An integrated workflow for phenazine-modifying enzyme characterization |
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| Authors: | 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 David Weller Trent R Northen Jan-Fang Cheng Nigel J Mouncey Samuel Deutsch Yasuo Yoshikuni |
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| Institution: | 1.US DOE Joint Genome Institute,Walnut Creek,USA;2.Environmental Genomics and Systems Biology Division,Lawrence Berkeley National Laboratory,Berkeley,USA;3.USDA Agricultural Research Service, Wheat Health, Genetics and Quality,Washington State University,Pullman,USA;4.Department of Plant Pathology,Washington State University,Pullman,USA;5.Department of Biology,University of North Carolina at Chapel Hill,Chapel Hill,USA;6.Howard Hughes Medical Institute,University of North Carolina at Chapel Hill,Chapel Hill,USA;7.Curriculum in Genetics and Molecular Biology,University of North Carolina at Chapel Hill,Chapel Hill,USA;8.Department of Microbiology and Immunology,University of North Carolina at Chapel Hill,Chapel Hill,USA;9.Carolina Center for Genome Sciences,University of North Carolina at Chapel Hill,Chapel Hill,USA;10.Biological Systems and Engineering Division,Lawrence Berkeley National Laboratory,Berkeley,USA |
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| 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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