Towards the Directed Evolution of Artificial Metalloenzymes.

Artificial metalloenzymes (ArMs) are a class of enzymes holding great promise. In contrast to natural enzymes, the core of ArMs is a synthetic metallocofactor, with potential for bio-orthogonal reactivity, incorporated within a host protein. Next to chemical optimization of the metallocofactor, genetic optimization of the protein allows the further improvement of the ArM. Genetic optimization through directed evolution requires extensive screening of a large sequence-scape to enable the optimization of a desired phenotype. The process is however mostly limited by the throughput of the tools and methods available for screening. In recent years, versatile methods based on droplet microfluidics have been developed to address the need for higher throughput. This article aims to give an introduction into ArMs and the recent technological developments allowing high-throughput directed evolution of enzymes.