Targeted metabolomic analyses at the pathway and organism level provide functional information for both pathway and host-engineering research. 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 the engineered pathway as well as central carbon metabolism aids identification of bottlenecks, and helps identify where increasing specific protein levels can yield dramatic improvements to the product titer or where allosteric regulation is limiting flux through the pathway. These assays are conducted by using an ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) system to rapidly acquire high-resolution, high-mass accuracy mass spectra and structural information of metabolites. High-throughput targeted metabolomic methods for engineered microbes utilize laboratory automation to reduce sample preparation variability, high-sensitivity UHPLC-MS detection, and leverage tight integration with the ABF ICE sequence/strain repository platform and Experiment Data Depot (EDD). Rapid quantification of metabolite levels in engineered microbes benefit the ABF by reducing DBTL cycle time. 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. Furthermore, metabolites have different physiochemical properties so chromatographic performance and limit of detection will vary depending on the metabolite assayed. Available internally to ABF researchers, as well as to ABF CRADA projects.
References and Additional Information:
Yaegashi, Junko, et al. “Rhodosporidium toruloides: a new platform organism for conversion of lignocellulose into terpene biofuels and bioproducts.” Biotechnology for biofuels 10.1 (2017): 241.
George, Kevin W., et al. “Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli.” Metabolic engineering (2018).
Brunk, Elizabeth, et al. “Characterizing strain variation in engineered E. coli using a multi-omics-based workflow.” Cell systems 2.5 (2016): 335-346.
Lawrence Berkeley National Laboratory
Pacific Northwest National Laboratory