Biosynthesis of 2,5-pyridinedicarboxylate from glucose via p-aminobenzoic acid in Escherichia coli

Pyridine carboxylic acids, because of their structural similarity to aromatic carboxylic acids, have garnered increasing attention as alternative compounds in chemical synthesis. However, their broader utilization has been limited by challenges in biosynthetic production. In this study, we developed a metabolic pathway for biosynthesizing 2,5-pyridinedicarboxylate (2,5-PDCA) from glucose from p-aminobenzoate (PABA). The heterologous expression of 4-amino-3-hydroxybenzoate 2,3-dioxygenase (AhdA) in Escherichia coli enabled the conversion of 0.5 g/L of 4-amino-3-hydroxybenzoate (4A3HBA) into 0.47 g/L of 2,5-PDCA. Subsequent systematic evaluation of p-hydroxybenzoate hydroxylase (PobA) variants and optimization of pobA and ahdA co-expression facilitated the development of a 2,5-PDCA biosynthetic module for efficient production from PABA. Incorporating this module into a PABA biosynthesis pathway enabled direct 2,5-PDCA production from glucose. Further enhancements were achieved by increasing metabolic flux through the shikimate pathway and optimizing sodium pyruvate supplementation. Under optimized conditions, we achieved a titer of 1.84 g/L in test-tube cultures after 72 h and 10.6 g/L in bioreactor fermentation after 144 h. Overall, this study introduces a valuable strategy for the microbial production of pyridine carboxylates and establishes a promising platform for broader applications in aromatic compound biosynthesis.