Utilization and valorization of lignin and lignin-derived compounds by Pseudomonas putida KT2440: A new role for glutathione peroxidase

Lignin, a complex natural aromatic polymer, poses significant challenges to its efficient degradation, hindering the utilization of biomass for many industrial applications. Bacterial degradation of lignin may offer a promising solution to this challenge. This project aimed at elucidating the function of secreted oxidative enzymes from Pseudomonas putida involved in degradation and utilization of lignin and lignin-derived compounds. Using CRISPR-Cas9 and CRISPR-Cas3 systems, the putative lignin-degrading versatile peroxidase gene (VP; PP _ 1686 , originally annotated as glutathione peroxidase GPx) and dye-decolorizing peroxidase gene ( PP_3248 ) were individually knocked out from P. putida KT2440. The ∆PP_1686 mutant exhibited impaired growth and utilization of lignin-derived compounds. This correlated with reduced expression of p-hydroxybenzoate hydroxylase pobA and of DNA repair modules, alongside compensatory upregulation of energy and redox supply pathways. This work expands our knowledge on bacterial glutathione peroxidase by presenting a role beyond ROS scavenging. This work revealed the importance of P. putida VP/GPx in maintaining redox balance while supporting lignin-derived aromatic metabolism, offering new targets for future investigation into stress–metabolism crosstalk and lignin valorization strategies. • Functional characterization of secreted oxidative enzymes of Pseudomonas putida in lignin degradation and utilization • CRISPR/Cas knockout revealed a novel role for P. putida glutathione peroxidase ( PP_1686 ) • Deletion of P. putida PP_1686 causes reduced lignin-derived compound utilization and growth • P. putida PP_1686 links oxidative stress regulation with aromatic compound catabolism