019). Defining the genes for the final steps in biosynthesis of the complex polyketide antibiotic mupirocin by Pseudomonas fluorescens NCIMB10586. Scientific Reports, 9(1), Article 1542. https://doi.org/10.1038/s41598-018-38038-9

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title = "Defining the genes for the final steps in biosynthesis of the complex polyketide antibiotic mupirocin by Pseudomonas fluorescens NCIMB10586",

abstract = "The mupirocin trans-AT polyketide synthase pathway, provides a model system for manipulation of antibiotic biosynthesis. Its final phase involves removal of the tertiary hydroxyl group from pseudomonic acid B, PA-B, producing the fully active PA-A in a complex series of steps. To further clarify requirements for this conversion, we fed extracts containing PA-B to mutants of the producer strain singly deficient in each mup gene. This additionally identified mupM and mupN as required plus the sequence but not enzymic activity of mupL and ruled out need for other mup genes. A plasmid expressing mupLMNOPVCFU+macpE together with a derivative of the producer P. fluorescens strain NCIMB10586 lacking the mup cluster allowed conversion of PA-B to PA-A . MupN converts apo-mAcpE to holo-form while MupM is a mupirocin-resistant isoleucyl tRNA synthase, preventing self-poisoning. Surprisingly, the expression plasmid failed to allow the closely related P. fluorescens strain SBW25 to convert PA-B to PA-A. ",

keywords = "antibiotic biosynthesis, polyketide synthase, pseudomonas fluorescens, mupirocin, pseudomonic acid, synthetic biology",

author = "Jack Connolly and Amber Wilson and Malgorzata Macioszek and Zhongshu Song and Luoyi Wang and Hadi Mohammad and Mukul Yadav and {di Martino}, Maura and Claire Litchfield and Joanne Hothersall and Anthony Haines and Elton Stephens and Crump, {Matthew P.} and Willis, {Christine L.} and Simpson, {Thomas J.} and Peter Winn and Christopher Thomas",

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Connolly, J, Wilson, A, Macioszek, M, Song, Z, Wang, L, Mohammad, H, Yadav, M, di Martino, M, Litchfield, C, Hothersall, J, Haines, A, Stephens, E, Crump, MP, Willis, CL, Simpson, TJ, Winn, P & Thomas, C 2019, 'Defining the genes for the final steps in biosynthesis of the complex polyketide antibiotic mupirocin by Pseudomonas fluorescens NCIMB10586', Scientific Reports, vol. 9, no. 1, 1542. https://doi.org/10.1038/s41598-018-38038-9

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AU - Wilson, Amber

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AU - Wang, Luoyi

AU - Mohammad, Hadi

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AU - Litchfield, Claire

AU - Hothersall, Joanne

AU - Haines, Anthony

AU - Stephens, Elton

AU - Crump, Matthew P.

AU - Willis, Christine L.

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AU - Winn, Peter

AU - Thomas, Christopher

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Defining the genes for the final steps in biosynthesis of the complex polyketide antibiotic mupirocin by Pseudomonas fluorescens NCIMB10586

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Keywords

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Novel hybrid anti-MRSA antibiotics from manipulation of the mupirocin and thiomarinol biosynthetic pathways

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