scholarly journals Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Emma Barahona ◽  
Emilio Jiménez-Vicente ◽  
Luis M. Rubio
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Rhodobacter Capsulatus ◽  
Random Mutagenesis ◽  
Fluorescence Emission ◽  
Inducible Promoter ◽  
Hydrogen Gas ◽  
Photosynthetic Organisms ◽  
Fe Protein ◽  
Protein Variants

Abstract When produced biologically, especially by photosynthetic organisms, hydrogen gas (H2) is arguably the cleanest fuel available. An important limitation to the discovery or synthesis of better H2-producing enzymes is the absence of methods for the high-throughput screening of H2 production in biological systems. Here, we re-engineered the natural H2 sensing system of Rhodobacter capsulatus to direct the emission of LacZ-dependent fluorescence in response to nitrogenase-produced H2. A lacZ gene was placed under the control of the hupA H2-inducible promoter in a strain lacking the uptake hydrogenase and the nifH nitrogenase gene. This system was then used in combination with fluorescence-activated cell sorting flow cytometry to screen large libraries of nitrogenase Fe protein variants generated by random mutagenesis. Exact correlation between fluorescence emission and H2 production levels was found for all automatically selected strains. One of the selected H2-overproducing Fe protein variants lacked 40% of the wild-type amino acid sequence, a surprising finding for a protein that is highly conserved in nature. We propose that this method has great potential to improve microbial H2 production by allowing powerful approaches such as the directed evolution of nitrogenases and hydrogenases.

Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

2019 ◽  
Author(s):  
Huifang Xu ◽  
Weinan Liang ◽  
Linlin Ning ◽  
Yuanyuan Jiang ◽  
Wenxia Yang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Fatty Acid ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Screening Method ◽  
Random Mutagenesis ◽  
Direct Production ◽  
Consumption Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient <i>Escherichia coli</i> host strain and report an HTS approach based on colorimetric detection of H<sub>2</sub>O<sub>2</sub>-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H<sub>2</sub>O<sub>2</sub> as co-substrate.

scholarly journals In Vivo, High-Throughput Selection of Thermostable Cyclohexanone Monooxygenase (CHMO)

Catalysts ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 935
Author(s):  
Sarah Maxel ◽  
Linyue Zhang ◽  
Edward King ◽  
Ana Paula Acosta ◽  
Ray Luo ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Rational Design ◽  
Random Mutagenesis ◽  
Residual Activity ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Coli Strain ◽  
Screening Tools ◽  
Cyclohexanone Monooxygenase

Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 is characterized as having wide substrate versatility for the biooxidation of (cyclic) ketones into esters and lactones with high stereospecificity. Despite industrial potential, CHMO usage is restricted by poor thermostability. Limited high-throughput screening tools and challenges in rationally engineering thermostability have impeded CHMO engineering efforts. We demonstrate the application of an aerobic, high-throughput growth selection platform in Escherichia coli (strain MX203) for the discovery of thermostability enhancing mutations for CHMO. The selection employs growth for the easy readout of CHMO activity in vivo, by requiring nicotinamide adenine dinucleotide phosphate (NADPH)-consuming enzymes to restore cellular redox balance. In the presence of the native substrate cyclohexanone, variant CHMO GV (A245G-A288V) was discovered from a random mutagenesis library screened at 42 °C. This variant retained native activity, exhibited ~4.4-fold improvement in residual activity after 30 °C incubation, and demonstrated ~5-fold higher cyclohexanone conversion at 37 °C compared to the wild type. Molecular modeling indicates that CHMO GV experiences more favorable residue packing and supports additional backbone hydrogen bonding. Further rational design resulted in CHMO A245G-A288V-T415C with improved thermostability at 45 °C. Our platform for oxygenase evolution enabled the rapid engineering of protein stability critical for industrial scalability.

scholarly journals Colloidal Aggregate Detection by Rapid Fluorescence Measurement of Liquid Surface Curvature Changes in Multiwell Plates

2007 ◽  
Vol 12 (7) ◽  
pp. 966-971 ◽  
Author(s):  
Lifeng Cai ◽  
Miriam Gochin
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Dynamic Range ◽  
Liquid Surface ◽  
Enzyme Inhibitor ◽  
Fluorescence Emission ◽  
Fluorescence Measurement ◽  
Screening Assay ◽  
Colloidal Aggregate ◽  
Multiwell Plates

A simple fluorescence method is reported for the detection of colloidal aggregate formation in solution, with specific applications to determine the critical micelle concentration (CMC) of surfactants and detect small-molecule promiscuous inhibitors. The method exploits the meniscus curvature changes in high-density multiwell plates associated with colloidal changes in solution. The shape of the meniscus has a significant effect on fluorescence intensity when detected using a top-read fluorescence plate reader because of the effect of total internal reflection on fluorescence emission through a curved liquid surface. A dynamic range of 60% is calculated and observed and is measured with a relative sensitivity of 2%. Facile determination of the CMC of a variety of surfactants is demonstrated, as well as a screening assay for aggregate forming properties of small drug-like compounds, a common cause of promiscuous inhibition in high-throughput screening (HTS) enzyme inhibitor assays. Our preliminary results show a potential HTS assay with Z′ factor of 0.76, with good separation between aggregating and nonaggregating small molecules. The method combines the high sensitivity and universality of classic surface tension methods with simplicity and high-throughput determination, enabling facile detection of molecular interactions involving a change in liquid or solid surface character. ( Journal of Biomolecular Screening 2007:966-971)

scholarly journals Optimized and Automated Protocols for High-Throughput Screening of Amylosucrase Libraries

2007 ◽  
Vol 12 (5) ◽  
pp. 715-723 ◽  
Author(s):  
Stéphane Emond ◽  
Gabrielle Potocki-Véronèse ◽  
Philippe Mondon ◽  
Khalil Bouayadi ◽  
Hakim Kharrat ◽  
...  
Keyword(s):  
Organic Solvents ◽  
High Throughput ◽  
High Throughput Screening ◽  
General Procedure ◽  
Random Mutagenesis ◽  
Culture Conditions ◽  
Screening Assay ◽  
High Production Rate ◽  
Automated Screening

This article describes the design and validation of a general procedure for the high-throughput isolation of amylosucrase variants displaying higher thermostability or increased resistance to organic solvents. This procedure consists of 2 successive steps: an in vivo selection that eliminates inactive variants followed by automated screening of active variants to isolate mutants displaying enhanced features. The authors chose an Escherichia coli expression vector, allowing a high production rate of the recombinant enzyme in miniaturized culture conditions. The screening assay was validated by minimizing variability for various parameters of the protocol, especially bacterial growth and protein production in cultures in 96-well microplates. Recombinant amylosucrase production was normalized by decreasing the coefficient of variance from 27% to 12.5%. Selective screening conditions were defined to select variants displaying higher thermostability or increased resistance to organic solvents. A first-generation amylosucrase variant library, constructed by random mutagenesis, was subjected to this procedure, yielding a mutant displaying a 25-fold increased stability at 50 °C compared to the parental wild-type enzyme. ( Journal of Biomolecular Screening 2007:715-723)

Application of directed evolution in the development of enantioselective enzymes

2000 ◽  
Vol 72 (9) ◽  
pp. 1615-1622 ◽  
Author(s):  
Manfred T. Reetz
Keyword(s):  
Organic Chemistry ◽  
Gene Expression ◽  
High Throughput ◽  
High Throughput Screening ◽  
Kinetic Resolution ◽  
Random Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Hydrolytic Kinetic Resolution ◽  
Novel Approach

A novel approach to developing enantioselective enzymes for use in organic chemistry has been devised which is independent of structural or mechanistic aspects. The underlying idea is to combine appropriate methods of random mutagenesis, gene expression, and high-throughput screening for enantioselectivity. If these actions are performed in repetitive cycles, an evolutionary pressure is created that leads to sequential improvements of the enantioselectivity of a given enzyme-catalyzed reaction. The concept is illustrated by an example involving the lipase-catalyzed hydrolytic kinetic resolution of an α-chiral ester, the enantio-selectivity increasing from ee = 2% (E =1.1) for a wild-type enzyme to ee = 90-93% (E = 25) for the best mutants.

scholarly journals Spectral Unmixing Plate Reader: High-Throughput, High-Precision FRET Assays in Living Cells

2016 ◽  
Vol 22 (3) ◽  
pp. 250-261 ◽  
Author(s):  
Tory M. Schaaf ◽  
Kurt C. Peterson ◽  
Benjamin D. Grant ◽  
David D. Thomas ◽  
Gregory D. Gillispie
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Structural Changes ◽  
Fluorescent Protein ◽  
Fluorescence Emission ◽  
Resonance Energy ◽  
Living Cells ◽  
Spectral Unmixing ◽  
Fluorescence Emission Spectrum ◽  
Plate Reader

We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 min. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded green fluorescent protein (GFP) donor and red fluorescent protein (RFP) acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells. Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of four component (basis) spectra (GFP emission, RFP emission, water Raman, and cell autofluorescence). Excitation and detection are both conducted from the top, allowing for thermoelectric control of the sample temperature from below. This spectral unmixing plate reader (SUPR) delivers an unprecedented combination of speed, precision, and accuracy for studying ensemble-averaged FRET in living cells. It complements our previously reported fluorescence lifetime plate reader, which offers the feature of resolving multiple FRET populations within the ensemble. The combination of these two direct waveform-recording technologies greatly enhances the precision and information content for HTS in drug discovery.

scholarly journals High-throughput synthetic rescue for exhaustive characterization of suppressor mutations in human genes

2020 ◽  
Vol 77 (21) ◽  
pp. 4209-4222
Author(s):  
Farah Kobaisi ◽  
Nour Fayyad ◽  
Eric Sulpice ◽  
Bassam Badran ◽  
Hussein Fayyad-Kazan ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Random Mutagenesis ◽  
Screening Methods ◽  
Knock Out ◽  
Suppressor Mutations ◽  
Human Genes ◽  
Cultured Human Cells

Abstract Inherited or acquired mutations can lead to pathological outcomes. However, in a process defined as synthetic rescue, phenotypic outcome created by primary mutation is alleviated by suppressor mutations. An exhaustive characterization of these mutations in humans is extremely valuable to better comprehend why patients carrying the same detrimental mutation exhibit different pathological outcomes or different responses to treatment. Here, we first review all known suppressor mutations’ mechanisms characterized by genetic screens on model species like yeast or flies. However, human suppressor mutations are scarce, despite some being discovered based on orthologue genes. Because of recent advances in high-throughput screening, developing an inventory of human suppressor mutations for pathological processes seems achievable. In addition, we review several screening methods for suppressor mutations in cultured human cells through knock-out, knock-down or random mutagenesis screens on large scale. We provide examples of studies published over the past years that opened new therapeutic avenues, particularly in oncology.

scholarly journals Syntrophic co-culture amplification of production phenotype for high-throughput screening of microbial strain libraries

2019 ◽  
Author(s):  
Tatyana E. Saleski ◽  
Alissa R. Kerner ◽  
Meng Ting Chung ◽  
Corine M. Jackman ◽  
Azzaya Khasbaatar ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Parent Strain ◽  
Dynamic Range ◽  
Random Mutagenesis ◽  
Spatial Separation ◽  
List Type ◽  
Target Molecule ◽  
Wide Range ◽  
Target Molecules

AbstractMicrobes can be engineered to synthesize a wide array of bioproducts, yet production phenotype evaluation remains a frequent bottleneck in the design-build-test cycle where strain development requires iterative rounds of library construction and testing. Here, we presentSyntrophicCo-cultureAmplification ofProduction phenotype (SnoCAP). Through a metabolic cross-feeding circuit, the production level of a target molecule is translated into highly distinguishable co-culture growth characteristics, which amplifies differences in production into highly distinguishable growth phenotypes. We demonstrate SnoCAP with the screening ofEscherichia colistrains for production of two target molecules: 2-ketoisovalerate, a precursor of the drop-in biofuel isobutanol, and L-tryptophan. The dynamic range of the screening can be tuned by employing an inhibitory analog of the target molecule. Screening based on this framework requires compartmentalization of individual producers with the sensor strain. We explore three formats of implementation with increasing throughput capability: confinement in microtiter plates (102-104assays/experiment), spatial separation on agar plates (104-105assays/experiment), and encapsulation in microdroplets (105-107assays/experiment). Using SnoCAP, we identified an efficient isobutanol production strain from a random mutagenesis library, reaching a final titer that is 5-fold higher than that of the parent strain. The framework can also be extended to screening for secondary metabolite production using a push-pull strategy. We expect that SnoCAP can be readily adapted to the screening of various microbial species, to improve production of a wide range of target molecules.HighlightsA high-throughput screening platform based on cross-feeding auxotrophs was developed.Compartmentalization was implemented in three formats: microplates, agar plates, and microdroplets.Utility of the screening was demonstrated for two proof-of-concept target molecules: 2-ketoisovalerate and L-tryptophan.The assay dynamic range was tuned by addition of an inhibitory analog.The screening was applied to identify a strain from a chemically mutagenized library that produces 5-fold higher isobutanol titer than the parent strain.

scholarly journals High-Throughput Screening of Higher Α-Amylase Producing Bacillus Licheniformis by Fluorescence-Activated Droplet Sorting

2021 ◽  
Author(s):  
Huiling Yuan ◽  
Ran Tu ◽  
Xinwei Tong ◽  
Yuping Lin ◽  
Qinhong Wang
Keyword(s):  
Bacillus Licheniformis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Single Cells ◽  
Random Mutagenesis ◽  
Strain Improvement ◽  
Industrial Applications ◽  
Cell Factory ◽  
Temperature Plasma ◽  
Droplet Sorting

Abstract Backgroundα-Amylases is one of the most important starch degrading enzymes and has the widest range of industrial applications. Bacillus licheniformis has been widely used as a cell factory for industrial production of amylase. However, difficulties in genetic modification of B. licheniformis have limited its widespread use. Directed evolution, based on the combination of random mutagenesis and high throughput screening (HTS), has been proven an effective strategy in strain improvement for increasing the productivity, but it requires a suitable HTS system to screen the desired mutants. Droplet-based microfluidics has emerged as a powerful tool for single-cell screening with ultra-high throughput, however, the accessibility of a droplet microfluidic HTS platform to users having no background in microfluidics is still an issue. ResultsHere, we first developed a microfluidic HTS platform based on fluorescence-activated droplet sorting (FADS) technology. This platform allowed (i) encapsulation of single cells in monodisperse water-in-oil droplets; (ii) cell growth and protein production in droplets; (iii) sorting of droplets based on their fluorescence intensities. To validate the platform, a model selection experiment of a binary mixture of Bacillus strains was performed and a 45.6-fold enrichment was achieved at a sorting rate of 300 droplets per second. Furthermore, we used the platform for the selection of higher α-amylase-producing strains from a library of B. licheniformis strains (a strain already used at industrial-scale). The B. licheniformis mutant library was generated by atmospheric and room temperature plasma (ARTP) mutagenesis. The clones displaying over 50% improvement in α-amylases productivity compared to the wild-type were isolated.ConclusionsWe established an efficient droplet microfluidic platform which consisted of droplet generation, droplet incubation, and sorting of droplets with a throughput of up to 1 × 106 droplets per h. The screening platform was demonstrated by successfully identifying B. licheniformis clones with improved α-amylase production. We believe that the droplet platform could be extended to the development of other industrially valuable strains.

scholarly journals High-throughput detection of ethanol-producing cyanobacteria in a microdroplet platform

2015 ◽  
Vol 12 (106) ◽  
pp. 20150216 ◽  
Author(s):  
Sara Abalde-Cela ◽  
Anna Gould ◽  
Xin Liu ◽  
Elena Kazamia ◽  
Alison G. Smith ◽  
...  
Keyword(s):  
Ethanol Production ◽  
High Throughput ◽  
High Throughput Screening ◽  
Growth Dynamics ◽  
Genetically Engineered ◽  
Quantitative Detection ◽  
Photosynthetic Organisms ◽  
High Throughput Detection ◽  
Plant Feedstock ◽  
Different Levels

Ethanol production by microorganisms is an important renewable energy source. Most processes involve fermentation of sugars from plant feedstock, but there is increasing interest in direct ethanol production by photosynthetic organisms. To facilitate this, a high-throughput screening technique for the detection of ethanol is required. Here, a method for the quantitative detection of ethanol in a microdroplet-based platform is described that can be used for screening cyanobacterial strains to identify those with the highest ethanol productivity levels. The detection of ethanol by enzymatic assay was optimized both in bulk and in microdroplets. In parallel, the encapsulation of engineered ethanol-producing cyanobacteria in microdroplets and their growth dynamics in microdroplet reservoirs were demonstrated. The combination of modular microdroplet operations including droplet generation for cyanobacteria encapsulation, droplet re-injection and pico-injection, and laser-induced fluorescence, were used to create this new platform to screen genetically engineered strains of cyanobacteria with different levels of ethanol production.

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