Multi-enzyme assemblies both in the cell membrane and cytoplasm boost intracellular lycopene production

The multi-enzyme assembly system demonstrates remarkable potential in enhancing both intracellular and extracellular enzyme catalysis. In this study, we employed a novel icosahedral protein cage, Mi3, as a protein scaffold and combined it with an ester bond-based peptide tagging system, ReverseTag/ReverseCatcher, to improve the enzymatic catalytic efficiency both in vitro and in vivo. In vitro, we fused ReverseTag to the N-terminal of exo-inulinase (EXINU) from Pseudomonas mucidolens, yielding ReverseTag-EXINU, which effectively bound to the surface of the ReverseCatcher-Mi3 protein cage. Following assembly, the K_m value decreased from 16.3 to 7.9 g/L, while k_cat/K_m value increased from 1.9 to 3.0 L s^-1 g^-1, indicating a significant enhancement in substrate affinity and enzymatic catalytic efficiency. In vivo, we constructed a protein-cage multi-enzyme assembly system located in the cytoplasm and cell membrane based on ReverseTag/ReverseCatcher/Mi3 system. In lycopene biosynthesis, the production of lycopene after assembly was increased by 2.97 times compared to free enzyme catalysis. This strategy holds profound implications for fields such as synthetic biology and enzyme engineering.