Engineered Pseudomonas Putida Kt2440 for Valorizing Lignin into Protocatechuic Acid

The biological valorization of lignin to aromatic platform compounds, specifically protocatechuic acid (PCA), has garnered significant interest in the field of bioeconomy. This study focuses on the engineering of Pseudomonas putida KT2440 to convert lignin and its derivatives into PCA. First, P. putida KT2440 was successfully designed to accumulate PCA by blocking the PCA degradation pathway and catabolite repression control protein. Distinct promoters were further utilized to control the integration expression of genes encoding the rate-limiting enzymes PobA and VanAB, which eliminated the accumulation of intermediates. Furthermore, the chromosomally integrated strain PCA1601 demonstrated the ability to produce 22.68 mM PCA from a mixture containing 20 mM p-coumaric acid and 4 mM ferulic acid, achieving a molar conversion yield of 94.5%. Notably, the implementation of fed-batch strategies led to a significant enhancement in the PCA production by strain PCA1601 at a record titer of 113.55 mM (17.5 g/L) from a considerably elevated lignin-derived aromatic concentration. Most importantly, the developed strain PCA1601 ultimately produced 15.27 mM (2.35 g/L) PCA when the real lignin streams were applied as substrates. The bioconversion of lignin to value-added PCA was thus realized by the genetically engineered plasmid-free P. putida KT2440, thereby representing a promising platform for the industrial bioproduction of high-valued aromatic compounds from renewable lignin.