4.7 Article

Organism Engineering for the Bioproduction of the Triaminotrinitrobenzene (TATB) Precursor Phloroglucinol (PG)

Journal

ACS SYNTHETIC BIOLOGY
Volume 8, Issue 12, Pages 2746-2755

Publisher

AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.9b00393

Keywords

synthetic biology; military environments; Tri-Service; metabolic engineering; enzyme mining; TX-TL; cell-free sensing

Funding

  1. U.S. Defense Advanced Research Projects Agency (DARPA) awards [HR0011-15-0084, FA8750-17-C-0229]
  2. Office of Naval Research Multidisciplinary University Research Initiative Award [N00014-16-1-2388]
  3. Army Research Office Multidisciplinary University Research Initiative Awards [W911NF-18-1-0200, W911NF-16-1-0372]
  4. Air Force Research Laboratory Center of Excellence Grant [FA8650-15-2-5518]
  5. Institute for Collaborative Biotechnologies [W911NF-09-0001, W911NF-19-2-0026]
  6. U.S. Army Research Office
  7. Office of Naval Research Vannevar Bush Faculty Fellowship [N00014-16-1-2509]
  8. Laboratory University Collaboration Initiative (LUCI) program
  9. Synthetic Biology for Military Environments Applied Research for the Advancement of S&T Priorities (ARAP) program of the U.S. Office of the Under Secretary of Defense for Research and Engineering

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Organism engineering requires the selection of an appropriate chassis, editing its genome, combining traits from different source species, and controlling genes with synthetic circuits. When a strain is needed for a new target objective, for example, to produce a chemical-of-need, the best strains, genes, techniques, software, and expertise may be distributed across laboratories. Here, we report a project where we were assigned phloroglucinol (PG) as a target, and then combined unique capabilities across the United States Army, Navy, and Air Force service laboratories with the shared goal of designing an organism to produce this molecule. In addition to the laboratory strain Escherichia coli, organisms were screened from soil and seawater. Putative PG-producing enzymes were mined from a strain bank of bacteria isolated from aircraft and fuel depots. The best enzyme was introduced into the ocean strain Marinobacter atlanticus CP1 with its genome edited to redirect carbon flux from natural fatty acid ester (FAE) production. PG production was also attempted in Bacillus subtilis and Clostridium acetobutylicum. A genetic circuit was constructed in E. coli that responds to PG accumulation, which was then ported to an in vitro paper-based system that could serve as a platform for future low-cost strain screening or for in-field sensing. Collectively, these efforts show how distributed biotechnology laboratories with domain-specific expertise can be marshalled to quickly provide a solution for a targeted organism engineering project, and highlights data and material sharing protocols needed to accelerate future efforts.

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