Lycopene is a natural carotenoid with strong antioxidant properties, widely used in fields such as food, health products, and medicine. In recent years, the production of lycopene through microbial fermentation based on the isopentenol utilization (IU) pathway has shown great potential. To address the toxicity of IU pathway substrates isoprenol and prenol to the host, the molecular chaperone protein SecB was overexpressed in Escherichia coli (E. coli) BL21 (DE3). A series of E. coli cell factories for lycopene production were constructed through modular engineering strategies, and the fermentation conditions were optimized by single factor experiments. The results showed that overexpression of SecB helped maintain cellular structural integrity, significantly reduced cell specific surface area (area/volume), and significantly enhanced cell membrane integrity and maintained membrane potential, thereby effectively alleviating the inhibition of substrate toxicity on cell growth and metabolism. Among all the constructed cell factories, the recombinant strain LYC3 containing IU pathway module, lycopene synthesis module, and SecB expression module had the highest lycopene yield of 8.99 mg/L, indicating that overexpression of SecB could enhance the synthesis of lycopene by E. coli. The optimized condition for the shake flask fermentation of recombinant strain LYC3 were as follows: LYC medium, 40 mmol/L substrate, simultaneous addition of substrate and inducer, and fermentation for 72 h. Under this optimized condition, the lycopene yield was further increased to 51.90 mg/L. This study laid a theoretical and technical foundation for the efficient synthesis of lycopene based on the IU pathway, and provided a reference for improving the efficiency of biomanufacturing through substrate tolerance engineering.