Skip to main content
ARS Home » Pacific West Area » Pullman, Washington » WHGQ » Research » Publications at this Location » Publication #352112

Research Project: Biology, Ecology, and Genomics of Pathogenic and Beneficial Microorganisms of Wheat, Barley, and Biofuel Brassicas

Location: Wheat Health, Genetics, and Quality Research

Title: An integrated workflow for phenazine biosynthetic gene cluster discovery and characterization

Author
item Coates, R. Cameron - Joint Genome Institute
item Bowen, Benjamin - Joint Genome Institute
item Oberortner, Ernst - Joint Genome Institute
item Thomashow, Linda
item Hadjithomas, Michalis - Joint Genome Institute
item Zhao, Zhiying - Joint Genome Institute
item Ke, Jing - Joint Genome Institute
item Silva, Leslie - Joint Genome Institute
item Louie, Katherine - Joint Genome Institute
item Wang, Gaoyan - Joint Genome Institute
item Robinson, David - Joint Genome Institute
item Tarver, Angela - Joint Genome Institute
item Hamilton, Matthew - Joint Genome Institute
item Lubbe, Andrea - Lawrence Berkeley National Laboratory
item Feltcher, Meghan - University Of North Carolina
item Dangl, Jeff - University Of North Carolina
item Pati, Amrita - Joint Genome Institute
item Weller, David
item Northen, Trent - Joint Genome Institute
item Cheng, Jan-fang - Joint Genome Institute
item Mouncey, Nigel - Joint Genome Institute
item Deutsch, Sam - Joint Genome Institute
item Yoshikuni, Yasuo - Joint Genome Institute

Submitted to: Journal of Industrial Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/9/2018
Publication Date: 7/20/2018
Citation: Coates, R., Bowen, B.P., Oberortner, E., Thomashow, L.S., Hadjithomas, M., Zhao, Z., Ke, J., Silva, L., Louie, K., Wang, G., Robinson, D., Tarver, A., Hamilton, M., Lubbe, A., Feltcher, M., Dangl, J., Pati, A., Weller, D.M., Northen, T.R., Cheng, J., Mouncey, N.J., Deutsch, S., Yoshikuni, Y. 2018. An integrated workflow for phenazine biosynthetic gene cluster discovery and characterization. Journal of Industrial Microbiology and Biotechnology. 45(7):567-577. https://doi.org/10.1007/s10295-018-2025-5.
DOI: https://doi.org/10.1007/s10295-018-2025-5

Interpretive Summary: Hundreds of new DNA sequences for microorganisms and biosynthetic gene clusters (BGCs) continue to be discovered, increasing the potential to develop and employ novel chemical diversity for agriculture, medicine, environmental and industrial purposes. However, technical difficulties associated with expression of such genes in the laboratory can make it difficult to characterize their function. To overcome these problems, we have developed a workflow for BGC characterization that integrates pathway identification, modular design, DNA synthesis, assembly and characterization. We used this workflow to characterize multiple phenazine BGCs. These pathways are well-suited to the workflow because all phenazines are derived from a common core scaffold that is subsequently modified by such enzymes as PhzM, PhzS, PhzH, and PhzO that lead to the production of well-characterized compounds. We expressed refactored synthetic modules of previously uncharacterized phenazine BGCs in the model bacterium Escherichia coli and were able to identify metabolic intermediates the strains produced, including a previously unidentified metabolite. Our results show how this approach can accelerate functional characterization of BGCs.

Technical 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 BGCs. Phenazine pathways are useful for this workflow because all phenazines are derived from a core scaffold for modification 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.