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 A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

2016 ◽  
Vol 60 (10) ◽  
pp. 5995-6002 ◽  
Author(s):  
Kristin R. Baker ◽  
Bimal Jana ◽  
Henrik Franzyk ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Throughput Screening ◽  
Gram Negative Bacteria ◽  
Chromogenic Substrate ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Screening Platform

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

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 Contribution of Membrane-Binding and Enzymatic Domains of Penicillin Binding Protein 5 to Maintenance of Uniform Cellular Morphology of Escherichia coli

2002 ◽  
Vol 184 (13) ◽  
pp. 3630-3639 ◽  
Author(s):  
David E. Nelson ◽  
Anindya S. Ghosh ◽  
Avery L. Paulson ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Site Directed Mutagenesis ◽  
Outer Face ◽  
Penicillin Binding Protein ◽  
Membrane Anchor ◽  
E Coli ◽  
Membrane Bound ◽  
Carboxy Terminal ◽  
Membrane Anchoring ◽  
Β Sheet

ABSTRACT Four low-molecular-weight penicillin binding proteins (LMW PBPs) of Escherichia coli are closely related and have similar dd-carboxypeptidase activities (PBPs 4, 5, and 6 and DacD). However, only one, PBP 5, has a demonstrated physiological function. In its absence, certain mutants of E. coli have altered diameters and lose their uniform outer contour, resulting in morphologically aberrant cells. To determine what differentiates the activities of these LMW PBPs, we constructed fusion proteins combining portions of PBP 5 with fragments of other dd-carboxypeptidases to see which hybrids restored normal morphology to a strain lacking PBP 5. Functional complementation occurred when truncated PBP 5 was combined with the terminal membrane anchor sequences of PBP 6 or DacD. However, complementation was not restored by the putative carboxy-terminal anchor of PBP 4 or by a transmembrane region of the osmosensor protein ProW, even though these hybrids were membrane bound. Site-directed mutagenesis of the carboxy terminus of PBP 5 indicated that complementation required a generalized amphipathic membrane anchor but that no specific residues in this region seemed to be required. A functional fusion protein was produced by combining the N-terminal enzymatic domain of PBP 5 with the C-terminal β-sheet domain of PBP 6. In contrast, the opposite hybrid of PBP 6 to PBP 5 was not functional. The results suggest that the mode of PBP 5 membrane anchoring is important, that the mechanism entails more than a simple mechanical tethering of the enzyme to the outer face of the inner membrane, and that the physiological differences among the LMW PBPs arise from structural differences in the dd-carboxypeptidase enzymatic core.

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 Directed Evolution of a Cp*Rh III ‐Linked Biohybrid Catalyst Based on a Screening Platform with Affinity Purification

ChemBioChem ◽  
2020 ◽  
Author(s):  
Shunsuke Kato ◽  
Akira Onoda ◽  
Naomasa Taniguchi ◽  
Ulrich Schwaneberg ◽  
Takashi Hayashi
Keyword(s):  
Directed Evolution ◽  
Affinity Purification ◽  
Screening Platform

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.

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