Directed Evolution of Artificial Metalloenzymes in Whole Cells

Abstract

Artificial metalloenzymes (ArMs), created by introducing synthetic cofactors into protein scaffolds,a re an emerging class of catalyst for non-natural reactions.Although many classes of ArMs are known, in vitro reconstitution of cofactors and proteins has been al imiting step in the highthroughput screening and directed evolution of ArMs because purification of individual host proteins is time-consuming. We describe the application of ap latform to combine mutants of the P450 enzyme CYP119 and the cofactor Ir(Me)MPIX in vivo,b yc oexpression of the CYP119 mutants with the heme transporter encoded by the hug operon, to the directed evolution of ArMs containing Ir(Me)MPIX in whole cells. We applied this platform to the development an ArMs catalyzing the insertion of the acyclic carbene from adiazopropanoate esters (Me-EDA) into the N À Hb onds of N-alkyl anilines,acombination of carbene and amine classes for which mutant enzymes of natural hemoproteins previously reacted with lowenantioselectivity.The mutants of the artificial metalloenzyme Ir(Me)CYP119 identified by an evolution campaign involving more than 4000 mutants are shown to catalyze the reaction of Me-EDAw ith N-methyl anilines to form chiral chiral amino esters with high TONa nd good enantioselectivity,t hereby demonstrating that the directed evolution of ArMs can rival that of natural enzymes in vivo.

A primer to directed evolution: current methodologies and future directions

“…This strategy was successfully applied for the evolution of a novel cytochrome P450 variant with an iridiumcontaining heme-like cofactor able to produce chiral amines enantioselectively. 112 Of increasing interest is also the evolution of entire metabolic pathways, where a set of enzymes work together towards the generation of a desired product (Fig. 8).…”

Spheroplasts preparation boosts the catalytic potential of a terpene cyclase

“…However, such approaches entail time-consuming molecular cloning, tedious downstream steps, and increased process costs. [10][11][12] Consequently, efficient strategies to circumvent mass transfer issues are of high interest in whole cell biocatalysis.…”