Journal
SCIENCE ADVANCES
Volume 5, Issue 6, Pages -Publisher
AMER ASSOC ADVANCEMENT SCIENCE
DOI: 10.1126/sciadv.aaw9180
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Funding
- Department of Energy (BER) [DE-SC0018249]
- Air Force Research Laboratory Center of Excellence Grant [FA8650-15-2-5518]
- David and Lucile Packard Foundation [2011-37152]
- Department of Defense, Defense Threat Reduction Agency [HDTRA1-15-1-0052]
- Dreyfus Teacher-Scholar Program
- U.S. Department of Energy (DOE) [DE-SC0018249] Funding Source: U.S. Department of Energy (DOE)
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Metabolic engineering uses enzymes to produce small molecules with industrial, pharmaceutical, and energy applications. However, efforts to optimize enzymatic pathways for commercial production are limited by the throughput of assays for quantifying metabolic intermediates and end products. We developed a multiplexed method for profiling CoA-dependent pathways that uses a cysteine-terminated peptide to covalently capture CoA-bound metabolites. Captured metabolites are then rapidly separated from the complex mixture by immobilization onto arrays of self-assembled monolayers and directly quantified by SAMDI mass spectrometry. We demonstrate the throughput of the assay by characterizing the cell-free synthesis of HMG-CoA, a key intermediate in the biosynthesis of isoprenoids, collecting over 10,000 individual spectra to map more than 800 unique reaction conditions. We anticipate that our rapid and robust analytical method will accelerate efforts to engineer metabolic pathways.
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