# Metabolic engineering of Escherichia coli BW25113 for the production of Vitamin K2 based on CRISPR/Cas9 mediated gene knockout and metabolic pathway modification

## TL;DR

Scientists engineered E. coli to produce more Vitamin K2 by modifying its genes and metabolic pathways, achieving high production levels in a lab setting.

## Contribution

A stepwise metabolic engineering strategy using CRISPR/Cas9 and pathway optimization to significantly increase Vitamin K2 production in E. coli.

## Key findings

- Overexpression of menA and ubiE genes led to 303 mg/L of MKH2-8 after 24 hours.
- Strain ΔB/MUWI achieved 859 mg/L in shake flasks and 1360 mg/L in a 5 L fermenter after 48 hours.
- Gene knockout and medium optimization significantly enhanced VK2 accumulation.

## Abstract

Vitamin K2 (VK2), as a derivative of the menaquinone family, plays an important role in the prevention of osteoporosis and cardiovascular calcification. The realization of the industrialization of VK2 and the reduction of its production cost have become the focus of attention.

In this work, an E. coli strain with high VK2 accumulation was constructed through rational metabolic engineering and stepwise improvement based on regulatory metabolic information and CRISPR/Cas9-mediated gene knockout. We first constructed a recombinant E. coli strain BW-T7/MU to produce menaquinol-8 (MKH2-8, a reduced form of VK2) by overexpressing menA and ubiE genes, which encoding the rate-limiting enzymes of the menaquinol pathway. After 24 h and 48 h of fermentation, this strain BW-T7/MU reach a titer of 303 mg/L and 232 mg/L. Secondly, we overexpressed different related genes wrbA (oxidative stress mitigation), qorB (reduction of quinones) and menF (conversion of chorismate to isochorismate), respectively. Among these recombinant strains, the strain BW-T7/MUW (overexpressing menA, ubiE and wrbA genes) reached the highest titer of VK2 after 48 h of fermentation. The optimization of the medium led to an increase in the accumulation of VK2. Subsequently, the rational metabolic engineering of gene knockout further increased the titer of VK2. The recombinant strain ΔB/MUW was selected as the dominant strain for further optimization, with a high VK2 titer of 724 mg/L. A final attempt is to overexpress ispB gene to increased flux of isoprenoid side chain synthesis, resulting in strain ΔB/MUWI with a titer of 859 mg/L in a shake flask and 1360 mg/L in a 5 L fermenter after 48 h cultivation.

The stepwise engineering strategy raised the VK2 titer from the initial 303 mg/L to 859 mg/L through rational pathway modification and systematic gene expression. Further optimization in batch fermentation increased the VK2 titer to 1360 mg/L, which highlights the strong engineering impact of our strategy.

The online version contains supplementary material available at 10.1186/s13036-025-00614-9.