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

## TL;DR

Scientists created a way to produce vitamin B12 in bacteria without using a costly and toxic chemical by using temperature control instead.

## Contribution

A thermal switch was developed to replace IPTG for inducing gene expression in vitamin B12 production in Escherichia coli.

## Key findings

- Culturing at 37°C followed by a shift to 32°C maximized vitamin B12 production.
- Adding an ssrA degradation tag to lacI increased vitamin B12 titer by 37.96%.
- The thermal switch strategy eliminates the need for IPTG in vitamin B12 biosynthesis.

## Abstract

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 PR 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.