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An integrated workflow for phenazine-modifying enzyme characterization

  • R. Cameron Coates
  • Benjamin P. Bowen
  • Ernst Oberortner
  • Linda Thomashow
  • Michalis Hadjithomas
  • Zhiying Zhao
  • Jing Ke
  • Leslie Silva
  • Katherine Louie
  • Gaoyan Wang
  • David Robinson
  • Angela Tarver
  • Matthew Hamilton
  • Andrea Lubbe
  • Meghan Feltcher
  • Jeffery L. Dangl
  • Amrita Pati
  • David Weller
  • Trent R. Northen
  • Jan-Fang Cheng
  • Nigel J. Mouncey
  • Samuel Deutsch
  • Yasuo YoshikuniEmail author
Metabolic Engineering and Synthetic Biology - Original Paper

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.

Keywords

Synthetic biology Biosynthesis Phenazine Pathway Refactored Pathway design 

Notes

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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Copyright information

© Society for Industrial Microbiology and Biotechnology 2018

Authors and Affiliations

  • R. Cameron Coates
    • 1
  • Benjamin P. Bowen
    • 1
    • 2
  • Ernst Oberortner
    • 1
  • Linda Thomashow
    • 3
    • 4
  • Michalis Hadjithomas
    • 1
  • Zhiying Zhao
    • 1
  • Jing Ke
    • 1
  • Leslie Silva
    • 1
  • Katherine Louie
    • 1
  • Gaoyan Wang
    • 1
  • David Robinson
    • 1
  • Angela Tarver
    • 1
  • Matthew Hamilton
    • 1
  • Andrea Lubbe
    • 2
  • Meghan Feltcher
    • 5
  • Jeffery L. Dangl
    • 5
    • 6
    • 7
    • 8
    • 9
  • Amrita Pati
    • 1
  • David Weller
    • 3
    • 4
  • Trent R. Northen
    • 1
    • 2
  • Jan-Fang Cheng
    • 1
    • 2
  • Nigel J. Mouncey
    • 1
  • Samuel Deutsch
    • 1
    • 2
    • 10
  • Yasuo Yoshikuni
    • 1
    • 2
    • 10
    Email author
  1. 1.US DOE Joint Genome InstituteWalnut CreekUSA
  2. 2.Environmental Genomics and Systems Biology DivisionLawrence Berkeley National LaboratoryBerkeleyUSA
  3. 3.USDA Agricultural Research Service, Wheat Health, Genetics and QualityWashington State UniversityPullmanUSA
  4. 4.Department of Plant PathologyWashington State UniversityPullmanUSA
  5. 5.Department of BiologyUniversity of North Carolina at Chapel HillChapel HillUSA
  6. 6.Howard Hughes Medical InstituteUniversity of North Carolina at Chapel HillChapel HillUSA
  7. 7.Curriculum in Genetics and Molecular BiologyUniversity of North Carolina at Chapel HillChapel HillUSA
  8. 8.Department of Microbiology and ImmunologyUniversity of North Carolina at Chapel HillChapel HillUSA
  9. 9.Carolina Center for Genome SciencesUniversity of North Carolina at Chapel HillChapel HillUSA
  10. 10.Biological Systems and Engineering DivisionLawrence Berkeley National LaboratoryBerkeleyUSA

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