Fig. 4: Modules 2 and 4 are functional together in an E. coli selection strain.

A Selection scheme to demonstrate CORE cycle functionality by coupling its activity to growth: Rational deletion of serA and gcvTHP achieves a selection strain (“C1S-Aux”) which is auxotrophic to serine and one-carbon units (CORE cycle-dependent biomass precursors highlighted in yellow). Its growth can be rescued by production of these precursors from formate via a heterologous pathway integrated in the chromosome (purple arrows), thus establishing a selection for formate-production via the CORE cycle. Expression of a β-alanine:pyruvate-transaminase (BPT) allows intracellular generation of malonate semialdehyde (MSA), while deletion of two native reductases (∆ydfG, ∆rutE) removes sink reactions converting it to 3-hydroxypropionate (3-HP). MSA and acetyl-CoA serve as substrates for BKACE, producing acetoacetate and formyl-CoA, which is hydrolyzed to formate spontaneously or converted via endogenous thioesterases/CoA-transferases. This metabolic scheme selects for the activity of module 2 and 4 together (blue, pink). B When expressed together with BPT, six out of seven tested BKACE-candidates rescue growth of the C1S-Aux strain. No growth is observed for a strain expressing only BPT but lacking BKACE or vice-versa. C Growth of the C1S-Aux + pZ-BKACE15-BPT strain is strictly dependent on supplementation of β-alanine in the medium, while the growth rate increases with the supplied β-alanine concentration. Shown data in (B) and (C) represent mean maximal growth rate values estimated from triplicate cultures (or quadruplicates for selected strains) grown in a 96-well plate. Error bars indicate standard deviation. Source data are provided as a Source Data file.