Fig. 2

Development and characterization of the thiosulfate-inducible IAA biosynthesis. (A) Schematic representation of the thiosulfate-inducible IAA-producing plasmids, comprising both the thiosulfate-responsive genetic circuit and the IAA biosynthesis pathway. The ThsS-ThsR proteins of the two-component regulatory system (TCS) are constitutively expressed from the J23114 promoter. The binding of thiosulfate to ThsS triggers a phosphorylation process, creating a complex that phosphorylates ThsR. This phosphorylated ThsR subsequently activates the PphsA promoter, which in turn triggers the expression of the IAA biosynthesis pathway. The pPhsA-IAA4 and pPhsA-IAA5 plasmids contain an operon of iad1, aspC, and ipdC genes with the B0034 RBS for each, while pPhsA-IAA6 and pPhsA-IAA7 additionally have a RiboJ insulator before the iad1 gene. The pPhsA-IAA5 and pPhsA-IAA7 plasmids include the ThsR(D57A) null mutant. (B) Cell growth (OD₆₀₀) and (C) IAA titer (mg/L) for pPhsA-IAA4 and pPhsA-IAA5 in the engineered EcN strains with increasing thiosulfate concentrations (0, 0.04, 0.2, and 1 mM). (D) Cell growth (OD₆₀₀) and (E) IAA yield (mg/L) for pPhsA-IAA6 and pPhsA-IAA7 plasmids in the engineered EcN strains in response to the increasing concentrations of thiosulfate (0, 0.04, 0.2, and 1 mM). The engineered EcN strains were cultivated in an IAA production medium with 2 g/L tryptophan and various thiosulfate levels for 24 h at 37 °C. Cell growth and IAA concentrations were measured using a UV-VIS spectrophotometer and HPLC analysis, respectively. Data represent the mean and SD from three biological replicates