Fed-batch fermentative bioconversion to valorize lignin aromatics into 2-pyrone-4,6-dicarboxylic acid through engineered Pseudomonas putida KT2440

Lignin is an abundant renewable carbon resource, accounting for approximately 30% of plant biomass. Its physicochemical properties arise from the irregular structure of aromatic monomers, which provide rigidity and protective functions in the plant cell wall. However, its heterogeneous aromatic composition limits efficient biological and chemical valorization. In this study, we demonstrate the highly selective fermentative bioconversion of lignin-derived compounds mixtures into 2-pyrone-4,6-dicarboxylic acid (PDC) using genetically engineered Pseudomonas putida KT2440 strains KT-PDC and KT-PDCV. The fermentative and selective production of PDC was investigated using mixtures of lignin-derived substrates comprising both model monomers and real lignin-derived aromatics. Optimizations of cultivation conditions, including appropriate phosphorus, nitrogen, and carbon source composition improved titer, rate, and yield of PDC production. The optimized strategy was further validated using real lignin-derived mixtures obtained via oxidative depolymerization. KT-PDC and KT-PDCV strains produced PDC from these mixtures, but KT-PDCV exhibited an excellent substrate consumption rate without hindering conversion efficiency or cell growth. Overall, this study highlights the importance of fermentation-based evaluation to assess the feasibility and potential for industrial scale-up of bioprocess systems, even when metabolic pathway engineering has already established strong potential at the flask-scale.