Bacillus subtilis as cell factory for enhanced production of the biopolymer precursor pyridine-2,6-dicarboxylic acid

Background: Pyridine-2,6-dicarboxylic acid (DPA) is a valuable dicarboxylic acid that has the potential to serve as a precursor for bio-sustainable and bio-degradable materials and self-healing polymers. It also plays a crucial role in the heat resistance of Bacillus subtilis spores. However, extracting DPA from spores is resource-intensive and technically complex, limiting its industrial application. To overcome these challenges, this study aims to engineer B. subtilis as a microbial cell factory for the direct production of free and soluble DPA.
Results: This study first demonstrated that blocking sporulation reduced DPA production due to the repression of dipicolinate synthase expression. To enhance extracellular DPA production, dipicolinate synthase expression was fine-tuned, increasing the extracellular DPA titer to 330 ± 10 mg/l in strain BSDYvyDVF. Transcriptomic analysis revealed that spore coat assembly genes modulate DPA production. By disrupting a spore coat assembly activator in strain BSDYvyDVF-gerE, sporulation was successfully inhibited, significantly boosting the DPA yield to 944 ± 3 mg/l. Further optimization of fermentation conditions was performed using an orthogonal design. The highest DPA titer of 1250 mg/l was achieved and validated through fed-batch fermentation in a 1.5-l bioreactor.
Conclusion: This study demonstrates the potential of engineered B. subtilis BSDYvyDVF-gerE as an efficient cell factory for sustainable DPA biosynthesis. In addition, it identifies key challenges in DPA production, including synthesis efficiency (regulation of key enzyme expression) and transport (intracellular-to-extracellular export), and proposes corresponding solutions.