Journal: bioRxiv

Article Title: Expanding the scope of redox-balance growth coupling techniques with a carbon cofeeding strategy

doi: 10.64898/2026.04.01.713023

Figure Lengend Snippet: Schematic depicting the genetic modifications to enable the overaccumulation of NADPH (Δ pgi , Δ edd , Δ qor , Δ sthA ) and the deletion of aceA to prohibit growth on acetate as a carbon source. Pathways enabling growth on a mixture of acetate and glucose are shown below and highlighted (red: acetaldehyde (strain APEQS_PduP), yellow: 3-HB (strain APEQS_3-HB), blue: mevalonate (strain APEQS_MEV_sa)). b, Simplified metabolic stoichiometries showing only bioavailable carbon and relevant reducing equivalents and assuming all carbon flows to acetyl-CoA (our pathways’ precursor molecule). Simplified stoichiometry of E. coli fermentative metabolism and APEQS metabolism when grown on glucose, showing redox-balanced fermentative or rescue pathways below. Ethanol fermentation requires less acetyl-CoA (green) than is produced from one glucose when redox balanced, reflecting its suitability as a fermentation pathway. Partially reducing pathways consume more acetyl-CoA (red) than is made available per unit glucose when redox balanced, indicating their inability to resolve redox balance in APEQS without acetate co-feeding. c, Unsuccessful growth coupling of strain APEQS_PduP in the absence of acetate (n=3). d, Unsuccessful growth coupling of strain APEQS_3-HB in the absence of acetate (n=3). e, Unsuccessful growth coupling of strain APEQS_MEV_sa in the absence of acetate (n=3). f, Successful growth coupling of strain APEQS_PduP when the strain is grown with additional 100 mM sodium acetate to satisfy stoichiometric constraints (n=3). g, Successful growth coupling of strain APEQS_3-HB when the strain is grown with additional 100 mM sodium acetate to satisfy stoichiometric constraints (n=3). h, Successful growth coupling of strain APEQS_MEV_sa when the strain is grown with additional 100 mM sodium acetate to satisfy stoichiometric constraints (n=3). All experiments were conducted in a MOPS medium containing 2% glucose with or without 100 mM acetate. Various concentrations of IPTG were added to modulate the induction of the three partially reducing pathways (high [IPTG]: blue (0.5 mM for p15A-based A5c backbone; 0.05 mM for ColE1-based pQE backbone), medium [IPTG]: purple (0.05 mM for p15A-based A5c backbone; 0.005 mM for ColE1-based pQE backbone), no IPTG: red). An empty vector control was included to demonstrate growth without leaky expression (orange). All growth experiments were repeated a minimum of 3 times and showed identical results.

Article Snippet: For growth coupling experiments and strain evolution, bacterial strains were grown in a minimal MOPS medium (Teknova M2106) containing 2% glucose (w/v) supplemented with the appropriate antibiotic based on the plasmid(s) present in the strain.

Techniques: Produced, Plasmid Preparation, Control, Expressing