In vivo Assembly of Artificial Metalloenzymes and Application in Whole-Cell Biocatalysis

Shreyans Chordia; Siddarth Narasimhan; Alessandra Lucini Paioni; Marc Baldus; Gerard Roelfes

doi:10.1002/anie.202014771

In vivo Assembly of Artificial Metalloenzymes and Application in Whole-Cell Biocatalysis

Shreyans Chordia, Siddarth Narasimhan, Alessandra Lucini Paioni, Marc Baldus, Gerard Roelfes

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

We report the supramolecular assembly of artificial metalloenzymes (ArMs), based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneous copper(II)–phenanthroline complex, in the cytoplasm of E. coli cells. A combination of catalysis, cell-fractionation, and inhibitor experiments, supplemented with in-cell solid-state NMR spectroscopy, confirmed the in-cell assembly. The ArM-containing whole cells were active in the catalysis of the enantioselective Friedel–Crafts alkylation of indoles and the Diels–Alder reaction of azachalcone with cyclopentadiene. Directed evolution resulted in two different improved mutants for both reactions, LmrR_A92E_M8D and LmrR_A92E_V15A, respectively. The whole-cell ArM system required no engineering of the microbial host, the protein scaffold, or the cofactor to achieve ArM assembly and catalysis. We consider this a key step towards integrating abiological catalysis with biosynthesis to generate a hybrid metabolism.

Original language	English
Pages (from-to)	5913-5920
Number of pages	8
Journal	Angewandte Chemie-International Edition
Volume	60
Issue number	11
Early online date	11 Jan 2021
DOIs	https://doi.org/10.1002/anie.202014771
Publication status	Published - 8 Mar 2021

Bibliographical note

Funding Information:
We thank L. Villarino for assistance with synthetic chemistry, Dr. A. Iyer for assistance with live cell confocal microscopy, R. Leveson‐Gower for assistance with the MS measurements, Prof. P. Tordo, Dr. O. Ouari (Aix‐Marseille Université) for providing AMUPol for the DNP experiments and Dr. C. Mayer for advice and discussions. We thank Prof. I. Shimada (U. of Tokyo) for the NMR assignments of the wild‐type LmrR and Dr. H. van Ingen for providing access to the solution‐state NMR instrument along with J. van der Zwan and Dr. S. Xiang for technical support and discussions. This work was supported by the Netherlands Organisation for Scientific Research (NWO, projects 700.26.121 and 700.10.443 to M.B. and 724.013.003 to G.R.). In addition, S.N. was supported by the Netherlands’ Magnetic Resonance Research School (NMARRS, project number 022.005.029). G.R. acknowledges support from the Netherlands Ministry of Education, Culture and Science (Gravitation program no. 024.001.035).

Publisher Copyright:
© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH

Keywords

artificial metalloenzymes
biocatalysis
copper
in cell NMR spectroscopy
in vivo catalysis

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title = "In vivo Assembly of Artificial Metalloenzymes and Application in Whole-Cell Biocatalysis",

abstract = "We report the supramolecular assembly of artificial metalloenzymes (ArMs), based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneous copper(II)–phenanthroline complex, in the cytoplasm of E. coli cells. A combination of catalysis, cell-fractionation, and inhibitor experiments, supplemented with in-cell solid-state NMR spectroscopy, confirmed the in-cell assembly. The ArM-containing whole cells were active in the catalysis of the enantioselective Friedel–Crafts alkylation of indoles and the Diels–Alder reaction of azachalcone with cyclopentadiene. Directed evolution resulted in two different improved mutants for both reactions, LmrR\_A92E\_M8D and LmrR\_A92E\_V15A, respectively. The whole-cell ArM system required no engineering of the microbial host, the protein scaffold, or the cofactor to achieve ArM assembly and catalysis. We consider this a key step towards integrating abiological catalysis with biosynthesis to generate a hybrid metabolism.",

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author = "Shreyans Chordia and Siddarth Narasimhan and \{Lucini Paioni\}, Alessandra and Marc Baldus and Gerard Roelfes",

note = "Funding Information: We thank L. Villarino for assistance with synthetic chemistry, Dr. A. Iyer for assistance with live cell confocal microscopy, R. Leveson‐Gower for assistance with the MS measurements, Prof. P. Tordo, Dr. O. Ouari (Aix‐Marseille Universit{\'e}) for providing AMUPol for the DNP experiments and Dr. C. Mayer for advice and discussions. We thank Prof. I. Shimada (U. of Tokyo) for the NMR assignments of the wild‐type LmrR and Dr. H. van Ingen for providing access to the solution‐state NMR instrument along with J. van der Zwan and Dr. S. Xiang for technical support and discussions. This work was supported by the Netherlands Organisation for Scientific Research (NWO, projects 700.26.121 and 700.10.443 to M.B. and 724.013.003 to G.R.). In addition, S.N. was supported by the Netherlands{\textquoteright} Magnetic Resonance Research School (NMARRS, project number 022.005.029). G.R. acknowledges support from the Netherlands Ministry of Education, Culture and Science (Gravitation program no. 024.001.035). Publisher Copyright: {\textcopyright} 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH",

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AU - Narasimhan, Siddarth

AU - Lucini Paioni, Alessandra

AU - Baldus, Marc

AU - Roelfes, Gerard

N1 - Funding Information: We thank L. Villarino for assistance with synthetic chemistry, Dr. A. Iyer for assistance with live cell confocal microscopy, R. Leveson‐Gower for assistance with the MS measurements, Prof. P. Tordo, Dr. O. Ouari (Aix‐Marseille Université) for providing AMUPol for the DNP experiments and Dr. C. Mayer for advice and discussions. We thank Prof. I. Shimada (U. of Tokyo) for the NMR assignments of the wild‐type LmrR and Dr. H. van Ingen for providing access to the solution‐state NMR instrument along with J. van der Zwan and Dr. S. Xiang for technical support and discussions. This work was supported by the Netherlands Organisation for Scientific Research (NWO, projects 700.26.121 and 700.10.443 to M.B. and 724.013.003 to G.R.). In addition, S.N. was supported by the Netherlands’ Magnetic Resonance Research School (NMARRS, project number 022.005.029). G.R. acknowledges support from the Netherlands Ministry of Education, Culture and Science (Gravitation program no. 024.001.035). Publisher Copyright: © 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH

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N2 - We report the supramolecular assembly of artificial metalloenzymes (ArMs), based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneous copper(II)–phenanthroline complex, in the cytoplasm of E. coli cells. A combination of catalysis, cell-fractionation, and inhibitor experiments, supplemented with in-cell solid-state NMR spectroscopy, confirmed the in-cell assembly. The ArM-containing whole cells were active in the catalysis of the enantioselective Friedel–Crafts alkylation of indoles and the Diels–Alder reaction of azachalcone with cyclopentadiene. Directed evolution resulted in two different improved mutants for both reactions, LmrR_A92E_M8D and LmrR_A92E_V15A, respectively. The whole-cell ArM system required no engineering of the microbial host, the protein scaffold, or the cofactor to achieve ArM assembly and catalysis. We consider this a key step towards integrating abiological catalysis with biosynthesis to generate a hybrid metabolism.

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KW - biocatalysis

KW - copper

KW - in cell NMR spectroscopy

KW - in vivo catalysis

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JO - Angewandte Chemie-International Edition

JF - Angewandte Chemie-International Edition

IS - 11

ER -

In vivo Assembly of Artificial Metalloenzymes and Application in Whole-Cell Biocatalysis

Abstract

Bibliographical note

Keywords

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