Directed Evolution of Artificial Metalloenzymes in Whole Cells
Dr. Yang Gu
Department of Chemistry, University of California, Berkeley, CA, 94720 USA
Chemical Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720 USA
Present address: CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China
Search for more papers by this authorBrandon J. Bloomer
Department of Chemistry, University of California, Berkeley, CA, 94720 USA
Chemical Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720 USA
Search for more papers by this authorDr. Zhennan Liu
Department of Chemistry, University of California, Berkeley, CA, 94720 USA
Chemical Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720 USA
Search for more papers by this authorReichi Chen
Department of Chemistry, University of California, Berkeley, CA, 94720 USA
Chemical Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720 USA
Search for more papers by this authorDouglas S. Clark
Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, 94720 USA
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720 USA
Search for more papers by this authorCorresponding Author
Prof. John F. Hartwig
Department of Chemistry, University of California, Berkeley, CA, 94720 USA
Chemical Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720 USA
Search for more papers by this authorDr. Yang Gu
Department of Chemistry, University of California, Berkeley, CA, 94720 USA
Chemical Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720 USA
Present address: CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China
Search for more papers by this authorBrandon J. Bloomer
Department of Chemistry, University of California, Berkeley, CA, 94720 USA
Chemical Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720 USA
Search for more papers by this authorDr. Zhennan Liu
Department of Chemistry, University of California, Berkeley, CA, 94720 USA
Chemical Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720 USA
Search for more papers by this authorReichi Chen
Department of Chemistry, University of California, Berkeley, CA, 94720 USA
Chemical Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720 USA
Search for more papers by this authorDouglas S. Clark
Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, 94720 USA
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720 USA
Search for more papers by this authorCorresponding Author
Prof. John F. Hartwig
Department of Chemistry, University of California, Berkeley, CA, 94720 USA
Chemical Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94720 USA
Search for more papers by this authorAbstract
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.
Conflict of interest
The authors declare no conflict of interest.
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