Engineering Strategies Enabled Protocatechuic Acid Production from Lignin by <i>Pseudomonas putida</i> KT2440

Bioconversion of lignin into fine aromatics offers new avenues for both lignin valorization and biomass utilization. However, the heterogeneity of lignin and the lack of an effective conversion route pose challenges in lignin valorization. Herein, advanced engineering strategies of Pseudomonas putida have been designed for the high-yield production of valuable protocatechuic acid (PCA) from both lignin-derived aromatics and real lignin hydrolysates. Blocking the PCA degradation pathway facilitated PCA production from both lignin-derived H- and G-type aromatics. The aromatic hydroxylation and O-demethylation steps were identified as the metabolic bottlenecks for producing PCA from H- and G-type aromatics, respectively. Modulating the expression of genes encoding the rate-limiting enzymes PobA and VanAB successfully eliminated the accumulation of intermediates, 4-hydroxybenzoic acid and vanillic acid, respectively. By integrating these beneficial gene modifications, the chromosomal strain PCA1601 showed a remarkable production performance of PCA, reaching a titer of 22.7 mM PCA and a molar yield of 94.5%. Implementing the fed-batch strategy further increased PCA production from mixed lignin-derived aromatics, yielding a record titer of 113.6 mM (17.5 g/L). Most importantly, strain PCA1601 showcased remarkable conversion capacity with real lignin hydrolysate, ultimately producing 15.3 mM (2.4 g/L) PCA. The engineered plasmid-free P. putida represents a promising platform for the industrial bioproduction of fine aromatics from renewable lignin.