Fig. 4. Activity of the GED shunt in a ∆PZF strain that enables a smooth transition into a GED cycle.

a Design of the ∆PZF (∆pfkAB ∆zwf ∆fsaAB ∆fruK) selection scheme. Xylose can be assimilated only via the activity of the GED shunt, where the biosynthesis of most biomass building blocks is dependent on the pathway (marked in yellow). Growth on gluconate (violet) is not dependent on reductive carboxylation via Gnd and thus serves as a positive control. Reaction directionalities are shown as predicted by flux balance analysis. b Growth on xylose upon overexpression of gnd, edd, and eda (pGED) was achieved only after adaptive evolution and was dependent on elevated CO₂ concentration. Values in parentheses indicate doubling times. Curves represent the average of technical quadruplicates, which differ from each other by <5%. Growth experiments were repeated independently three times to ensure reproducibility. c ¹³C-labeling experiments confirm the operation of the GED shunt (mutant ‘C’). Cells were cultivated with xylose (1-¹³C) and ¹³CO₂. Observed labeling fits the expected pattern and differs from that of a WT strain cultured under the same conditions. Results from additional labeling experiments are shown in Supplementary Fig. 3. 3PG 3-phosphoglycerate, ALA Alanine, GAP glyceraldehyde-3-phosphate, GLY Glycine, HIS Histidine, PYR pyruvate, SER Serine, VAL Valine. Source data underlying b and c are provided as a Source Data file.