Assembly and Evolution of Artificial Metalloenzymes within E. coli Nissle 1917 for Enantioselective and Site-Selective Functionalization of C–H and C=C Bonds

Liu, Zhennan; Huang, Jing; Gu, Yang; Clark, Douglas S.; Mukhopadhyay, Aindrila; Keasling, Jay D.; Hartwig, John F.

doi:10.1021/jacs.1c10975

Assembly and Evolution of Artificial Metalloenzymes within E. coli Nissle 1917 for Enantioselective and Site-Selective Functionalization of C–H and C=C Bonds

Journal Article · Tue Jan 04 23:00:00 EST 2022 · Journal of the American Chemical Society

DOI:https://doi.org/10.1021/jacs.1c10975· OSTI ID:1961807

Liu, Zhennan ^[1]; Huang, Jing ^[2]; Gu, Yang ^[1]; ^[1]; ^[1]; Keasling, Jay D. ^[3]; Hartwig, John F. ^[1]

Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Joint BioEnergy Institute (JBEI), Emeryville, CA (United States)
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Univ. of California, Berkeley, CA (United States); Shenzhen Institutes for Advanced Technologies, Shenzhen (China); Danish Technical University, Lyngby (Denmark)

The potential applications afforded by the generation and reactivity of artificial metalloenzymes (ArMs) in microorganisms are vast. Here we show that a non-pathogenic E. coli strain, Nissle 1917 (EcN), is a suitable host for the creation of ArMs from cytochrome P450s and artificial heme cofactors. An outer-membrane receptor in EcN transports an iridium porphyrin into the cell, and the Ir-CYP119 (CYP119 containing iridium porphyrin) assembled in vivo catalyzes carbene insertions into benzylic C-H bonds enantioselectively and site-selectively. The application of EcN as a whole-cell screening platform eliminates the need for laborious processing procedures, drastically increases the ease and throughput of screening, and accelerates the development of Ir-CYP119 with improved catalytic properties. Studies to identify the transport machinery suggest that a transporter different from the previously assumed ChuA receptor serves to usher the iridium porphyrin into the cytoplasm.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences & Biosciences Division; USDOE Laboratory Directed Research and Development (LDRD) Program; National Science Foundation (NSF); National Institutes of Health (NIH); Joint Bioenergy Institute Project

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1961807

Journal Information:: Journal of the American Chemical Society, Journal Name: Journal of the American Chemical Society Journal Issue: 2 Vol. 144; ISSN 0002-7863

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

References (34)

Towards the Evolution of Artificial Metalloenzymes—A Protein Engineer's Perspective Markel, Ulrich; Sauer, Daniel F.; Schiffels, Johannes Angewandte Chemie International Edition, Vol. 58, Issue 14 https://doi.org/10.1002/anie.201811042	journal	February 2019
Site‐Selective Functionalization of (sp ³ )C−H Bonds Catalyzed by Artificial Metalloenzymes Containing an Iridium‐Porphyrin Cofactor Gu, Yang; Natoli, Sean N.; Liu, Zhennan Angewandte Chemie International Edition, Vol. 58, Issue 39 https://doi.org/10.1002/anie.201907460	journal	September 2019
Orthogonal Expression of an Artificial Metalloenzyme for Abiotic Catalysis Reynolds, Evan W.; Schwochert, Timothy D.; McHenry, Matthew W. ChemBioChem, Vol. 18, Issue 24 https://doi.org/10.1002/cbic.201700397	journal	November 2017
Cytochrome P450 oxygenases of Taxol biosynthesis Kaspera, Rüdiger; Croteau, Rodney Phytochemistry Reviews, Vol. 5, Issue 2-3 https://doi.org/10.1007/s11101-006-9006-4	journal	October 2006
Structural biology of bacterial iron uptake Krewulak, Karla D.; Vogel, Hans J. Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1778, Issue 9 https://doi.org/10.1016/j.bbamem.2007.07.026	journal	September 2008
Intramolecular C(sp3)H amination of arylsulfonyl azides with engineered and artificial myoglobin-based catalysts Bordeaux, Melanie; Singh, Ritesh; Fasan, Rudi Bioorganic & Medicinal Chemistry, Vol. 22, Issue 20 https://doi.org/10.1016/j.bmc.2014.05.015	journal	October 2014
Selective C–H bond functionalization using repurposed or artificial metalloenzymes Upp, David M.; Lewis, Jared C. Current Opinion in Chemical Biology, Vol. 37 https://doi.org/10.1016/j.cbpa.2016.12.027	journal	April 2017
Selective C H bond functionalization with engineered heme proteins: new tools to generate complexity Zhang, Ruijie K.; Huang, Xiongyi; Arnold, Frances H. Current Opinion in Chemical Biology, Vol. 49 https://doi.org/10.1016/j.cbpa.2018.10.004	journal	April 2019
The Siderophore Binding Protein FeuA Shows Limited Promiscuity toward Exogenous Triscatecholates Peuckert, Florian; Ramos-Vega, Ana Laura; Miethke, Marcus Chemistry & Biology, Vol. 18, Issue 7 https://doi.org/10.1016/j.chembiol.2011.05.006	journal	July 2011
Cytochrome P450: taming a wild type enzyme Jung, Sang Taek; Lauchli, Ryan; Arnold, Frances H. Current Opinion in Biotechnology, Vol. 22, Issue 6 https://doi.org/10.1016/j.copbio.2011.02.008	journal	December 2011
Artificial Metalloenzymes on the Verge of New-to-Nature Metabolism Jeschek, Markus; Panke, Sven; Ward, Thomas R. Trends in Biotechnology, Vol. 36, Issue 1 https://doi.org/10.1016/j.tibtech.2017.10.003	journal	January 2018
Improved Method for the Incorporation of Heme Cofactors into Recombinant Proteins Using Escherichia coli Nissle 1917 Fiege, Kerstin; Querebillo, Christine Joy; Hildebrandt, Peter Biochemistry, Vol. 57, Issue 19 https://doi.org/10.1021/acs.biochem.8b00242	journal	April 2018
Artificial Metalloenzymes: Reaction Scope and Optimization Strategies Schwizer, Fabian; Okamoto, Yasunori; Heinisch, Tillmann Chemical Reviews, Vol. 118, Issue 1 https://doi.org/10.1021/acs.chemrev.7b00014	journal	May 2017
Chemical Optimization of Whole-Cell Transfer Hydrogenation Using Carbonic Anhydrase as Host Protein Rebelein, Johannes G.; Cotelle, Yoann; Garabedian, Brett ACS Catalysis, Vol. 9, Issue 5 https://doi.org/10.1021/acscatal.9b01006	journal	April 2019
Beyond Iron: Iridium-Containing P450 Enzymes for Selective Cyclopropanations of Structurally Diverse Alkenes Key, Hanna M.; Dydio, Paweł; Liu, Zhennan ACS Central Science, Vol. 3, Issue 4 https://doi.org/10.1021/acscentsci.6b00391	journal	April 2017
Artificial Metalloenzymes: Challenges and Opportunities Davis, Holly J.; Ward, Thomas R. ACS Central Science, Vol. 5, Issue 7 https://doi.org/10.1021/acscentsci.9b00397	journal	July 2019
A Simple Combinatorial Codon Mutagenesis Method for Targeted Protein Engineering Belsare, Ketaki D.; Andorfer, Mary C.; Cardenas, Frida S. ACS Synthetic Biology, Vol. 6, Issue 3 https://doi.org/10.1021/acssynbio.6b00297	journal	January 2017
Ru-Porphyrin Protein Scaffolds for Sensing O ₂ Winter, Michael B.; McLaurin, Emily J.; Reece, Steven Y. Journal of the American Chemical Society, Vol. 132, Issue 16 https://doi.org/10.1021/ja101527r	journal	April 2010
Metal-Substituted Protein MRI Contrast Agents Engineered for Enhanced Relaxivity and Ligand Sensitivity Lelyveld, Victor S.; Brustad, Eric; Arnold, Frances H. Journal of the American Chemical Society, Vol. 133, Issue 4 https://doi.org/10.1021/ja107936d	journal	February 2011
Chemoselective, Enzymatic C–H Bond Amination Catalyzed by a Cytochrome P450 Containing an Ir(Me)-PIX Cofactor Dydio, Paweł; Key, Hanna M.; Hayashi, Hiroki Journal of the American Chemical Society, Vol. 139, Issue 5 https://doi.org/10.1021/jacs.6b11410	journal	January 2017
Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants the Keio collection Baba, Tomoya; Ara, Takeshi; Hasegawa, Miki Molecular Systems Biology, Vol. 2, Article No. 2006.0008 https://doi.org/10.1038/msb4100050	journal	February 2006
Directed evolution of artificial metalloenzymes for in vivo metathesis Jeschek, Markus; Reuter, Raphael; Heinisch, Tillmann Nature, Vol. 537, Issue 7622 https://doi.org/10.1038/nature19114	journal	August 2016
Enantioselective, intermolecular benzylic C–H amination catalysed by an engineered iron-haem enzyme Prier, Christopher K.; Zhang, Ruijie K.; Buller, Andrew R. Nature Chemistry, Vol. 9, Issue 7 https://doi.org/10.1038/nchem.2783	journal	May 2017
Unnatural biosynthesis by an engineered microorganism with heterologously expressed natural enzymes and an artificial metalloenzyme Huang, Jing; Liu, Zhennan; Bloomer, Brandon J. Nature Chemistry, Vol. 13, Issue 12 https://doi.org/10.1038/s41557-021-00801-3	journal	October 2021
Enzymatic assembly of carbon–carbon bonds via iron-catalysed sp3 C–H functionalization Zhang, Ruijie K.; Chen, Kai; Huang, Xiongyi Nature, Vol. 565, Issue 7737 https://doi.org/10.1038/s41586-018-0808-5	journal	December 2018
Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import Buchanan, Susan K.; Lukacik, Petra; Grizot, Sylvestre The EMBO Journal, Vol. 26, Issue 10 https://doi.org/10.1038/sj.emboj.7601693	journal	April 2007
Enzymatic functionalization of carbon–hydrogen bonds Lewis, Jared C.; Coelho, Pedro S.; Arnold, Frances H. Chem. Soc. Rev., Vol. 40, Issue 4 https://doi.org/10.1039/C0CS00067A	journal	January 2011
E. coli surface display of streptavidin for directed evolution of an allylic deallylase Heinisch, Tillmann; Schwizer, Fabian; Garabedian, Brett Chemical Science, Vol. 9, Issue 24 https://doi.org/10.1039/C8SC00484F	journal	January 2018
Combining enzymes and organometallic complexes: novel artificial metalloenzymes and hybrid systems for C–H activation chemistry Perez-Rizquez, Carlos; Rodriguez-Otero, Alba; Palomo, Jose M. Organic & Biomolecular Chemistry, Vol. 17, Issue 30 https://doi.org/10.1039/C9OB01091B	journal	January 2019
Non-iron metalloporphyrins: potent antibacterial compounds that exploit haem/Hb uptake systems of pathogenic bacteria Stojiljkovic, Igor; Kumar, Veena; Srinivasan, Nithya Molecular Microbiology, Vol. 31, Issue 2 https://doi.org/10.1046/j.1365-2958.1999.01175.x	journal	January 1999
An artificial metalloenzyme with the kinetics of native enzymes Dydio, P.; Key, H. M.; Nazarenko, A. Science, Vol. 354, Issue 6308 https://doi.org/10.1126/science.aah4427	journal	October 2016
Analysis of the Genome Structure of the Nonpathogenic Probiotic Escherichia coli Strain Nissle 1917 Grozdanov, Lubomir; Raasch, Carsten; Schulze, Jürgen Journal of Bacteriology, Vol. 186, Issue 16 https://doi.org/10.1128/JB.186.16.5432-5441.2004	journal	August 2004
Bacterial cytochrome P450-catalyzed regio- and stereoselective steroid hydroxylation enabled by directed evolution and rational design Zhang, Xiaodong; Peng, Yaqin; Zhao, Jing Bioresources and Bioprocessing, Vol. 7, Issue 1 https://doi.org/10.1186/s40643-019-0290-4	journal	January 2020
Regioselective hydroxylation of steroid hormones by human cytochromes P450 Niwa, Toshiro; Murayama, Norie; Imagawa, Yurie Drug Metabolism Reviews, Vol. 47, Issue 2 https://doi.org/10.3109/03602532.2015.1011658	journal	February 2015

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