Directed Evolution of Protein Catalysts

2018 ◽  
Vol 87 (1) ◽  
pp. 131-157 ◽  
Author(s):  
Cathleen Zeymer ◽  
Donald Hilvert
Keyword(s):  
Directed Evolution ◽  
Rational Design ◽  
Chemical Transformations ◽  
Catalytic Promiscuity ◽  
Catalytic Activities ◽  
Effective Strategies ◽  
Wide Range ◽  
Protein Properties ◽  
New Protein ◽  
Modify Protein

Directed evolution is a powerful technique for generating tailor-made enzymes for a wide range of biocatalytic applications. Following the principles of natural evolution, iterative cycles of mutagenesis and screening or selection are applied to modify protein properties, enhance catalytic activities, or develop completely new protein catalysts for non-natural chemical transformations. This review briefly surveys the experimental methods used to generate genetic diversity and screen or select for improved enzyme variants. Emphasis is placed on a key challenge, namely how to generate novel catalytic activities that expand the scope of natural reactions. Two particularly effective strategies, exploiting catalytic promiscuity and rational design, are illustrated by representative examples of successfully evolved enzymes. Opportunities for extending these approaches to more complex biocatalytic systems are also considered.

scholarly journals Laboratory-Directed Protein Evolution

2005 ◽  
Vol 69 (3) ◽  
pp. 373-392 ◽  
Author(s):  
Ling Yuan ◽  
Itzhak Kurek ◽  
James English ◽  
Robert Keenan
Keyword(s):  
Protein Evolution ◽  
Selective Pressure ◽  
Evolutionary Potential ◽  
Effective Strategies ◽  
Scientific Disciplines ◽  
Wide Range ◽  
Protein Functions ◽  
Evolution Of Proteins ◽  
Efficient Screening ◽  
New Protein

SUMMARY Systematic approaches to directed evolution of proteins have been documented since the 1970s. The ability to recruit new protein functions arises from the considerable substrate ambiguity of many proteins. The substrate ambiguity of a protein can be interpreted as the evolutionary potential that allows a protein to acquire new specificities through mutation or to regain function via mutations that differ from the original protein sequence. All organisms have evolutionarily exploited this substrate ambiguity. When exploited in a laboratory under controlled mutagenesis and selection, it enables a protein to “evolve” in desired directions. One of the most effective strategies in directed protein evolution is to gradually accumulate mutations, either sequentially or by recombination, while applying selective pressure. This is typically achieved by the generation of libraries of mutants followed by efficient screening of these libraries for targeted functions and subsequent repetition of the process using improved mutants from the previous screening. Here we review some of the successful strategies in creating protein diversity and the more recent progress in directed protein evolution in a wide range of scientific disciplines and its impacts in chemical, pharmaceutical, and agricultural sciences.

scholarly journals Improved yellow-green split fluorescent proteins for protein labeling and signal amplification

2020 ◽  
Author(s):  
Shuqin Zhou ◽  
Siyu Feng ◽  
David Brown ◽  
Bo Huang
Keyword(s):  
Directed Evolution ◽  
Signal Amplification ◽  
Mammalian Cells ◽  
Rational Design ◽  
Fluorescent Proteins ◽  
Protein Labeling ◽  
Small Fragment ◽  
Wide Range ◽  
Split System ◽  
Low Expression

AbstractThe flexibility and versatility of self-complementing split fluorescent proteins (FPs) have enabled a wide range of applications. In particular, the FP1-10/11 split system contains a small fragment that facilitates efficient generation of endogenous-tagged cell lines and animals as well as signal amplification using tandem FP11 tags. To improve the FP1-10/11 toolbox we previously developed, here we used a combination of directed evolution and rational design approaches, resulting in two mNeonGreen (mNG)-based split FPs (mNG3A1-10/11 and mNG3K1-10/11) and one mClover-based split FP (CloGFP1-10/11). mNG3A1-10/11 and mNG3K1-10/11 not only enhanced the complementation efficiency at low expression levels, but also allowed us to demonstrate signal amplification using tandem mNG211 fragments in mammalian cells.

scholarly journals Improved yellow-green split fluorescent proteins for protein labeling and signal amplification

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0242592
Author(s):  
Shuqin Zhou ◽  
Siyu Feng ◽  
David Brown ◽  
Bo Huang
Keyword(s):  
Directed Evolution ◽  
Signal Amplification ◽  
Mammalian Cells ◽  
Rational Design ◽  
Fluorescent Proteins ◽  
Protein Labeling ◽  
Small Fragment ◽  
Wide Range ◽  
Split System ◽  
Low Expression

The flexibility and versatility of self-complementing split fluorescent proteins (FPs) have enabled a wide range of applications. In particular, the FP1-10/11 split system contains a small fragment that facilitates efficient generation of endogenous-tagged cell lines and animals as well as signal amplification using tandem FP11 tags. To improve the FP1-10/11 toolbox we previously developed, here we used a combination of directed evolution and rational design approaches, resulting in two mNeonGreen (mNG)-based split FPs (mNG3A1-10/11 and mNG3K1-10/11) and one mClover-based split FP (CloGFP1-10/11). mNG3A1-10/11 and mNG3K1-10/11 not only enhanced the complementation efficiency at low expression levels, but also allowed us to demonstrate signal amplification using tandem mNG211 fragments in mammalian cells.

Strongly Coupled Redox-Linked Conformational Switching at the Active Site of the Non-Heme Iron-Dependent Dioxygenase, TauD

2019 ◽  
Author(s):  
Christopher John ◽  
Greg M. Swain ◽  
Robert P. Hausinger ◽  
Denis A. Proshlyakov
Keyword(s):  
Active Site ◽  
Structural Model ◽  
Heme Iron ◽  
Chemical Transformations ◽  
Experimental Conditions ◽  
Strongly Coupled ◽  
Non Heme Iron ◽  
Reversible Redox ◽  
Wide Range ◽  
Complex Structural

2-Oxoglutarate (2OG)-dependent dioxygenases catalyze C-H activation while performing a wide range of chemical transformations. In contrast to their heme analogues, non-heme iron centers afford greater structural flexibility with important implications for their diverse catalytic mechanisms. We characterize an <i>in situ</i> structural model of the putative transient ferric intermediate of 2OG:taurine dioxygenase (TauD) by using a combination of spectroelectrochemical and semi-empirical computational methods, demonstrating that the Fe (III/II) transition involves a substantial, fully reversible, redox-linked conformational change at the active site. This rearrangement alters the apparent redox potential of the active site between -127 mV for reduction of the ferric state and 171 mV for oxidation of the ferrous state of the 2OG-Fe-TauD complex. Structural perturbations exhibit limited sensitivity to mediator concentrations and potential pulse duration. Similar changes were observed in the Fe-TauD and taurine-2OG-Fe-TauD complexes, thus attributing the reorganization to the protein moiety rather than the cosubstrates. Redox difference infrared spectra indicate a reorganization of the protein backbone in addition to the involvement of carboxylate and histidine ligands. Quantitative modeling of the transient redox response using two alternative reaction schemes across a variety of experimental conditions strongly supports the proposal for intrinsic protein reorganization as the origin of the experimental observations.

scholarly journals Rational design of photocatalysts for ammonia production from water and nitrogen gas

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Seokwoo Choe ◽  
Sung Min Kim ◽  
Yeji Lee ◽  
Jin Seok ◽  
Jiyong Jung ◽  
...  
Keyword(s):  
Energy Source ◽  
Rational Design ◽  
Future Research ◽  
Ammonia Production ◽  
Significant Progress ◽  
Nitrogen Gas ◽  
Research Directions ◽  
Effective Strategies ◽  
Future Research Directions ◽  
Made In

AbstractPhotocatalytic N2 reduction has emerged as one of the most attractive routes to produce NH3 as a useful commodity for chemicals used in industries and as a carbon-free energy source. Recently, significant progress has been made in understanding, exploring, and designing efficient photocatalyst. In this review, we outline the important mechanistic and experimental procedures for photocatalytic NH3 production. In addition, we review effective strategies on development of photocatalysts. Finally, our analyses on the characteristics and modifications of photocatalysts have been summarized, based on which we discuss the possible future research directions, particularly on preparing more efficient catalysts. Overall, this review provides insights on improving photocatalytic NH3 production and designing solar-driven chemical conversions.

scholarly journals Fluorescent thermal shift-based method for detection of NF-κB binding to double-stranded DNA

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Peter D. Leitner ◽  
Ilja Vietor ◽  
Lukas A. Huber ◽  
Taras Valovka
Keyword(s):  
Dna Binding ◽  
Rational Design ◽  
Dissociation Constants ◽  
Inflammatory Diseases ◽  
Dimethyl Fumarate ◽  
Bioinformatic Analysis ◽  
Quantitative Detection ◽  
Regulatory Sequences ◽  
Wide Range ◽  
Inhibitory Drug

AbstractThe nuclear factor kappa B (NF-κB) family of dimeric transcription factors regulates a wide range of genes by binding to their specific DNA regulatory sequences. NF-κB is an important therapeutic target linked to a number of cancers as well as autoimmune and inflammatory diseases. Therefore, effective high-throughput methods for the detection of NF-κB DNA binding are essential for studying its transcriptional activity and for inhibitory drug screening. We describe here a novel fluorescence-based assay for quantitative detection of κB consensus double-stranded (ds) DNA binding by measuring the thermal stability of the NF-κB proteins. Specifically, DNA binding proficient NF-κB probes, consisting of the N-terminal p65/RelA (aa 1–306) and p50 (aa 1–367) regions, were designed using bioinformatic analysis of protein hydrophobicity, folding and sequence similarities. By measuring the SYPRO Orange fluorescence during thermal denaturation of the probes, we detected and quantified a shift in the melting temperatures (ΔTm) of p65/RelA and p50 produced by the dsDNA binding. The increase in Tm was proportional to the concentration of dsDNA with apparent dissociation constants (KD) of 2.228 × 10–6 M and 0.794 × 10–6 M, respectively. The use of withaferin A (WFA), dimethyl fumarate (DMF) and p-xyleneselenocyanate (p-XSC) verified the suitability of this assay for measuring dose-dependent antagonistic effects on DNA binding. In addition, the assay can be used to analyse the direct binding of inhibitors and their effects on structural stability of the protein probe. This may facilitate the identification and rational design of new drug candidates interfering with NF-κB functions.

scholarly journals Enamine/Transition Metal Combined Catalysis: Catalytic Transformations Involving Organometallic Electrophilic Intermediates

2019 ◽  
Vol 377 (6) ◽  
Author(s):  
Samson Afewerki ◽  
Armando Córdova
Keyword(s):  
Transition Metal ◽  
Metal Complex ◽  
Carbonyl Compounds ◽  
Transition Metal Catalysis ◽  
Chemical Transformations ◽  
Considerable Effort ◽  
Metal Catalysis ◽  
First Report ◽  
Wide Range ◽  
Selective Chemical

AbstractThe concept of merging enamine activation catalysis with transition metal catalysis is an important strategy, which allows for selective chemical transformations not accessible without this combination. The amine catalyst activates the carbonyl compounds through the formation of a reactive nucleophilic enamine intermediate and, in parallel, the transition metal activates a wide range of functionalities such as allylic substrates through the formation of reactive electrophilic π-allyl-metal complex. Since the first report of this strategy in 2006, considerable effort has been devoted to the successful advancement of this technology. In this chapter, these findings are highlighted and discussed.

Experiences with alluvial foundations for earth dams in the Prairie provinces

1979 ◽  
Vol 16 (2) ◽  
pp. 255-271 ◽  
Author(s):  
N. Peters ◽  
K. N. Lamb
Keyword(s):  
Rational Design ◽  
Preliminary Design ◽  
Empirical Relationships ◽  
Design Concepts ◽  
Theoretical Design ◽  
Pore Pressures ◽  
Wide Range ◽  
Thick Layers ◽  
Limited Testing ◽  
Prairie Provinces

The foundations for numerous dams in proglacial and interglacial valleys in the Prairie provinces consist of soft alluvial soils. The deposits are up to 60 m deep, and contain thick layers of clay interspersed with lenses and layers of silt, sand, and gravel.This paper describes the damsite investigation and laboratory testing required, the design methods and construction procedures used, and the foundation performance observed during and after construction. A number of empirical relationships between index tests and physical properties of the soils, which provide useful guidelines for preliminary design, are presented.The design approach has gradually evolved from an empirical design with limited testing to a more rational design based on detailed investigations and thorough instrumentation. Increased reliance is placed on observational apparatus to monitor movements and pore pressures to confirm design assumptions as construction proceeds. The theoretical design is always checked with former designs of dams that have performed satisfactorily.Safe economical dams have been constructed in spite of large deformations and high pore pressures. Two case histories illustrate the wide range in dam design for alluvial foundations. The first shows an older design cross section with modifications required to ensure a stable dam, and the second describes a recently constructed dam that incorporates many of the latest design concepts.

scholarly journals Protein Engineering Approaches to Enhance Fungal Laccase Production in S. cerevisiae

2021 ◽  
Vol 22 (3) ◽  
pp. 1157
Author(s):  
Pablo Aza ◽  
Felipe de Salas ◽  
Gonzalo Molpeceres ◽  
David Rodríguez-Escribano ◽  
Iñigo de la Fuente ◽  
...  
Keyword(s):  
Protein Engineering ◽  
Directed Evolution ◽  
Signal Peptide ◽  
Laccase Activity ◽  
Laccase Production ◽  
Wide Range ◽  
Mating Factor ◽  
Conserved Amino Acids ◽  
Basidiomycete Fungi

Laccases secreted by saprotrophic basidiomycete fungi are versatile biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the sole requirement. Saccharomyces cerevisiae is a preferred host for engineering fungal laccases. To assist the difficult secretion of active enzymes by yeast, the native signal peptide is usually replaced by the preproleader of S. cerevisiae alfa mating factor (MFα1). However, in most cases, only basal enzyme levels are obtained. During directed evolution in S. cerevisiae of laccases fused to the α-factor preproleader, we demonstrated that mutations accumulated in the signal peptide notably raised enzyme secretion. Here we describe different protein engineering approaches carried out to enhance the laccase activity detected in the liquid extracts of S. cerevisiae cultures. We demonstrate the improved secretion of native and engineered laccases by using the fittest mutated α-factor preproleader obtained through successive laccase evolution campaigns in our lab. Special attention is also paid to the role of protein N-glycosylation in laccase production and properties, and to the introduction of conserved amino acids through consensus design enabling the expression of certain laccases otherwise not produced by the yeast. Finally, we revise the contribution of mutations accumulated in laccase coding sequence (CDS) during previous directed evolution campaigns that facilitate enzyme production.

Automation and Management of Design Process of the Main Drive for an Innovative Fruits and Vegetables Washer

2021 ◽  
Vol 27 (1) ◽  
pp. 9-17
Author(s):  
V. P. Bui ◽  
◽  
S. S. Gavruishin ◽  
V. B. Phung ◽  
H. M. Dang ◽  
...  
Keyword(s):  
Design Process ◽  
Rational Design ◽  
Fruits And Vegetables ◽  
Rational Solution ◽  
Quality Criteria ◽  
Pareto Optimal ◽  
Optimal Domain ◽  
Wide Range ◽  
A New Technique ◽  
New Generation

A new technique is described, used by the authors to automate the design process of the main drive of a new generation machine intended for industrial washing of fruits and vegetables. To solve the problem of multi-criteria design, the original approach is proposed that uses interconnected mathematical models describing the dynamic behavior, strength reliability and functional characteristics of the machine in a unified information space. The generalized mathematical model includes 12 controlled parameters, 16 functional constraints, and 3 quality criteria. A genetic algorithm was used to find the space of Pareto-optimal solutions. The situational approach was used to select the final rational solution from a set of solutions belonging to the Pareto-optimal domain. The rational design of option the washer found using the proposed approach is compared with the existing ones. The proposed design methodology can be recommended for the design of a wide range of similar mechanical structures.

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