Investigating the enzymatic dark matter in <i>Pseudomonas </i>secretome reveals the synergistic interactions in the DypB-based catalytic network for bio-design of an efficient lignin degrading synthetic enzyme cocktail

Biocatalytic lignin degradation is a key strategy to develop green, low-carbon biorefining technology. This process involves the synergy between lignin oxidizing 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 characterized. Moreover, it's a significant challenge to reveal the synergistic mechanism in enzyme consortiums.<b> </b>In this study, network analysis of the periplasmic proteome revealed the potential accessory enzymes for B-type dye-decolorizing peroxidases (DypBs) in <i>Pseudomonas putida</i> A514. The catalytic network in DypBs-based multi-enzyme consortium was characterized, which couple with quinone reductase, nitroreductases and superoxide dismutase to drive quinone redox cycling for enhancement of Fenton reaction. The synthetic enzyme cocktail (SEC), recruiting 16 enzymes, was further designed with four functions. Its lignin degradation capacity was 2-fold higher than the DypBs alone and showed comparable with that of the native periplasmic secretome, which itself consisted of thousands of proteins. Importantly, we developed the SEC-A514 cell system, which incorporated the advantages of <i>in vitro</i> enzyme catalysis and <i>in vivo </i>cell catabolism. Chemical analysis showed this system not only extended the degradation spectra for lignin functional groups, but also efficiently metabolized lignin depolymerization products. As a result, 25% of lignin was utilized and its weight average molecular weight was reduced by 30%. Our study advances the knowledge of bacterial lignin degrading multi-enzyme consortiums and provides a promising route for lignin valorization.