Xcalibur 3.0 quan browser

Independent ¹³C-tracer experiments, as described in “Metabolite monitoring and quantification,” confirmed that extracellular depletion of the substrates correlated with substrate consumption. The extracellular depletion of each carbohydrate substrate (three biological replicates) was determined throughout 24 h of cell growth. Culture aliquots were pelleted by centrifugation, and the supernatants were stored at −20°C until further analysis. According to previously reported LC methods for carbohydrate analysis (21 (link)), we applied an analytical method using LC-HRMS for monitoring the carbohydrate concentration in the extracellular solution. Peak identification and quantification of carbohydrate concentrations were conducted with Thermo Scientific Xcalibur 3.0 Quan Browser. Carbohydrate consumption rates (in millimoles per gram [dry weight] per hour) were obtained by combining the regression analyses of carbohydrate depletion over time with biomass growth rates (Table S2).

Aliquots of cell cultures were collected periodically throughout growth, filtered (Costar Spin-X 0.22-μm-pore-size filter), and stored at −20°C until liquid chromatography–high-resolution mass spectrometry (LC-HRMS) analysis. Concentrations of substrates and extracellular metabolites were determined from standards prepared from commercial chemicals (Millipore-Sigma, St. Louis, MO, or Fisher Scientific, Pittsburgh, PA). Extracellular samples were diluted to maintain concentrations within the standard range. Quantification of metabolite concentrations was conducted with a Thermo Scientific Xcalibur 3.0 Quan browser. Regression analysis on the substrate depletion and metabolite production over time was performed to determine consumption and secretion rates, respectively, normalized to biomass growth. Biomass yield (Y_X/S) was determined from the linear fit of the substrate concentration and g_CDW determined from sample aliquots corresponding to the same time point throughout exponential growth.