Crosslinking of T6SS and T0SS for the secretion of unconventional peroxidase contributes to bacterial lignin utilization

<p>S<span>ecretion</span>&nbsp;of lignolytic enzymes is a competitive advantage for microbial survival in the heterogeneous environments. They are commonly by signal peptides to be transported to the&nbsp;extracellular milieu for lignin catabolism, as the highly complex polymer cannot be translocated through the cell membrane. However, some bacterial lignolytic enzymes lack signal peptides, yet, can still be secreted. It remains unclear how these leaderless proteins cross the inner and outer membranes. Here, we reveal a novel secretion mechanism for the unconventional B-type dye-decolorizing peroxidases (DypB) in<em>&nbsp;Pseudomonas putida</em>. Type VI secretion system (T6SS) mediates the inner membrane channel, where interaction of DypB with VgrG and Hcp accounts for periplasmic delivery. Once in the periplasm, DypB is allocated into outer membrane vesicles (OMVs), also called T0SS, and released to extracellular space. The crosslinked translocation model for DypB delivery represents a novel and ingenious mechanism by which bacteria harness extant systems to thrive in a dynamically changing environment. Moreover, we developed 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 provides multiple new perspectives on unconventional protein secretion and its biotechnological applications.</p>