An integrated workflow for phenazine-modifying enzyme characterization
Abstract
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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- 0000 0004 0449 479X grid.451309.a US DOE Joint Genome Institute Walnut Creek CA USA
- 0000 0004 0449 479X grid.451309.a US DOE Joint Genome Institute Walnut Creek CA USA, 0000 0001 2231 4551 grid.184769.5 Environmental Genomics and Systems Biology Division Lawrence Berkeley National Laboratory Berkeley CA USA
- 0000 0001 2157 6568 grid.30064.31 USDA Agricultural Research Service, Wheat Health, Genetics and Quality Washington State University Pullman WA USA, 0000 0001 2157 6568 grid.30064.31 Department of Plant Pathology Washington State University Pullman WA USA
- 0000 0001 2231 4551 grid.184769.5 Environmental Genomics and Systems Biology Division Lawrence Berkeley National Laboratory Berkeley CA USA
- 0000000122483208 grid.10698.36 Department of Biology University of North Carolina at Chapel Hill Chapel Hill NC USA
- 0000000122483208 grid.10698.36 Department of Biology University of North Carolina at Chapel Hill Chapel Hill NC USA, 0000000122483208 grid.10698.36 Howard Hughes Medical Institute University of North Carolina at Chapel Hill Chapel Hill NC USA, 0000000122483208 grid.10698.36 Curriculum in Genetics and Molecular Biology University of North Carolina at Chapel Hill Chapel Hill NC USA, 0000000122483208 grid.10698.36 Department of Microbiology and Immunology University of North Carolina at Chapel Hill Chapel Hill NC USA, 0000000122483208 grid.10698.36 Carolina Center for Genome Sciences University of North Carolina at Chapel Hill Chapel Hill NC USA
- 0000 0004 0449 479X grid.451309.a US DOE Joint Genome Institute Walnut Creek CA USA, 0000 0001 2231 4551 grid.184769.5 Environmental Genomics and Systems Biology Division Lawrence Berkeley National Laboratory Berkeley CA USA, 0000 0001 2231 4551 grid.184769.5 Biological Systems and Engineering Division Lawrence Berkeley National Laboratory Berkeley CA USA
- 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)
- Contributing Org.:
- Murdoch Univ., WA (Australia)
- OSTI Identifier:
- 1773508
- Alternate Identifier(s):
- OSTI ID: 1465460
- Grant/Contract Number:
- AC02-05CH11231
- Resource Type:
- Published Article
- Journal Name:
- Journal of Industrial Microbiology and Biotechnology
- Additional Journal Information:
- Journal Name: Journal of Industrial Microbiology and Biotechnology Journal Volume: 45 Journal Issue: 7; Journal ID: ISSN 1367-5435
- Publisher:
- Oxford University Press
- Country of Publication:
- Germany
- 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. Germany: 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. Germany. https://doi.org/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. Sun .
"An integrated workflow for phenazine-modifying enzyme characterization". Germany. https://doi.org/10.1007/s10295-018-2025-5.
@article{osti_1773508,
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 = {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.},
doi = {10.1007/s10295-018-2025-5},
journal = {Journal of Industrial Microbiology and Biotechnology},
number = 7,
volume = 45,
place = {Germany},
year = {Sun Jul 01 00:00:00 EDT 2018},
month = {Sun Jul 01 00:00:00 EDT 2018}
}
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https://doi.org/10.1007/s10295-018-2025-5
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