The Impact of Structure Refinement in the Construction of Whole-Cell Catalysts with Artificial Metalloenzymes Assembled in Artificial Sanctuaries

Le résumé fourni par la source

The strategic integration of artificial metalloenzymes (ArMs) into intracellular artificial sanctuaries created from liquid–liquid phase separation (LLPS) offers a promising approach to expand whole-cell catalysts’ biocatalytic capabilities. However, the structural parameters governing the design of cross-linkers (LLPS initiators) and metal cofactors in such systems remain underexplored. In this report, we systematically evaluate the impact of ligand-cross-linker’s arm length on the assembly and catalytic performance of ArMs in E. coli, using the recently reported HaloTag-SNAPTag (HS) ArM scaffold as the model LLPS-able protein for sanctuary creation. A series of tridentate ligand-cross-linkers (L0–L5) with tunable oligo(ethylene glycol) (OEG) spacers in their connecting arms were synthesized as triggers for HS multimerization and LLPS. It is observed that while the cross-linkers’ arm length minimally influenced LLPS initiation, it affects protein multimerization yields and catalytic activity. For ArMs assembled via route i, in which cofactors are independently introduced, such as the artificial metathesis Ru-HS-ArMs, the impact of cross-linkers on the catalytic activity is relatively small. However, for ArMs assembled via route ii, in which metal ions bind ligand-cross-linkers to form cofactors, the impact of cross-linkers is larger, and an optimized structure can lead to a significantly more effective catalytic system. These findings establish critical guidelines for future designs using the ArMAS-LLPS platform, highlighting the necessity of structure refinement to maximize catalytic performance in compartmentalized whole-cell systems for various applications.

Ce résumé expose les affirmations des auteurs. BNTIC ne l’interprète pas comme une validation indépendante des résultats.