Fig. 3: Experimental investigation of methanol-dependent growth.

a, b Central metabolism of the methanol-dependent E. coli strains ΔfrmAΔfbp (a) and ΔfrmAΔtpiA (b) expressing the RuMP cycle genes mdh, hps and phi. Synthetic pathway reactions and enzymes are depicted in blue, the endogenous metabolic reactions are in gray and gene deletions are in black. The number of dots represents the number of carbons of the specific metabolite and the coloring. The dots are colored according to thepredicted methanol-derived metabolite fraction, whereby the part of the metabolite that stoichiometrically must originate from methanol is illustrated in blue, the one that is produced from pyruvate in red. c, d Methanol-dependent growth of ΔfrmAΔfbp (c) (n = 5) and ΔfrmAΔtpiA (d) (n = 4) expressing Cupriavidus necator mdh2 CT4-1 from pSEVA424 and Methylobacillus flagellatus hps and phi from pSEVA131. Methanol-dependent strains were cultivated in minimal medium supplemented with both methanol (500 mM) plus pyruvate (20 mM) (purple), methanol (500 mM) as the sole carbon source (blue) and pyruvate (20 mM) as the sole carbon source (red). The biological replicates in the multi-substrate condition (purple) are plotted separately. Error bars represent the standard deviation and are not visible because they are smaller than the size of the markers. For abbreviations, see Fig. 1. Source data are provided as a Source Data file.