Artificial metalloenzymes with different protein scaffolds, cofactors and functions have been prepared to expand the natural enzymatic repertoire with abiotic reactions. However, due to the sensitivity of metal centers toward various biomolecules, especially glutathione, low activity and turnover of artificial metalloenzymes in vivo are systematic problems not fully solved. Apart from straightforward routes such as the use of neutralizing agents, metal cofactors modification and directed evolution, one may notice that nature can create isolated microenvironments for diverse biological processes within cells. Following this way, here we report the in vivo assembly of artificial metalloenzymes based on HaloTag-SNAPTag fusion protein. These metalloenzymes have metal cofactors bound on protein interfaces, and can trigger liquid-liquid phase separation to form liquid condensates inside Escherichia coli. These condensates serve as membraneless, isolated compartments for artificial metalloenzymes to efficiently perform intracellular catalysis, mediating abiotic unmasking, coupling and polymerization reactions. The cellular compartmentalization also enables spatial control of reactions, either facilitating a cascade reaction within the confined spaces, or concurrent reactions with spatial separation. Such engineered Escherichia coli can work as whole-cell catalyst with confined metal species, colonizing at mice intestine to effect in vivo abiotic transformations with a lower chance of heavy metal poisoning. These results represent a systematic strategy to stabilize and potentiate artificial metalloenzyme in vivo, with potential applications in fields such as non-natural metabolism, fermentation and drug delivery.
Designer Compartment for Artificial Metalloenzymes through Controlled Liquid-Liquid Phase Separation
01 November 2021, Version 1
This content is a preprint and has not undergone peer review at the time of posting.