Summary Lignin is one of the largest carbon reservoirs in the environment, playing an important role in the global carbon cycle. However, lignin degradation in bacteria, especially non-model organisms, has not been well characterized either enzymatically or genetically. Here, a lignin-degrading bacterial strain, Pseudomonas putida A514, was used as the research model. Genomic and proteomic analyses suggested that two B subfamily dye-decolorizing peroxidases (DypBs) were prominent in lignin depolymerization, while the classic O 2 -dependent ring cleavage strategy was utilized in central pathways to catabolize lignin-derived aromatic compounds that were funnelled by peripheral pathways. These enzymes, together with a range of transporters, sequential and expression-dose dependent regulation and stress response systems coordinated for lignin metabolism. Catalytic assays indicated these DypBs show unique Mn 2+ independent lignin depolymerization activity, while Mn 2+ oxidation activity is absent. Furthermore, a high synergy between DypB enzymes and A514 cells was observed to promote cell growth (5 × 10 12 cfus/ml) and lignin degradation (27%). This suggested DypBs are competitive lignin biocatalysts and pinpointed limited extracellular secretion capacity as the rate-limiting factor in bacterial lignin degradation. DypB production was, therefore, optimized in recombinant strains and a 14,141-fold increase in DypB activity (56,565 U/l) was achieved, providing novel insights for lignin bioconversion.
The secretion of ligninolytic enzyme provides a competitive advantage for microbial survival. These enzymes are commonly transported to the extracellular milieu via signal peptides for the catabolism of lignin, which cannot be translocated through the cell membrane. However, some bacterial ligninolytic enzymes lack signal peptides, yet they can still be secreted. It remains unclear how these unconventional proteins cross the cell membrane. Here, we reveal a secretion mechanism for the unconventional B-type dye-decolorizing peroxidase (DypB) in Pseudomonas putida A514. Type VI secretion system (T6SS) mediates the inner membrane channel, where interaction of DypB with the T6SS components VgrG and Hcp accounts for periplasmic delivery. Once in the periplasm, DypB is allocated into outer membrane vesicles (OMVs) and released to the extracellular space. The crosslinked translocation model for DypB delivery represents an ingenious mechanism by which bacteria harness extant systems to thrive in a nutrient-poor environment. Moreover, we develop an OMV-surface display platform to improve DypB secretion and further enhance lignin utilization, exemplifying the applicability of OMVs as a lignin biocatalytic nanoreactor. Our study demonstrates the previously unrecognized crosslinking of T6SS and OMVs for the secretion of unconventional ligninolytic peroxidase and provides new perspectives on its biotechnological applications.
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