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An integrated workflow for phenazine-modifying enzyme characterization

  • Metabolic Engineering and Synthetic Biology - Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Increasing availability of new genomes and putative biosynthetic gene clusters (BGCs) has extended the opportunity to access novel chemical diversity for agriculture, medicine, environmental and industrial purposes. However, functional characterization of BGCs through heterologous expression is limited because expression may require complex regulatory mechanisms, specific folding or activation. We developed an integrated workflow for BGC characterization that integrates pathway identification, modular design, DNA synthesis, assembly and characterization. This workflow was applied to characterize multiple phenazine-modifying enzymes. Phenazine pathways are useful for this workflow because all phenazines are derived from a core scaffold for modification by diverse modifying enzymes (PhzM, PhzS, PhzH, and PhzO) that produce characterized compounds. We expressed refactored synthetic modules of previously uncharacterized phenazine BGCs heterologously in Escherichia coli and were able to identify metabolic intermediates they produced, including a previously unidentified metabolite. These results demonstrate how this approach can accelerate functional characterization of BGCs.

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Acknowledgements

The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, operated by Lawrence Berkeley National Laboratory under Contract no. DE-AC02-05CH11231. We would like to thank Dr. Wayne Reeve of Murdoch University for kindly providing us with a live strain of Rhizobium leguminosarum bv. trifolii CC283b.

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Authors and Affiliations

  1. US DOE Joint Genome Institute, Walnut Creek, CA, USA

    R. Cameron Coates, Benjamin P. Bowen, Ernst Oberortner, Michalis Hadjithomas, Zhiying Zhao, Jing Ke, Leslie Silva, Katherine Louie, Gaoyan Wang, David Robinson, Angela Tarver, Matthew Hamilton, Amrita Pati, Trent R. Northen, Jan-Fang Cheng, Nigel J. Mouncey, Samuel Deutsch & Yasuo Yoshikuni

  2. Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Benjamin P. Bowen, Andrea Lubbe, Trent R. Northen, Jan-Fang Cheng, Samuel Deutsch & Yasuo Yoshikuni

  3. USDA Agricultural Research Service, Wheat Health, Genetics and Quality, Washington State University, Pullman, WA, USA

    Linda Thomashow & David Weller

  4. Department of Plant Pathology, Washington State University, Pullman, WA, USA

    Linda Thomashow & David Weller

  5. Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Meghan Feltcher & Jeffery L. Dangl

  6. Howard Hughes Medical Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Jeffery L. Dangl

  7. Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Jeffery L. Dangl

  8. Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Jeffery L. Dangl

  9. Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Jeffery L. Dangl

  10. Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Samuel Deutsch & Yasuo Yoshikuni

Authors
  1. R. Cameron Coates
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  2. Benjamin P. Bowen
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  3. Ernst Oberortner
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  4. Linda Thomashow
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  5. Michalis Hadjithomas
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  6. Zhiying Zhao
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  7. Jing Ke
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  8. Leslie Silva
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  9. Katherine Louie
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  10. Gaoyan Wang
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  11. David Robinson
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  12. Angela Tarver
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  13. Matthew Hamilton
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  14. Andrea Lubbe
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  15. Meghan Feltcher
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  16. Jeffery L. Dangl
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  17. Amrita Pati
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  18. David Weller
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  19. Trent R. Northen
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  20. Jan-Fang Cheng
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  21. Nigel J. Mouncey
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  22. Samuel Deutsch
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  23. Yasuo Yoshikuni
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Corresponding author

Correspondence to Yasuo Yoshikuni.

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Coates, R.C., Bowen, B.P., Oberortner, E. et al. An integrated workflow for phenazine-modifying enzyme characterization. J Ind Microbiol Biotechnol 45, 567–577 (2018). https://doi.org/10.1007/s10295-018-2025-5

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  • DOI: https://doi.org/10.1007/s10295-018-2025-5

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