Tailoring the Tag/Catcher System by Integrating Covalent Bonds and Noncovalent Interactions for Highly Efficient Protein Self-Assembly

Covalent bonds and noncovalent interactions play crucial roles in enzyme self-assembly. Here, we designed a Tag/Catcher system named NGTag/NGCatcher in which the Catcher is a highly charged protein that can bind proteins with positively charged tails and rapidly form a stable isopeptide bond with NGTag. In this study, we present a multienzyme strategy based on covalent bonds and noncovalent interactions. In vitro, mCherry, YFP, and GFP can form protein-rich three-dimensional networks based on NGCatcher, NGTag, and RK (Arginine/Lysine) tails, respectively. Furthermore, this technology was applied to improve lycopene production in Escherichia coli. Three key enzymes were involved in lycopene production variants from Deinococcus wulumugiensis R12 of NGCatcher_CrtE, NGTag_Idi, and RKIspARK, where the multienzyme complexes were clearly observed in vivo and in vitro, and the lycopene production in vivo was 17.8-fold higher than that in the control group. The NGTag/NGCatcher system will provide new opportunities for in vivo and in vitro multienzyme catalysis.