Fig. 7: Validation of the ability of ReaL-MGE 2.0 to mediate simultaneous editing of multiple kilobase-scale sequences in S. brevitalea DSM7029.

a Flow chart of the screening of S. brevitalea DSM7029 mutants with modified malonyl-CoA metabolic network by glidobactin yield. Using ReaL-MGE 2.0 to mediate 22 kilobase-scale sequence insertion in S. brevitalea DSM7029 (i), the bacterial solution was spread on the plate containing an elevated concentration of gentamicin (30 μg mL⁻¹) (i). Colonies were selected from plates and inoculated into 96-well plates for culture (iii). The 254 nm absorption intensity of the selected mutants was detected (iv). b Absorbance at 254 nm, intracellular malonyl-CoA concentrations, and glidobactins’ yield determined for 103 selected mutants leading to the choice of clones 43# (S. brevitalea DSM7029.C43) and 89# (S. brevitalea DSM7029.C89) for further detection. c Whole genome sequencing of S. brevitalea DSM7029.C89 showing mutated sites. Green arrows, malonyl-CoA metabolic pathway promoter replacements; purple arrows, LVA insertion.