The Directed Evolution of Radiation Resistance in E. coli

1995 ◽  
Vol 216 (2) ◽  
pp. 549-553 ◽  
Author(s):  
D. Ewing
Keyword(s):  
Directed Evolution ◽  
Radiation Resistance ◽  
E Coli

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

scholarly journals CotA laccase: high-throughput manipulation and analysis of recombinant enzyme libraries expressed in E. coli using droplet-based microfluidics

The Analyst ◽  
2014 ◽  
Vol 139 (13) ◽  
pp. 3314-3323 ◽  
Author(s):  
Thomas Beneyton ◽  
Faith Coldren ◽  
Jean-Christophe Baret ◽  
Andrew D. Griffiths ◽  
Valérie Taly
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
Recombinant Enzyme ◽  
Cell Analysis ◽  
E Coli ◽  
Cota Laccase

A high-throughput cell analysis and sorting platform using droplet-based microfluidics is introduced for directed evolution of recombinant CotA laccase expressed in E. coli.

scholarly journals Directed Evolution of Ionizing Radiation Resistance in Escherichia coli

2009 ◽  
Vol 191 (16) ◽  
pp. 5240-5252 ◽  
Author(s):  
Dennis R. Harris ◽  
Steve V. Pollock ◽  
Elizabeth A. Wood ◽  
Reece J. Goiffon ◽  
Audrey J. Klingele ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ionizing Radiation ◽  
Directed Evolution ◽  
Radiation Resistance ◽  
Bacterial Species ◽  
Repair System ◽  
Multiple Alleles ◽  
Evolutionary Pathways ◽  
K 12 ◽  
Ionizing Radiation Resistance

ABSTRACT We have generated extreme ionizing radiation resistance in a relatively sensitive bacterial species, Escherichia coli, by directed evolution. Four populations of Escherichia coli K-12 were derived independently from strain MG1655, with each specifically adapted to survive exposure to high doses of ionizing radiation. D37 values for strains isolated from two of the populations approached that exhibited by Deinococcus radiodurans. Complete genomic sequencing was carried out on nine purified strains derived from these populations. Clear mutational patterns were observed that both pointed to key underlying mechanisms and guided further characterization of the strains. In these evolved populations, passive genomic protection is not in evidence. Instead, enhanced recombinational DNA repair makes a prominent but probably not exclusive contribution to genome reconstitution. Multiple genes, multiple alleles of some genes, multiple mechanisms, and multiple evolutionary pathways all play a role in the evolutionary acquisition of extreme radiation resistance. Several mutations in the recA gene and a deletion of the e14 prophage both demonstrably contribute to and partially explain the new phenotype. Mutations in additional components of the bacterial recombinational repair system and the replication restart primosome are also prominent, as are mutations in genes involved in cell division, protein turnover, and glutamate transport. At least some evolutionary pathways to extreme radiation resistance are constrained by the temporally ordered appearance of specific alleles.

scholarly journals Improved precursor-directed biosynthesis in E. coli via directed evolution

2010 ◽  
Vol 64 (1) ◽  
pp. 59-64 ◽  
Author(s):  
Ho Young Lee ◽  
Colin J B Harvey ◽  
David E Cane ◽  
Chaitan Khosla
Keyword(s):  
Directed Evolution ◽  
E Coli ◽  
Directed Biosynthesis

scholarly journals Click-chemistry enabled directed evolution of glycosynthases for bespoke glycans synthesis

2020 ◽  
Author(s):  
Ayushi Agrawal ◽  
Chandra Kanth Bandi ◽  
Tucker Burgin ◽  
Youngwoo Woo ◽  
Heather B. Mayes ◽  
...  
Keyword(s):  
Single Cell ◽  
Click Chemistry ◽  
Directed Evolution ◽  
Cell Sorting ◽  
Screening Methods ◽  
Carbohydrate Active Enzymes ◽  
E Coli ◽  
Highly Sensitive ◽  
Increased Activity

AbstractEngineering of carbohydrate-active enzymes like glycosynthases for chemoenzymatic synthesis of bespoke oligosaccharides has been limited by the lack of suitable directed evolution based protein engineering methods. Currently there are no ultrahigh-throughput screening methods available for rapid and highly sensitive single cell-based screening of evolved glycosynthase enzymes employing azido sugars as substrates. Here, we report a fluorescence-based approach employing click-chemistry for the selective detection of glycosyl azides (versus free inorganic azides) that facilitated ultrahigh-throughput in-vivo single cell-based assay of glycosynthase activity. This discovery has led to the development of a directed evolution methodology for screening and sorting glycosynthase mutants for synthesis of desired fucosylated oligosaccharides. Our screening technique facilitated rapid fluorescence activated cell sorting of a large library of glycosynthase variants (>106 mutants) expressed in E. coli to identify several novel mutants with increased activity for β-fucosyl-azide activated donor sugars towards desired acceptor sugars, demonstrating the broader applicability of this methodology.

scholarly journals Creating Hybrid Genes by Homologous Recombination

2000 ◽  
Vol 16 (1-2) ◽  
pp. 3-13 ◽  
Author(s):  
Peter L. Wang
Keyword(s):  
Directed Evolution ◽  
High Efficiency ◽  
Genomic Sequence ◽  
Sequence Data ◽  
Strand Breaks ◽  
E Coli ◽  
Hybrid Genes ◽  
Distinct Features

Recombination of homologous genes is a powerful mechanism for generating sequence diversity, and can be applied to protein analysis and directed evolution.In vitrorecombination methods such as DNA shuffling are very flexible and can give hybrid genes with multiple crossovers; they have been used extensively to evolve proteins with improved and novel properties.In vivorecombination in bothE. coliand yeast is greatly enhanced by double-strand breaks; forE. coli, mutant strains are often necessary to obtain high efficiency. Intra- and inter-molecular recombinationIn vivohave distinct features; both give hybrids with one or two crossovers, and have been used to study structure-function relationships of many proteins. Recentlyin vivorecombination has been used to generate diversity for directed evolution, creating a large phage display antibody library. Recombination methods will become increasingly useful in light of the explosion in genomic sequence data and potential for engineered proteins.

scholarly journals A fluorogenic screening platform enables directed evolution of an alkyne biosynthetic tool

2016 ◽  
Vol 52 (75) ◽  
pp. 11239-11242 ◽  
Author(s):  
Xuejun Zhu ◽  
Peyton Shieh ◽  
Michael Su ◽  
Carolyn R. Bertozzi ◽  
Wenjun Zhang
Keyword(s):  
Directed Evolution ◽  
E Coli ◽  
Membrane Bound ◽  
Screening Platform

A fluorogenic screening platform enabled the engineering of a membrane-bound bifunctional desaturase/acetylenase for improved activity in E. coli.

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 Diverse engineered heme proteins enable stereodivergent cyclopropanation of unactivated alkenes

2017 ◽  
Author(s):  
Anders M. Knight ◽  
S. B. Jennifer Kan ◽  
Russell D. Lewis ◽  
Oliver F. Brandenberg ◽  
Kai Chen ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Heme Proteins ◽  
Cytochromes P450 ◽  
Enantiomeric Excess ◽  
Saturation Mutagenesis ◽  
Protein Diversity ◽  
Site Saturation ◽  
E Coli ◽  
Carbene Transfer ◽  
Aqueous Conditions

<p>Stereodivergent syntheses leading to the different stereoisomers of a product are useful in the discovery and testing of drugs and agrochemicals. A longstanding challenge in catalysis, developing sets of stereodivergent catalysts is often solved for enzymes by screening Nature’s diversity for biocatalysts with complementary stereoselectivities. Here, Nature’s protein diversity has been leveraged to develop stereodivergent catalysts for a reaction not known in biology, cyclopropanation via carbene transfer. By screening diverse native and engineered heme proteins, we identified globins and serine-ligated cytochromes P450 with promiscuous activity for cyclopropanation of unactivated alkene substrates. Their activities and stereoselectivities were enhanced by directed evolution: 1-3 rounds of site-saturation mutagenesis and screening generated enzymes that catalyze the stereodivergent cyclopropanation to form each of the four stereoisomers of unactivated alkenes and electron-deficient alkenes with up to 5,400 total turnovers and 98% enantiomeric excess. These fully genetically encoded biocatalysts function in whole <i>E. coli</i> cells in mild, aqueous conditions and provide the first example of enantioselective, intermolecular iron-catalyzed cyclopropanation of unactivated alkenes via carbene transfer.</p>

Sign in / Sign up

Export Citation Format

Share Document