Fig. 4: Evidence of cytosolic PDH and glyoxylate shunt activities inferred from 13C metabolic flux and 2H isotope tracing analyses.
Improvement in A SA production flux, B cytosolic PDH flux, and C cytosolic ICL flux in the strain EcGS-FF-gltA compared to the strain SA suggested by MFA. Error bars denote the 95% confidence intervals computationally determined by parameter continuation. D Schematic of [4-2H]glucose tracing. [4-2H]glucose labels cytosolic NADH (NAD2H) produced through glycolysis, while NADH produced through cytosolic PDH is unlabeled. FBP fructose-1,6-bisphosphate, DHAP dihydroxyacetone phosphate, GAP glyceraldehyde-3-phosphate, GAPDH glyceraldehyde-3-phosphate dehydrogenase, OAA oxaloacetate, MDH malate dehydrogenase, FUMR fumarase, FRD fumarate reductase, CIT citrate synthase, PDH pyruvate dehydrogenase. E 2H-labeled M + 1 fraction of SA in the wild-type strain (WT), the parental strain SA, and the improved strain EcGS-FF-gltA. The wild-type strain showed no labeling of SA that was synthesized only in mitochondria. The parental strain SA showed 2H-labeling of SA produced through the rTCA pathway driven by NAD2H produced from GAPDH. The improved strain EcGS-FF-gltA showed lower 2H-labeling of SA because NAD2H was diluted with unlabeled NADH produced by cytosolic IoPDH and the glyoxylate shunt produced unlabeled SA without requiring NADH. Error bars, mean ± SD (n = 3 biologically independent samples). Two-tailed unpaired t-test was used to calculate p value. Source data are provided as a Source Data file.
