Fig. 4: Design and construction of dark reaction in E. coli.
a Designing a synthetic CO2-fixation pathway-ADRP. Abbreviation: DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate; ATP, adenosine triphosphate; NADH, nicotinamide adenine dinucleotide reduced; C1, C1 module; C3, C3 module; ADRP, artificial dark reaction pathway. b The specific activity of stAckA, ecASD, bsMgsA, and ecLDH. c SDS-PAGE of purified enzymes (ClFDH, FR, FLS*, scDHAK, bsMgsA, and ecLDH) in ADRP. The unit is kDa. d Schematic diagram of ADRP reconstruction in vitro for one-pot catalysis. Abbreviation: ATP, adenosine triphosphate; NADH, nicotinamide adenine dinucleotide reduced; TPP, thiamine pyrophosphate. e Time curve of pyruvate accumulation in reaction supernatant with full ADRP enzymes and the control without ClFDH. f Schematic diagram of carbon atoms transit to pyruvate via glycolysis pathway and ADRP. The labeled (13C) and unlabeled carbon atoms were represented by closed and open circles, respectively. m + 0 represents metabolites without 13C atom and m + 3 represents metabolites with three 13C atoms. Abbreviation: DHAP, dihydroxyacetone phosphate. g Relative abundance of mass isotopomers for intracellular formate, DHA, MET, and pyruvate in strains TD-F0 and TD-F1 under 24 h anaerobic fermentation (NaH13CO3 and glucose were used as co-substrates; 5 mM ATP, 5 mM NADH, and glucose were exogenously added to medium to provide additional metabolic energy in this experiment). m + 0 represented metabolites without 13C atom, m + 1 represented metabolites with one 13C atom, m + 2 represented metabolites with two 13C atoms, and m + 3 represented metabolites with three 13C atoms. Abbreviation: DHA, dihydroxyacetone; MET, methylglyoxal. Values were shown as mean ± s.d. from three biological replicates (b, e, g). All experiments were repeated independently three times with similar results (c). *p < 0.05, **p < 0.01, ***p < 0.001, n.s., no significance, as determined by two-sided t test. Source data are provided as a Source Data file.
