High-throughput mapping of CoA metabolites by SAMDI-MS to optimize the cell-free biosynthesis of HMG-CoA.

ABSTRACT: 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

SUBMITTER: O'Kane PT 

PROVIDER: S-EPMC6551189 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature