Engineering Bacillus Subtilis and Fermentation Process Optimization for High-Level Production of Pulcherriminic Acid

Area of Science:

• Microbial Natural Products
• Metabolic Engineering
• Synthetic Biology

Background:

• Pulcherriminic acid (PA) is a microbial cyclic dipeptide and precursor to pulcherrimin, an iron-chelating siderophore with agricultural biocontrol potential.
• Low natural biosynthesis limits the practical application of PA as a bioactive compound.

Purpose of the Study:

• To develop a metabolic engineering strategy for enhancing pulcherriminic acid (PA) production in *Bacillus subtilis*.
• To establish an efficient biosynthetic platform for high-level production of PA.

Main Methods:

• Screening of *Bacillus subtilis* strains to identify a suitable metabolic engineering chassis (164T7P).
• Genetic modification including promoter replacement (T7 promoter) and gene knockout (*abrB*, *spoIVB*, *codY*).
• Optimization of fermentation conditions (carbon source, l-leucine, FeCl3 concentration) and fed-batch fermentation.

Main Results:

• Engineered strain achieved a 7.18-fold increase in PA titer (0.201 g/L) after promoter replacement.
• Gene knockouts of *abrB* or *spoIVB* enhanced PA production, while *codY* deletion suppressed it.
• Optimized fermentation and fed-batch processes in a 5-L bioreactor yielded a record 3.91 g/L PA titer.

Conclusions:

• A robust metabolic engineering strategy was established for significantly boosting PA biosynthesis in *Bacillus subtilis*.
• The developed platform enables high-level production of pulcherriminic acid, facilitating its further development and application.