A novel LPS-dependent outer membrane-anchoring mechanism for T9SS substrates enables engineered enzyme display and whole-cell PET degradation in Cytophaga hutchinsonii.

Robust surface display systems are crucial for engineering Gram-negative bacteria as whole-cell biocatalysts. In the efficient cellulose degrader Cytophaga hutchinsonii, cellulases are secreted by the Type IX Secretion System (T9SS), yet their mechanism for outer membrane anchoring remained unknown. Here, we report a novel lipopolysaccharide (LPS)-dependent anchoring mechanism for T9SS substrates. We initially found that the anchoring of T9SS substrates to the outer membrane involves a modification, evident as a characteristic ladder-like pattern on PVDF membrane. By expressing heterologous proteins fused to the CTDs of specific cellulases, we demonstrated that this modification and anchoring are strictly CTD-dependent. Using bioinformatic analysis and gene deletion, we identified WaaL, which encodes a key enzyme involved in LPS biosynthesis. LC-MS/MS proteomics demonstrated that the ΔwaaL mutant fails to modify and anchor T9SS substrates. Therefore, we conclude that outer membrane anchoring depends on the combined action of LPS and the substrate CTDs. Leveraging this mechanism, we developed a novel surface display platform by fusing heterologous enzymes to T9SS substrate CTDs. As a proof-of-concept, we successfully displayed a functional polyethylene terephthalate hydrolase (PETase) on the surface of C. hutchinsonii, enabling the degradation of PET. Our work not only uncovers a fundamental mechanism for protein anchoring in C. hutchinsonii but also establishes an LPS-CTD-based platform for programmable surface display in Gram-negative bacteria, significantly expanding the toolbox for synthetic biology and biotechnological applications.