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Title: An integrated workflow for phenazine-modifying enzyme characterization

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 have 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.

Authors:
 [1];  [2];  [1];  [3];  [1];  [1];  [1];  [1];  [1];  [1];  [1];  [1];  [1];  [4];  [5];  [6];  [1];  [3];  [2];  [2] more »;  [1];  [7];  [7] « less
  1. USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States)
  2. USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Environmental Genomics and Systems Biology Division
  3. Washington State Univ., Pullman, WA (United States). USDA-ARS Wheat Health, Genetics, and Quality Research Program and Dept. of Plant Pathology
  4. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Environmental Genomics and Systems Biology Division
  5. Univ. of North Carolina, Chapel Hill, NC (United States). Dept. of Biology
  6. Univ. of North Carolina, Chapel Hill, NC (United States). Howard Hughes Medical Inst., Carolina Center for Genome Sciences, Dept. of Genetics and Molecular Biology, Dept. of Biology, and Dept. of Microbiology and Immunology
  7. USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division and Environmental Genomics and Systems Biology Division
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Contributing Org.:
Murdoch Univ., WA (Australia)
OSTI Identifier:
1465460
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Industrial Microbiology and Biotechnology
Additional Journal Information:
Journal Volume: 45; Journal Issue: 7; Related Information: © 2018, Society for Industrial Microbiology and Biotechnology.; Journal ID: ISSN 1367-5435
Publisher:
Springer
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Synthetic biology; Biosynthesis; Phenazine; Pathway; Refactored; Pathway design

Citation Formats

Coates, R. Cameron, Bowen, Benjamin P., Oberortner, Ernst, Thomashow, Linda, Hadjithomas, Michalis, Zhao, Zhiying, Ke, Jing, Silva, Leslie, Louie, Katherine, Wang, Gaoyan, Robinson, David, Tarver, Angela, Hamilton, Matthew, Lubbe, Andrea, Feltcher, Meghan, Dangl, Jeffery L., Pati, Amrita, Weller, David, Northen, Trent R., Cheng, Jan-Fang, Mouncey, Nigel J., Deutsch, Samuel, and Yoshikuni, Yasuo. An integrated workflow for phenazine-modifying enzyme characterization. United States: N. p., 2018. Web. doi:10.1007/s10295-018-2025-5.
Coates, R. Cameron, Bowen, Benjamin P., Oberortner, Ernst, Thomashow, Linda, Hadjithomas, Michalis, Zhao, Zhiying, Ke, Jing, Silva, Leslie, Louie, Katherine, Wang, Gaoyan, Robinson, David, Tarver, Angela, Hamilton, Matthew, Lubbe, Andrea, Feltcher, Meghan, Dangl, Jeffery L., Pati, Amrita, Weller, David, Northen, Trent R., Cheng, Jan-Fang, Mouncey, Nigel J., Deutsch, Samuel, & Yoshikuni, Yasuo. An integrated workflow for phenazine-modifying enzyme characterization. United States. doi:10.1007/s10295-018-2025-5.
Coates, R. Cameron, Bowen, Benjamin P., Oberortner, Ernst, Thomashow, Linda, Hadjithomas, Michalis, Zhao, Zhiying, Ke, Jing, Silva, Leslie, Louie, Katherine, Wang, Gaoyan, Robinson, David, Tarver, Angela, Hamilton, Matthew, Lubbe, Andrea, Feltcher, Meghan, Dangl, Jeffery L., Pati, Amrita, Weller, David, Northen, Trent R., Cheng, Jan-Fang, Mouncey, Nigel J., Deutsch, Samuel, and Yoshikuni, Yasuo. Thu . "An integrated workflow for phenazine-modifying enzyme characterization". United States. doi:10.1007/s10295-018-2025-5. https://www.osti.gov/servlets/purl/1465460.
@article{osti_1465460,
title = {An integrated workflow for phenazine-modifying enzyme characterization},
author = {Coates, R. Cameron and Bowen, Benjamin P. and Oberortner, Ernst and Thomashow, Linda and Hadjithomas, Michalis and Zhao, Zhiying and Ke, Jing and Silva, Leslie and Louie, Katherine and Wang, Gaoyan and Robinson, David and Tarver, Angela and Hamilton, Matthew and Lubbe, Andrea and Feltcher, Meghan and Dangl, Jeffery L. and Pati, Amrita and Weller, David and Northen, Trent R. and Cheng, Jan-Fang and Mouncey, Nigel J. and Deutsch, Samuel and Yoshikuni, Yasuo},
abstractNote = {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 have 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.},
doi = {10.1007/s10295-018-2025-5},
journal = {Journal of Industrial Microbiology and Biotechnology},
number = 7,
volume = 45,
place = {United States},
year = {2018},
month = {3}
}

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