Fig. 5: Application of ReaL-MGE 1.0 in the construction of E. coli BL21 malonyl-CoA chassis cells.

a Scheme of the FapR biosensor. At low malonyl-CoA (left) the FapR repressor binds to fapO and prevents expression of the gfp/gentamicin resistance operon, rendering the host gentamicin sensitive. Elevated malonyl-CoA binds to FapR, inactivating its DNA binding to fapO and releasing expression of the gfp/gentamicin resistance operon. b Scheme of selection procedure to identify the elevated malonyl-CoA E. coli host. The second recovery in ReaL-MGE 1.0 (i) before FACS sorting for GFP expression (ii) and plating on gentamicin to isolate single colonies (iii) that were picked into microplates for GFP quantitation (iv) and HP-LC determination of intracellular malonyl-CoA concentration (v). c FACS analysis from step (ii) SSC - side scatter, FSC - forward scatter. d Intracellular malonyl-CoA concentrations were determined for 150 candidates, leading to the choice of clone 33# (E. coli BL21.C33) for further work. e Evaluation of malonyl-CoA in E. coli BL21 and the E. coli BL21.C33 derivative. f Whole genome sequencing of E. coli BL21.C33 showing mutated sites. Green arrows, malonyl-CoA metabolic pathway promoter replacements; purple arrow, LVA insertion; orange blocks, transposase/prophage deletions. The xseA gene was also mutated by the plasmid-borne inactivation cassette (Fig. 3a) as intended. g Schematic of the mutated metabolic genes in E. coli BL21.C33. Values are means of the biological replicates, and the error bars indicate the standard deviations of all (n = 5) biological replicates. P-values were obtained using the two-tailed Student’s t test: ****P < 0.0001.