Fig. 2

Introduction and optimization of (S)-2,3-oxidosqualene biosynthesis pathway in E. coli and exhibits decreased membrane leakage and increased intracellular ATP level. a Full biosynthetic pathway of (S)-2,3-oxidosqualene consisting of native MEP pathway and heterologous pathway; b integration of heterologous S. cerevisiae terg9 gene, Methylococcus capsulatus smo gene with M1–93 artificial promoter into genomic DNA of E. coli MG1655 at the mgsA site and pta site, respectively, based on the two pathways genes, the native promoter of idi was replaced with artificial promoter M1–46. c GC–MS detection of (S)-2,3-oxidosqualene for the engineered strains. The engineered strain with the heterologous pathway and promoter replacement of rate-limiting enzyme idi synthesized the representative (S)-2,3-oxidosqualene, TMS derivative while control strain not. d Engineered strain had a 24% decrease in membrane leakage relative to the control strain when challenged with 5 mM H₂O₂. Membrane leakage was assessed using the SYTOX Green nucleic acid stain. e Engineered strain had a 70% increase in ATP content relative to the control strain during challenge with 5 mM H₂O₂. ATP content was assessed using an ATP assay kit (Beyotime). Error bars indicate standard deviation of at least three biological replicates. f We termed the engineered strain Iron Man E. coli (IME) with a figurative analogy of wearing “Iron Man’s armor”-like suit for E. coli cell