Production of Vitamin B₁₂ in Escherichia coli Using a Thermal Switch to Control Pathway Genes

Isopropyl β-D-thiogalactopyranoside (IPTG), while widely utilized for inducing gene expression in systems governed by T7lac and related promoters, poses significant challenges due to its toxicity and expense, prompting the exploration of alternative induction strategies. In this study, we developed a series of inducer-free vitamin B₁₂-producing strains featuring thermally regulated pathway genes. We engineered a thermal switch by replacing the lacI promoter with the P_R promoter, which is regulated by the temperature-sensitive repressor cI857 from the λ bacteriophage. As a result, target genes driven by T7lac or other lac-derived promoters containing lac operators were expressed upon lowering the temperature. Our findings indicate that culturing at 37°C and then shifting to 32°C when the optical density at 600 nm reaches 2 is the most effective strategy for vitamin B₁₂ production. Additionally, the vitamin B12 titer increased by 37.96% after introducing an ssrA degradation tag at the C-terminus of lacI. This study introduces a novel strategy for vitamin B₁₂ production that circumvents the need for IPTG by implementing a thermal switch. This approach may have significant implications for chemical bioproduction processes that have traditionally relied on IPTG for gene induction.