Production of Vitamin B₁₂ in <i>Escherichia coli</i> Using a Thermal Switch to Control Pathway Genes

Isopropyl β-D-thiogalactopyranoside (IPTG), while widely utilized for inducing gene expression in systems governed by T7<i>lac</i> 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 <i>lacI</i> 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 T7<i>lac</i> or other <i>lac</i>-derived promoters containing <i>lac</i> 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 <i>lacI</i>. 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.