Fig. 3

Validation of the hemiautotrophic phenotype in the reconstructed strain. a We grew the reconstructed, evolved, and ancestral clones with isotopically labeled ¹³CO₂ as an inorganic carbon source and non-labeled pyruvate as an energy source, and performed isotopic labeling analysis. Amino acids derived from the CBB cycle (e.g., serine) are labeled green, whereas amino acids derived from the energy module (e.g., alanine) are in blue. As predicted for a hemiautotrophic growth mode, amino acids derived from the intermediates of the CBB module are almost fully labeled in the reconstructed and evolved strain but not in the ancestor strain. In marked contrast, amino acids derived from the TCA and lower glycolysis  (energy module) show low levels of labeling. This validates that the reconstructed and evolved strains synthesize CBB module biomass precursors from CO₂ using the non-native CBB cycle. b We propagated cells in minimal media supplemented with non-labeled pyruvate (5 g/L) and labeled ¹³CO₂ atmosphere (0.1 atm). We used an elemental mass analyzer to measure the isotopic carbon composition in the total cell biomass (mean ± SD; n = 4). The reconstructed strain shows one-third of cellular carbon originated from fixed inorganic carbon, a value similar to the evolved strains and in line with the expected fraction of biomass precursors originating from the CBB module. The ancestor strain required the addition of xylose (non-labeled, 0.2 g/L) for growth. Labeled carbon in the ancestor strain results from the RuBisCO-dependent catabolism of xylose and from the assimilation of inorganic carbon via anaplerotic reactions