Directed Evolution of Artificial Metalloenzymes in Whole Cells

• Published in: Angewandte Chemie International Edition Vol. 61; no. 5; pp. e202110519 - n/a
• Main Authors: Gu, Yang, Bloomer, Brandon J., Liu, Zhennan, Chen, Reichi, Clark, Douglas S., Hartwig, John F.
• Format: Journal Article
• Language: English
• Published: WEINHEIM Wiley 26.01.2022; Wiley Subscription Services, Inc
• Edition: International ed. in English
• Subjects: Aniline; artificial metalloenzymes; Catalysts; Chemistry; Chemistry, Multidisciplinary; Cofactors; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Directed evolution; Directed Molecular Evolution; Enantiomers; Enzymes; Esters; Evolution; Heme; Metalloproteins - chemistry; Metalloproteins - genetics; Metalloproteins - metabolism; Methane - analogs & derivatives; Methane - chemistry; Methane - metabolism; Mutants; N−H insertion; P450; Physical Sciences; Protein purification; Proteins; Science & Technology; whole cell; CYTOCHROME-P450; AMINATION; DISPLAY; STREPTAVIDIN; N-H insertion; whole cell; P450; artificial metalloenzymes; directed evolution; N−H insertion
• ISSN: 1433-7851; 1521-3773; 1521-3773
• DOI: 10.1002/anie.202110519

Artificial metalloenzymes (ArMs), created by introducing synthetic cofactors into protein scaffolds, are 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 a limiting step in the high‐throughput screening and directed evolution of ArMs because purification of individual host proteins is time‐consuming. We describe the application of a platform to combine mutants of the P450 enzyme CYP119 and the cofactor Ir(Me)MPIX in vivo, by coexpression 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 α‐diazopropanoate esters (Me‐EDA) into the N−H bonds of N‐alkyl anilines, a combination of carbene and amine classes for which mutant enzymes of natural hemoproteins previously reacted with low enantioselectivity. 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‐EDA with N‐methyl anilines to form chiral chiral amino esters with high TON and good enantioselectivity, thereby demonstrating that the directed evolution of ArMs can rival that of natural enzymes in vivo. Whole‐cell E. coli platform enables directed evolution campaigns with ArMs containing an abiotic Ir‐porphyrin cofactor involving the generation of more than 4000 mutants to develop an enantioselective insertion of a simple acyclic carbene from a diazo ester into the N−H bond of N‐methyl aniline that has formed product with poor enantioselectivity when catalyzed by prior enzyme systems.