Advances in ultrahigh-throughput screening for directed enzyme evolution

2020 ◽  
Vol 49 (1) ◽  
pp. 233-262 ◽  
Author(s):  
Ulrich Markel ◽  
Khalil D. Essani ◽  
Volkan Besirlioglu ◽  
Johannes Schiffels ◽  
Wolfgang R. Streit ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Enzyme Evolution ◽  
Screening Methods

This review summarizes how ultrahigh-throughput screening methods employ cells and biomimetic compartments to access the vast, unexplored diversity of biocatalysts with novel functions derived from directed evolution and metagenomics libraries.

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.

Multi-substrate–activity space and quasi-species in enzyme evolution: Ohno's dilemma, promiscuity and functional orthogonality

2009 ◽  
Vol 37 (4) ◽  
pp. 740-744 ◽  
Author(s):  
Bengt Mannervik ◽  
Arna Runarsdottir ◽  
Sanela Kurtovic
Keyword(s):  
Multivariate Analysis ◽  
Directed Evolution ◽  
Enzyme Evolution ◽  
Activity Space ◽  
Glutathione Transferases ◽  
Multidimensional Space ◽  
Alternative Substrates ◽  
Catalytic Activities ◽  
Evolutionary Trajectories

A functional enzyme displays activity with at least one substrate and can be represented by a vector in substrate–activity space. Many enzymes, including GSTs (glutathione transferases), are promiscuous in the sense that they act on alternative substrates, and the corresponding vectors operate in multidimensional space. The direction of the vector is governed by the relative activities of the diverse substrates. Stochastic mutations of already existing enzymes generate populations of variants, and clusters of functionally similar mutants can serve as parents for subsequent generations of enzymes. The proper evolving unit is a functional quasi-species, which may not be identical with the ‘best’ variant in its generation. The manifestation of the quasi-species is dependent on the substrate matrix used to explore catalytic activities. Multivariate analysis is an approach to identifying quasi-species and to investigate evolutionary trajectories in the directed evolution of enzymes for novel functions.

scholarly journals Multiplexed Screening of Population-level Synthetic Gene Oscillator Libraries

2021 ◽  
Author(s):  
Andrew Lezia ◽  
Nicholas Csicsery ◽  
Jeff Hasty
Keyword(s):  
Directed Evolution ◽  
High Throughput Screening ◽  
Population Level ◽  
Synthetic Gene ◽  
Screening Methods ◽  
Gene Circuits ◽  
Synthetic Gene Circuits ◽  
Invaluable Tool ◽  
Long Time ◽  
Screening Approaches

Directed evolution has become an invaluable tool in protein engineering and has greatly influenced the construction of synthetic gene circuits. The ability to generate diversity at precise targets for directed evolution approaches has improved vastly, allowing researchers to create large, specific mutant libraries with relative ease. Screening approaches for large mutant libraries have similarly come a long way, especially when the desired behavior can easily be tested for with static, single time-point assays. For more complex gene circuits with dynamic phenotypes that change over time, directed evolution approaches to controlling and tuning circuit behavior have been hindered by the lack of sufficiently high-throughput screening methods to isolate variants with desired characteristics. Here we utilize directed mutagenesis and multiplexed microfluidics to develop a workflow for creating, screening and tuning dynamic gene circuits that operate at the population level. Specifically, we create a mutant library of an existing oscillator, the synchronized lysis circuit, and tune its dynamics while uncovering principles regarding its behavior. Lastly, we utilize this directed evolution workflow to construct a new synchronized genetic oscillator that exhibits robust dynamics over long time scales.

Teaching old enzymes new tricks: engineering and evolution of glycosidases and glycosyl transferases for improved glycoside synthesisThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Systems and Chemical Biology, and has undergone the Journal's usual peer review process.

2008 ◽  
Vol 86 (2) ◽  
pp. 169-177 ◽  
Author(s):  
Fathima Aidha Shaikh ◽  
Stephen G. Withers
Keyword(s):  
Directed Evolution ◽  
Chemical Biology ◽  
Review Process ◽  
Therapeutic Potential ◽  
Peer Review Process ◽  
Alternative Methods ◽  
Screening Methods ◽  
Special Issue ◽  
New Class ◽  
Selection Of

The therapeutic potential of glycosides has made them an attractive target for drug development. The biological extraction and chemical synthesis of these molecules is often challenging and low yielding, thus alternative methods for the synthesis of polysaccharides are being pursued. A new class of enzymes, glycosynthases, which are nucleophile mutants of glycosidases, can perform the transglycosylation reaction without hydrolyzing the product, and thus provide a valuable resource for polysaccharide and glycan synthesis. Directed evolution of glycosynthases has expanded the repertoire of glycosidic linkages formed and the donors and acceptors (both sugar and nonsugar) that can be used by the glycosynthase. The application of new screening methods, such as FACS, to the directed evolution of glycosynthases will aid in the development of enzymes that are able to efficiently synthesize new, and therapeutically relevant glycosidic linkages.

scholarly journals "Multi-Agent" Screening Improves the Efficiency of Directed Enzyme Evolution

2021 ◽  
Author(s):  
Tian Yang ◽  
Zhixia Ye ◽  
Michael D Lynch
Keyword(s):  
Directed Evolution ◽  
Tertiary Structure ◽  
Fitness Landscape ◽  
Combinatorial Libraries ◽  
Enzyme Engineering ◽  
Enzyme Evolution ◽  
Combinatorial Search ◽  
Screening Process ◽  
Multiple Substrates ◽  
Multi Agent

Enzyme evolution has enabled numerous advances in biotechnology. However, directed evolution programs can still require many iterative rounds of screening to identify optimal mutant sequences. This is due to the sparsity of the fitness landscape, which in turn, is due to hidden mutations that only offer improvements synergistically in combination with other mutations. These hidden mutations are only identified by evaluating mutant combinations, necessitating large combinatorial libraries or iterative rounds of screening. Here, we report a multi-agent directed evolution approach that incorporates diverse substrate analogues in the screening process. With multiple substrates acting like multiple agents navigating the fitness landscape, we are able to identify hidden mutant residues that impact substrate specificity without a need for testing numerous combinations. We initially validate this approach in engineering a malonyl-CoA synthetase for improved activity with a wide variety of non-natural substrates. We found that hidden mutations are often distant from the active site, making them hard to predict using popular structure-based methods. Interestingly, many of the hidden mutations identified in this case are expected to destabilize interactions between elements of tertiary structure, potentially affecting protein flexibility. This approach may be widely applicable to accelerate enzyme engineering. Lastly, multi-agent system inspired approaches may be more broadly useful in tackling other complex combinatorial search problems in biology.

Data Screening Methods – Application to Differential Diagnosis in Pancreatic Pathology from Radiological Signs

1978 ◽  
Vol 17 (01) ◽  
pp. 36-40 ◽  
Author(s):  
J.-P. Durbec ◽  
Jaqueline Cornée ◽  
P. Berthezene
Keyword(s):  
Differential Diagnosis ◽  
Mutual Information ◽  
Data Processing ◽  
Screening Methods ◽  
Automatic Data ◽  
Chronic Calcifying Pancreatitis ◽  
Number Of Patients ◽  
Calcifying Pancreatitis ◽  
Pancreatic Pathology ◽  
Data Screening

The practice of systematic examinations in hospitals and the increasing development of automatic data processing permits the storing of a great deal of information about a large number of patients belonging to different diagnosis groups.To predict or to characterize these diagnosis groups some descriptors are particularly useful, others carry no information. Data screening based on the properties of mutual information and on the log cross products ratios in contingency tables is developed. The most useful descriptors are selected. For each one the characterized groups are specified.This approach has been performed on a set of binary (presence—absence) radiological variables. Four diagnoses groups are concerned: cancer of pancreas, chronic calcifying pancreatitis, non-calcifying pancreatitis and probable pancreatitis. Only twenty of the three hundred and forty initial radiological variables are selected. The presence of each corresponding sign is associated with one or more diagnosis groups.

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