Combinatorial Methylerythritol Phosphate Pathway Engineering and Process Optimization for Increased Menaquinone-7...

Area of Science:

• Microbial Biotechnology
• Metabolic Engineering
• Vitamin Synthesis

Background:

• Vitamin K2 (menaquinone), particularly menaquinone-7 (MK-7), is vital for health and found in daily diets.
• Previous work engineered MK-7 synthesis modules in *Bacillus subtilis* (strain BS20), achieving 360 mg/l, but the precursor-supplying MEP pathway remained unoptimized.

Purpose of the Study:

• To enhance MK-7 production by engineering the methylerythritol phosphate (MEP) pathway in *Bacillus subtilis*.
• To optimize fermentation conditions and scale up MK-7 production for potential industrial application.

Main Methods:

• Overexpression of five key genes in the MEP pathway within the engineered *Bacillus subtilis* strain BS20.
• Optimization of fermentation parameters (pH, temperature, aeration) using a parallel bioreactor system.
• Scale-up of MK-7 production in a 50-l bioreactor.

Main Results:

• The engineered strain (BS20DFHG) achieved a titer of 415 mg/l MK-7 in shake flasks, a significant improvement over the previous strain.
• Optimized fermentation conditions led to a maximum MK-7 titer of 242 mg/l in a 50-l bioreactor scale-up.

Conclusions:

• Engineering the MEP pathway is an effective strategy to increase MK-7 production in *Bacillus subtilis*.
• The developed strain BS20DFHG and optimized fermentation process show promise for future industrial-scale MK-7 manufacturing.