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 BSD04. Transcriptomic analysis revealed that spore coat assembly genes modulate DPA production. By disrupting a spore coat assembly activator in strain BSD05, 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 BSD05 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.