Bacterial Surface-Assembled Chitinosome for Dismantling Chitin into N-Acetyl Glucosamine

The construction of an efficient multienzyme catalystfor dismantlingrecalcitrant polymers into high-value-added chemicals is appealingin sustainable biomanufacturing. Colocalization of cascade enzymeshas been broadly adopted by natural and synthetic multienzyme systemsfor chemical synthesis, while there has been rare research on thedecomposition of polymeric substrates. In this study, we constructedan E. coli surface-assembly (ESA) systemto colocalize a synthetic chitinosome composed of two chitin hydrolases,BpChiA and BlNagZ, via the SpyCatcher/SpyTag (SpyC/SpyT) conjugation.In the ESA-chitinosome complexes, the loading efficiency andspecific activity of enzymes were improved by tuning the copies ofSpyC and the linker lengths between the SpyT and enzymes, respectively.The ratio of BpChiA and BlNagZ was also optimized. The ESA-chitinosomecomplexes exhibited higher productivity of N-acetylglucosamine than the mixture of separately assembled enzymes on cellsurfaces. This work demonstrates that the enzyme colocalization andthe formation of the ternary ESA-chitinosome-chitincomplexes are critical for the catalytic synergism of cascade enzymes,providing new insights into the decomposition of recalcitrant polymericsubstrates. The ESA-chitinosomecomplex was constructed to produceGlcNAc by decomposing renewable chitin biopolymers.