Investigating the auxiliary enzymes in Pseudomonas secretome reveals the synergistic DypB-based catalytic network and Pseudomonas cells for biocatalytic degradation of lignin

Biocatalytic lignin degradation is a key strategy to develop green, low-carbon biorefining technology. This process involves the synergy between ligninolytic enzymes and auxiliary enzymes. However, the auxiliary enzymes within secretomes, which are composed of thousands of enzymes, remain enigmatic, although several lignin oxidizing enzymes have been well-characterized. Moreover, it's a challenge to understand synergistic lignin degradation via diverse array of enzymes, especially for bacterial oxidoreductases. In this study, the co-expression network of periplasmic proteome revealed the potential accessory enzymes for B-type dye-decolorizing peroxidases (DypBs) in Pseudomonas putida A514. The catalytic network of DypBs-based multi-enzyme complex was characterized. DypBs couple with the quinone reductases, nitroreductase and superoxide dismutase to drive quinone redox cycling for enhancing the production of hydroxyl radical. The synthetic enzyme cocktail (SEC), recruiting 15 enzymes, was further designed with four functions. The lignin degradation capacity of SEC showed comparable with that of the native periplasmic secretome, overcoming the limitation of lignin spontaneous repolymerization. Importantly, we revealed the synergistic mechanism of SEC-A514 cell system, which incorporated the advantages of in vitro enzyme catalysis and in vivo microbial catabolism. Chemical analysis showed this system substantially reduced the molecular weight of lignin, extended the degradation spectra for lignin functional groups, and efficiently metabolized lignin depolymerization products. As a result, 25% of lignin was utilized and its average molecular weight was reduced by 27%. Our study advances the knowledge of bacterial lignin degrading multi-enzymes and provides a promising route for lignin valorization.