Figure 1. Quantification of alternative routes of PEP generation during growth on acetate.

(a) Schematic showing two routes of PEP synthesis during growth on acetate. After malate is produced via glyoxylate shunt, malic enzyme (maeAB) can convert malate to PYR, from which PEP can be formed by the activity of ppsA or ptsI. Alternatively, pck can convert oxaloacetate (OAC) to PEP directly. (b) Growth rates of four strains during growth on acetate, wild-type (WT), ΔppsA, ΔptsI and the double-knockout ΔppsAΔptsI. (c) Labelling of valine from [1-¹³C]alanine, reflecting PYR labelling. Labelling is M1 (from tracer) and M0 (from unlabelled precursors in central metabolism). (d) Labelling of aspartate from [1-¹³C]alanine, reflecting OAC labelling. Aspartate is almost entirely unlabelled (M0). (e) Labelling of the first two (C1–C2) carbons of phenylalanine, reflecting the labelling of the first two carbons of PEP. (f) Schematic depicting the conversion of [1-¹³C]alanine to PEP and the measured amino acids. Opened and filled circles represent unlabelled (¹²C) and labelled (¹³C) carbons, respectively. (g) Percentage of PEP generated from PYR. Approximately 60% of PEP is generated from PYR in the WT and each single knockout strain; however, the flux is completely eliminated in the double knockout, indicating dual responsibility of ppsA and ptsI for the conversion of PYR to PEP. Data presented in b are mean±s.e.m. of two biological replicates. Labelling data in c–e have been corrected for natural abundances and unlabelled biomass present before tracer introduction. The error presented in g reflects the propagation of GC-MS measurement error through the calculation.