Tools for strain improvement, such as metabolic flux analysis, rely heavily on accurate metabolite data and carbon-flux measurements to restrict parameters for model predictions. 

Monitoring metabolites that are part of the engineered pathway as well as central carbon metabolism aids in identifying bottlenecks. We can identify where increasing specific protein levels can yield dramatic improvements to the product titer, or where allosteric regulation is limiting flux through the pathway. 

We conduct targeted metabolomic assays using ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) and gas chromatography-mass spectrometry (GC-MS) to rapidly acquire high-resolution, high-mass accuracy mass spectra and structural information of metabolites as well as their absolute concentrations if standards are available. 

Our high-throughput targeted metabolomic methods for engineered microbes utilize laboratory automation to reduce sample preparation variability, high-sensitivity UHPLC-MS and GC-MS detection, and leverage tight integration with the Agile BioFoundry’s ICE sequence/strain repository platform and Experiment Data Depot. 

By rapidly quantifying metabolite levels in engineered microbes, we can reduce DBTL cycle times. The sensitivity of targeted metabolomic assays are limited by the number of cells used to extract the metabolites, the amount of the metabolites inside the cell, and the availability of metabolite standards. Metabolites have different physicochemical properties, so chromatographic performance and limit of detection will vary depending on the metabolite assayed.