Directed Evolution of Artificial Metalloenzymes in Whole Cells

2021 ◽  
Author(s):  
Yang Gu ◽  
Brandon Bloomer ◽  
Zhennan Liu ◽  
Douglas Clark ◽  
John F. Hartwig
Keyword(s):  
Directed Evolution ◽  
Whole Cells ◽  
Artificial Metalloenzymes

scholarly journals In Vivo Assembly of Artificial Metalloenzymes and Application in Whole‐Cell Biocatalysis

2020 ◽  
Author(s):  
Shreyans Chordia ◽  
Siddarth Narasimhan ◽  
Alessandra Lucini Paioni ◽  
Marc Baldus ◽  
Gerard Roelfes
Keyword(s):  
Directed Evolution ◽  
Cell Fractionation ◽  
Whole Cell ◽  
Whole Cells ◽  
E Coli ◽  
Whole Cell Biocatalysis ◽  
Catalytically Active ◽  
Active Transition ◽  
Artificial Metalloenzymes

Artificial metalloenzymes (ArMs), which are hybrids of catalytically active transition metal complexes and proteins, have emerged as promising approach to the creation of biocatalysts for reactions that have no equivalent in nature. Here we report the assembly and application in catalysis of ArMs in the cytoplasm of E. coli cells based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneously added copper(II)‐phenanthroline (Cu(II)‐phen) complex. The ArMs are spontaneously assembled by addition of Cu(II)‐phen to E. coli cells that express LmrR and it is shown that the ArM containing whole cells are active in the catalysis of the enantioselective vinylogous Friedel‐Crafts alkylation of indoles. The ArM assembly in E. coli is further supported by a combination of cell‐ fractionation and inhibitor experiments and confirmed by in‐cell solid‐state NMR. A mutagenesis study showed that the same trends in catalytic activity and enantioselectivity in response to mutations of LmrR were observed for the ArM containing whole cells and the isolated ArMs. This made it possible to perform a directed evolution study using ArMs in whole cells, which gave rise to a mutant, LmrR_A92E_M8D that showed increased activity and enantioselectivity in the catalyzed vinylogous Friedel‐Crafts alkylation of a variety of indoles. The unique aspect of this whole‐cell ArM system is that no engineering of the microbial host, the protein scaffold or the cofactor is required to achieve ArM assembly and catalysis. This makes this system attractive for applications in whole cell biocatalysis and directed evolution, as demonstrated here. Moreover, our findings represent important step forward towards achieving the challenging goal of a hybrid metabolism by integrating artificial metalloenzymes in biosynthetic pathways.

scholarly journals In Vivo Assembly of Artificial Metalloenzymes and Application in Whole‐Cell Biocatalysis

2020 ◽  
Author(s):  
Shreyans Chordia ◽  
Siddarth Narasimhan ◽  
Alessandra Lucini Paioni ◽  
Marc Baldus ◽  
Gerard Roelfes
Keyword(s):  
Directed Evolution ◽  
Cell Fractionation ◽  
Whole Cell ◽  
Whole Cells ◽  
E Coli ◽  
Whole Cell Biocatalysis ◽  
Catalytically Active ◽  
Active Transition ◽  
Artificial Metalloenzymes

Artificial metalloenzymes (ArMs), which are hybrids of catalytically active transition metal complexes and proteins, have emerged as promising approach to the creation of biocatalysts for reactions that have no equivalent in nature. Here we report the assembly and application in catalysis of ArMs in the cytoplasm of E. coli cells based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneously added copper(II)‐phenanthroline (Cu(II)‐phen) complex. The ArMs are spontaneously assembled by addition of Cu(II)‐phen to E. coli cells that express LmrR and it is shown that the ArM containing whole cells are active in the catalysis of the enantioselective vinylogous Friedel‐Crafts alkylation of indoles. The ArM assembly in E. coli is further supported by a combination of cell‐ fractionation and inhibitor experiments and confirmed by in‐cell solid‐state NMR. A mutagenesis study showed that the same trends in catalytic activity and enantioselectivity in response to mutations of LmrR were observed for the ArM containing whole cells and the isolated ArMs. This made it possible to perform a directed evolution study using ArMs in whole cells, which gave rise to a mutant, LmrR_A92E_M8D that showed increased activity and enantioselectivity in the catalyzed vinylogous Friedel‐Crafts alkylation of a variety of indoles. The unique aspect of this whole‐cell ArM system is that no engineering of the microbial host, the protein scaffold or the cofactor is required to achieve ArM assembly and catalysis. This makes this system attractive for applications in whole cell biocatalysis and directed evolution, as demonstrated here. Moreover, our findings represent important step forward towards achieving the challenging goal of a hybrid metabolism by integrating artificial metalloenzymes in biosynthetic pathways.

Directed Evolution of Artificial Metalloenzymes in Whole Cells

2021 ◽  
Author(s):  
Yang Gu ◽  
Brandon Bloomer ◽  
Zhennan Liu ◽  
Douglas Clark ◽  
John F. Hartwig
Keyword(s):  
Directed Evolution ◽  
Whole Cells ◽  
Artificial Metalloenzymes

scholarly journals Artificial Biosynthetic Pathway for an Unnatural Terpenoid with an Iridiumcontaining P450

2020 ◽  
Author(s):  
Jing Huang ◽  
Zhennan Liu ◽  
brandon bloomer ◽  
Douglas Clark ◽  
Aindrila Mukhopadhyay ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Chemical Reactions ◽  
Biosynthetic Pathway ◽  
Metabolic Networks ◽  
Complex Molecules ◽  
Whole Cells ◽  
Porphyrin Complex ◽  
Heme Transport ◽  
Artificial Metalloenzymes

<div>Synthetic biology enables microbial hosts to produce complex molecules that are</div><div>otherwise produced by organisms that are rare or difficult to cultivate, but the structures of these</div><div>molecules are limited to chemical reactions catalyzed by natural enzymes. The integration of</div><div>artificial metalloenzymes (ArMs) that catalyze abiotic reactions into metabolic networks could</div><div>broaden the cache of molecules produced biosynthetically by microorgansms. We report the</div><div>assembly of an ArM containing an iridium-porphyrin complex in the cytoplasm of a terpene</div><div>producing Escherichia coli by a heterologous heme transport machinery, and insertion of this ArM</div><div>into a natural biosynthetic pathway to produce an unnatural terpenoid. This work shows that</div><div>synthetic biology and synthetic chemistry, incorporated together in whole cells, can produce</div><div>molecules previously inaccessible to nature.</div>

Directed Evolution of Artificial Metalloenzymes

2014 ◽  
Vol 55 (1) ◽  
pp. 51-60 ◽  
Author(s):  
Adriana Ilie ◽  
Manfred T. Reetz
Keyword(s):  
Directed Evolution ◽  
Artificial Metalloenzymes

scholarly journals Towards the Directed Evolution of Artificial Metalloenzymes

2021 ◽  
Vol 75 (4) ◽  
pp. 257-260
Author(s):  
Jaicy Vallapurackal
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
Droplet Microfluidics ◽  
Host Protein ◽  
Great Promise ◽  
Genetic Optimization ◽  
The Core ◽  
Technological Developments ◽  
Artificial Metalloenzymes ◽  
Tools And Methods

Artificial metalloenzymes (ArMs) are a class of enzymes holding great promise. In contrast to natural enzymes, the core of ArMs is a synthetic metallocofactor, with potential for bio-orthogonal reactivity, incorporated within a host protein. Next to chemical optimization of the metallocofactor, genetic optimization of the protein allows the further improvement of the ArM. Genetic optimization through directed evolution requires extensive screening of a large sequence-scape to enable the optimization of a desired phenotype. The process is however mostly limited by the throughput of the tools and methods available for screening. In recent years, versatile methods based on droplet microfluidics have been developed to address the need for higher throughput. This article aims to give an introduction into ArMs and the recent technological developments allowing high-throughput directed evolution of enzymes.

scholarly journals Artificial Biosynthetic Pathway for an Unnatural Terpenoid with an Iridiumcontaining P450

2020 ◽  
Author(s):  
Jing Huang ◽  
Zhennan Liu ◽  
brandon bloomer ◽  
Douglas Clark ◽  
Aindrila Mukhopadhyay ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Chemical Reactions ◽  
Biosynthetic Pathway ◽  
Metabolic Networks ◽  
Complex Molecules ◽  
Whole Cells ◽  
Porphyrin Complex ◽  
Heme Transport ◽  
Artificial Metalloenzymes

<div>Synthetic biology enables microbial hosts to produce complex molecules that are</div><div>otherwise produced by organisms that are rare or difficult to cultivate, but the structures of these</div><div>molecules are limited to chemical reactions catalyzed by natural enzymes. The integration of</div><div>artificial metalloenzymes (ArMs) that catalyze abiotic reactions into metabolic networks could</div><div>broaden the cache of molecules produced biosynthetically by microorgansms. We report the</div><div>assembly of an ArM containing an iridium-porphyrin complex in the cytoplasm of a terpene</div><div>producing Escherichia coli by a heterologous heme transport machinery, and insertion of this ArM</div><div>into a natural biosynthetic pathway to produce an unnatural terpenoid. This work shows that</div><div>synthetic biology and synthetic chemistry, incorporated together in whole cells, can produce</div><div>molecules previously inaccessible to nature.</div>

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