Center for Synthetic Biology, Northwestern University, Evanston, IL, USA.
Department of Chemistry, Northwestern University, Evanston, IL, USA.
Sci Adv. 2019 Jun 5;5(6):eaaw9180. doi: 10.1126/sciadv.aaw9180. eCollection 2019 Jun.
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.
代谢工程利用酶来生产具有工业、制药和能源应用的小分子。然而，为了实现商业生产而优化酶途径的努力受到了用于定量代谢中间产物和终产物的分析方法通量的限制。我们开发了一种用于分析 CoA 依赖性途径的多重分析方法，该方法使用末端为半胱氨酸的肽来共价捕获与 CoA 结合的代谢物。然后，通过将捕获的代谢物固定在自组装单层的阵列上，与复杂混合物快速分离，并通过 SAMDI 质谱直接定量。我们通过对异戊二烯生物合成中关键中间体 HMG-CoA 的无细胞合成进行特征分析，展示了该分析方法的通量，收集了超过 10000 张单独的光谱，绘制了 800 多个独特的反应条件。我们预计，我们快速而稳健的分析方法将加速代谢途径的工程改造。
Zotero 插件
