January 18, 2023
Understanding of bacterial lignin extracellular degradation mechanisms by Pseudomonas putida KT2440 via secretomic analysis
AbstractBacterial lignin depolymerization is believed to be primarily achieved by a secreted enzyme system. Effects of such extracellular enzyme systems on lignin structural changes and depolymerization pathways are still not clearly understood, which remains as a bottleneck in the bacterial lignin bioconversion process. This study investigated lignin depolymerization using an isolated secretome secreted by Pseudomonas putida KT2440 that grew on glucose as the only carbon source. Enzyme assays revealed that the secretome harbored oxidase and peroxidase/Mn2+-peroxidase capacity and reached the highest activity at 120 h of the fermentation time. The degradation rate of alkali lignin was found to be only 8.1% by oxidases, but increased to 14.5% with the activation of peroxidase/Mn2+-peroxidase. Gas chromatography-mass spectrometry (GC-MS) and two-dimensional 1H-13C heteronuclear single-quantum coherence (HSQC) NMR analysis revealed that the oxidases exhibited strong C-C bond (ß-ß, ß-5, and ß-1) cleavage. The activation of peroxidases enhanced lignin degradation by stimulating C-O bond (ß-O-4) cleavage, resulting in increased yields of aromatic monomers and dimers. Further mass spectrometry-based quantitative proteomics measurements comprehensively identified different groups of enzymes particularly oxidoreductases in P. putida secretome, including reductases, peroxidases, monooxygenases, dioxygenases, oxidases, and dehydrogenases, potentially contributed to the lignin degradation process. Overall, we discovered that bacterial depolymerization of alkali lignin to vanillin, vanillic acid, and other lignin derived-aromatics involved a series of oxidative cleavage, catalyzed by active DyP-type peroxidase, multicopper oxidase, and other accessory enzymes. These resutls will guide further metabolic engineering design to improve the efficiency of lignin bioconversion.
Published: January 18, 2023