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Isotope-assisted metabolic pathway analysis in the marine diatom Phaeodactylum tricornutum reveals evidence for flux through the Entner-Doudoroff (ED) pathway . (a) Four different Metabolic Models (I-IV) were constructed to explain isotope labeling data from a 1-¹³C labeling experiment; each column represents a different model. Pathways included in a model are denoted by a “Y”, color-coded according to the color of the pathway in Figure 3. (b) Vertical bars represent the sum of squared residuals (SSR) of each model and horizontal lines represent the acceptable SSR corresponding to the number of redundant isotopomer measurements in each model. Models II and IV, both containing the ED pathway show a significantly decreased SSR compared to otherwise identical models lacking the ED pathway. (c) The carbon rearrangements of the Embden-Meyerhof-Parnas (EMP) and ED pathways are shown, with ¹³C atoms shown as blue squares and ¹²C atoms shown as white squares. The EMP pathway transfers ¹³C from glucose C-1 to pyruvate C-3, whereas the ED pathway transfers ¹³C from glucose C-1 to pyruvate C-1 (red boxes). (d) The isotopomers of the amino acid alanine reflect those of pyruvate. The measured abundances of alanine isotopomers are compared against the simulated enrichments of Models I and II. Model I that lacks the ED pathway over-simulates the abundance Ala{123}, and under-simulates the abundance of Ala{123}. These errors are corrected in Model II, which utilizes the ED pathway. Abbreviations not defined above: PPP, pentose phosphate pathway; TCA, tricarboxylic acid cycle. Isotopomer notation: bold=¹³C, regular font=¹²C. See our publication for further pathway analysis and more detail.