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Results for “directed evolution”

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At least 703 records · Page 39Linked to original sources
PubMed2000

Analysis of genes involved in 6-deoxyhexose biosynthesis and transfer in Saccharopolyspora erythraea.

Glycosylation represents an attractive target for protein engineering of novel antibiotics, because specific attachment of one or more deoxysugars is required for the bioactivity of many antibiotic and antitumour polyketides. However, proper assessment of the potential of these enzymes for such combinatorial biosynthesis requires both more precise information on the enzymology of the pathways and also improved Escherichia coli-actinomycete shuttle vectors. New replicative vectors have been constructed and used to express independently the dnmU gene of Streptomyces peucetius and the eryBVII gene of Saccharopolyspora erythraea in an eryBVII deletion mutant of Sac. erythraea. Production of erythromycin A was obtained in both cases, showing that both proteins serve analogous functions in the biosynthetic pathways to dTDP-L-daunosamine and dTDP-L-mycarose, respectively. Over-expression of both proteins was also obtained in S. lividans, paving the way for protein purification and in vitro monitoring of enzyme activity. In a further set of experiments, the putative desosaminyltransferase of Sac. erythraea, EryCIII, was expressed in the picromycin producer Streptomyces sp. 20032, which also synthesises dTDP-D-desosamine. The substrate 3-alpha-mycarosylerythronolide B used for hybrid biosynthesis was found to be glycosylated to produce erythromycin D only when recombinant EryCIII was present, directly confirming the enzymatic role of EryCIII. This convenient plasmid expression system can be readily adapted to study the directed evolution of recombinant glycosyltransferases.

Anti-Bacterial Agents↗
PubMed2025

Decoding the distribution, structure-function-redox potential relationship and recent advances in fungal laccases: a systematic approach.

Laccases, categorized as multicopper oxidases, are recognized for their multifaceted roles in ecosystems and their utility in diverse industrial applications. Laccases from higher fungi, specifically Ascomycota and Basidiomycota, have garnered significant research interest due to their elevated redox potentials and their capacity to degrade lignin in decaying wood, alongside other industrial uses. Here, we have conducted a comprehensive and systematic analysis on fungal laccases using Web of Science, Scopus, PubMed, and ScienceDirect. The genomic distribution, phylogenetic affiliation, and structural organization of laccase-encoding genes in higher fungal species were investigated, as were the catalytic mechanisms of the corresponding enzymes. Additionally, the study explores the correlation between structural domains and redox potential, as well as the impact of post-translational modifications like glycosylation on enzyme activity. Furthermore, the recent advancements in laccase engineering, employing strategies such as rational design, directed evolution, and heterologous expression are discussed. The review also explores the scope of "artificial intelligence and machine learning" in deducing the structure-function relationships, optimizing codon usage, predicting signal peptides, enhancing enzymatic performance, and developing host-specific genetic engineering techniques is also discussed for tailoring fungal laccases to meet the demands of industrial biocatalysis for improved activity and stability.

Laccase↗
PubMed2006

Pro-antibiotic substrates for the identification of enantioselective hydrolases.

A new growth-based screening method for the identification of enantioselective hydrolases, such as lipases and esterases, using pro-antibiotic substrates was devised. An enantioselective hydrolase could be identified by measuring growth rates of cells in liquid media containing (R)- or (S)-2-phenylbutyric chloramphenicol esters. This method can be applied to the screening of novel enantioselective microbes and to the high-throughput screening for the directed evolution of enantioselective hydrolytic enzymes.

Anti-Bacterial Agents↗
PubMed1988

Oral granular cell tumors: a clinicopathologic and immunocytochemical study.

To investigate the histogenesis of the granular cell, a large series of granular cell tumors was studied for clinical and histopathologic features with emphasis on immunocytochemical markers. The nongingival granular cell tumors (NGGCT) were found to be more prevalent among females than males by a ratio of 2:1 and arose on the tongue (67%), the buccal mucosa (13%), the lips (8%), the soft palate (6%), and other sites (6%). With the use of the avidin-biotin-peroxidase method, polyclonal rabbit antisera were employed. The antisera were directed to the following antigens: S-100 protein, myoglobin, myosin, actin, desmin, alpha-1-antitrypsin, and muramidase. Results indicated that granular cell tumors are not homogenous for immunocytochemical markers. Nongingival granular cell tumors were universally positive for S-100 protein and failed to exhibit immunoreactivity for myogenous or histiocytic markers. Alternatively, the gingival granular cell tumor of infancy was negative for all markers, whereas rhabdomyoma was reactive with myogenous markers and a subpopulation of tumor cells displayed S-100 protein immunoreactivity. The granular cell ameloblastoma was reactive only with antiserum to alpha-1-antitrypsin. Ultrastructurally, granular cells from one of two NGGCT showed a direct evolution from skeletal muscle fibers. It is concluded that the oral NGGCT is a tumor positive for S-100 protein that may arise from muscle or nerve sheath.

Adolescent↗
PubMed1982

On a global representation of dynamical characteristics in ESS-models.

ESS-models are considered in the frame of adequate dynamical systems. This leads to a method of global representation of the dynamical characteristics, which allows for a comprehensive view on the relations among ESS-matrices. As an extension of ESS-models, the concept of ESS-models with changing matrix is introduced, which, in applications, makes use of such global representations. In particular, structurally stable and "catastrophic" cases can be identified and their significance in the context of ESS-models can be clarified. As a complete result for the case of two strategies a surface representation is obtained, which is an analogue to the elementary "cusp catastrophe". These concepts are applied in a model that describes directed evolution of a behavioural component to a certain limit.

Animals↗
PubMed2025

Ultrahigh-throughput screening assay for PET-degrading enzymes.

In recent years, several PET-degrading enzymes have been identified from both known microorganisms and metagenomic sources in response to the growing environmental issue of polyethylene terephthalate (PET) accumulation. Despite this progress, there is a limited number of (ultra)high-throughput screening methods for assessing PET-hydrolyzing activity without relying on surrogate substrates. This method utilizes the coupled activity of ketoreductases (KREDs) and diaphorase to produce a fluorescent compound (resorufin) in the presence of PET degradation products, offering a more direct and efficient screening approach. A metagenomic KRED was coupled with the diaphorase from Clostridium kluyveri to enable the detection of the hydrolysis of PET degradation products catalyzed by the Bacillus subtilis BS2 esterase. The coupled reaction was established in water-in-oil microdroplets, encapsulating a single E. coli cell per droplet, demonstrating its potential for use in the ultrahigh-throughput screening of metagenomic libraries or randomized libraries for directed evolution campaigns.

High-Throughput Screening Assays↗
PubMed2004

In vitro compartmentalization by double emulsions: sorting and gene enrichment by fluorescence activated cell sorting.

Water-in-oil (w/o) emulsions can be used to compartmentalize and select large gene libraries for a predetermined function. The aqueous droplets of the w/o emulsion function as cell-like compartments in each of which a single gene is transcribed and translated to give multiple copies of the protein (e.g., an enzyme) it encodes. While compartmentalization ensures that the gene, the protein it encodes, and the products of the activity of this protein remain linked, it does not directly afford a way of selecting for the desired activity. Here we show that re-emulsification of w/o emulsions gives water-in-oil-in-water (w/o/w) emulsions with an external (continuous) water phase through which droplets containing fluorescent markers can be isolated by fluorescence-activated cell sorting (FACS). These w/o/w emulsions can be sorted by FACS, while the content of the aqueous droplets of the primary w/o emulsion remains intact. Consequently, genes embedded in these water droplets together with a fluorescent marker can be isolated and enriched from an excess of genes embedded in water droplets without a fluorescent marker. The ability of FACS instruments to sort up to 40000 events per second may endow this technology a wide potential in the area of high-throughput screening and the directed evolution of enzymes.

DNA Modification Methylases↗
PubMed2006

Engineering of beta-propeller protein scaffolds by multiple gene duplication and fusion of an idealized WD repeat.

The ability to design specific amino acid sequences that fold into desired structures is central to engineering novel proteins. Protein design is also a good method to assess our understanding of sequence-structure and structure-function relationships. While beta-sheet structures are important elements of protein architecture, it has traditionally been more difficult to design beta-proteins than alpha-helical proteins. Taking advantage of the tandem repeated sequences that form the structural building blocks in a group of beta-propeller proteins; we have used a consensus design approach to engineer modular and relatively large scaffolds. An idealized WD repeat was designed from a structure-based sequence alignment with a set of structural guidelines. Using a plasmid sequential ligation strategy, artificial concatemeric genes with up to 10 copies of this idealized repeat were then constructed. Corresponding proteins with 4 through to 10 WD repeats were soluble when over-expressed in Escherichia coli. Notably, they were sufficiently stable in vivo surviving attack from endogenous proteases, and maintained a homogeneous, non-aggregated form in vitro. The results show that the beta-propeller scaffold is an attractive platform for future engineering work, particularly in experiments in which directed evolution techniques might improve the stability of the molecules and/or tailor them for a specific function.

Amino Acid Motifs↗
PubMed2006

Dual selection of a genetic switch by a single selection marker.

Forward engineering of synthetic genetic circuits in living cells is expected to deliver various applications in biotechnology and medicine and to provide valuable insights into the design principles of natural gene networks. However, lack of biochemical data and complexity of biological environment complicate rational design of such circuits based on quantitative simulation. Previously, we have shown that directed evolution can complement our weakness in designing genetic circuits by screening or selecting functional circuits from a large pool of nonfunctional ones. Here we describe a dual selection strategy that allows selection of both ON and OFF states of genetic circuits using tetA as a single selection marker. We also describe a successful demonstration of a genetic switch selection from a 2000-fold excess background of nonfunctional switches in three rounds of iterative selection. The dual selection system is more robust than the previously reported selection system employing three genes, with no observed false positive mutants during the simulated selections.

Computer Simulation↗
PubMed2006

Site-specific recombination of asymmetric lox sites mediated by a heterotetrameric Cre recombinase complex.

Previous reports have demonstrated that new Cre recombinase specificities can be developed for symmetrically designed lox mutants through directed evolution. The development of Cre variants that allow the recombination of true asymmetric lox mutant sites has not yet been addressed, however. In the present study, we demonstrate that a mixture of two different site-specific Cre recombinase molecules (wt Cre and a mutant Cre) catalyzes efficient recombination between two asymmetric lox sites in vitro, presumably via formation of a functionally active heterotetrameric complex. The results may broaden the application of site-specific recombination in basic and applied research, including the custom-design of recombinases for natural, asymmetric, and lox-related target sequences present in the genome. Future applications may potentially include genomic manipulations, for example, site-specific integrations, deletions or substitutions within precise regions of the genomes of mammalians and other organisms.

Animals↗
PubMed2003

Evolutionary markers in the (beta/alpha)8-barrel fold.

Enzymes with the (beta/alpha)(8)-barrel fold are involved in the catalysis of a wide variety of biochemical reactions. The active sites of these enzymes are located on the C-terminal face of the central beta-barrel. Conserved amino acid sequence, as well as secondary, tertiary and quaternary structure patterns are providing a rich body of data to support the premise of a common ancestry of many members of the (beta/alpha)(8)-barrel fold family of enzymes. Recent data indicate that there is at least one example of a bienzyme that functions as an ammonia channel, adding a new level of functional diversity to the (beta/alpha)(8)-barrel fold. These proteins have become ideal tools that can be used in conjunction with directed evolution techniques to engineer novel catalytic activities.

Amino Acid Motifs↗
PubMed2004

Synthesis and modifications of carbohydrates, using biotransformations.

Enzymes continue to be used as important catalysts, for the generation of rare and 'unnatural' monosaccharides and for the selective formation of glycosidic linkages. Multi-enzyme systems have been employed in one-pot strategies for multistep reaction sequences and for co-factor regeneration. The efficiency of glycosidases for glycosylation reactions has been dramatically increased by active-site mutagenesis to generate glycosynthases. First reports have detailed the expansion and optimization of glycosynthase substrate specificity by directed evolution. Novel glycosyltransferases are being identified from genomic databases and have been shown to glycosylate complex metabolites, such as glycopeptide antibiotics, with exquisite selectivity and in good yields. An emerging field is the application of glycosynthases and glycosyltransferases to reactions on solid support, generating potential applications in microarrays.

Biotransformation↗
PubMed2006

Engineering of glycosidases and glycosyltransferases.

In recent years, substantial advances have been made in the engineering of glycosidases and glycosyltransferases for the synthesis and degradation of glycan structures. Key developments include improvement of the thermostability of xylanase through comprehensive saturation mutagenesis, creation of the first glycosynthase derived from an inverting glycosidase and the emergence of a new class of modified glycosidases capable of efficiently synthesizing thioglycosidic linkages. Of particular note is the increased use of random mutagenesis and directed evolution tactics for tailoring glycosidase activity. Although the engineering of glycosyltransferases is still in its early stages, recent work on the structure-based alteration of substrate specificity and the manipulation of glycosyltransferase profiles in whole cells to effect complex changes in in vivo glycobiology probably foreshadows a wave of considerable innovation in this area.

Animals↗
PubMed2005

Focusing mutations into the P. fluorescens esterase binding site increases enantioselectivity more effectively than distant mutations.

Rational design of enzymes with improved properties, such as enantioselectivity, usually focuses mutations within the substrate binding site. On the other hand, directed evolution of enzymes usually targets the entire protein and discovers beneficial mutations far from the substrate binding site. In this paper, we propose an explanation for this discrepancy and show that a combined approach--random mutagenesis within the substrate binding site--is better. To increase the enantioselectivity (E) of a Pseudomonas fluorescens esterase (PFE) toward methyl 3-bromo-2-methylpropionate, we focused mutagenesis into the substrate binding site at Trp28, Val121, Phe198, and Val225. Five of the catalytically active mutants (13%) showed better enantioselectivity than wild-type PFE. The increases in enantioselectivity were higher (up to 5-fold, reaching E = 61) than with mutants identified by random mutagenesis of the entire enzyme.

Binding Sites↗
PubMed2025

A base editor facilitates simultaneous purine and pyrimidine substitutions for ex vivo and in vivo mutagenesis screens.

Genetic mutations are closely linked to human diseases, yet the relationship between many mutations and their corresponding phenotypes remains poorly understood. Furthermore, tools to study the connection between nucleotide variations and phenotypes are limited. To address this issue, we developed ACGBEmax by fusing the dual-functional deaminase, engineered N-methylpurine DNA glycosylase, and evolved SOS response associated peptidase domain with nCas9(D10A). ACGBEmax enables the precise conversion of A, C, and G to other bases in mammalian cells, thereby generating an extensive range of base mutations types. We used ACGBEmax to generate HPRT variants, identifying mutations conferring resistance to 6-thioguanine. Additionally, we performed in situ mutagenesis of Ctnnb1 in mouse liver, identifying both known and potential oncogenic mutations. Our results prove that ACGBEmax is a powerful tool for generating a wide spectrum of mutation types at specific gene loci, highlighting its significant potential for applications in functional screening and the directed evolution of protein variants.

Animals↗
PubMed2006

Identifying the interacting positions of a protein using Boolean learning and support vector machines.

It is known that in the three-dimensional structure of a protein, certain amino acids can interact with each other in order to provide structural integrity or aid in its catalytic function. If these positions are mutated the loss of this interaction usually leads to a non-functional protein. Directed evolution experiments, which probe the sequence space of a protein through mutations in search for an improved variant, frequently result in such inactive sequences. In this work, we address the use of machine learning algorithms, Boolean learning and support vector machines (SVMs), to find such pairs of amino acid positions. The recombination method of imparting mutations was simulated to create in silico sequences that were used as training data for the algorithms. The two algorithms were combined together to develop an approach that weighs the structural risk as well as the empirical risk to solve the problem. This strategy was adapted to a multi-round framework of experiments where the data generated in the present round is used to design experiments for the next round to improve the generated library, as well as the estimation of the interacting positions. It is observed that this strategy can greatly improve the number of functional variants that are generated as well as the average number of mutations that can be made in the library.

Algorithms↗
PubMed2006

Manipulating proteins for neuroscience.

The design and manipulation of proteins has created many tools that have become popular in neurobiological studies, and new developments in protein science will be the fuel for future research. Genetically encoded protein-based biosensors have been developed with a wider range of sensing moieties, enabling detection of changes in localized protein synthesis, voltage, glutamate and/or glucose levels. Existing sensors, such as cameleon, have been modified and improved. Heterologous expression of Channelrhodopsin-2 and other light-gated methods for controlling cellular polarization enable action potentials to be non-invasively evoked, facilitating the study and modulation of behavior in intact animals. Finally, new methods in protein manipulation, including the site-specific incorporation of unnatural amino acids in vivo and the directed evolution of proteins, show promise in elucidating neural function with greater precision and flexibility.

Amino Acids↗
PubMed2003

Protein engineering in bioelectrocatalysis.

Electrochemistry of redox proteins is a broadly applicable technology with important applications in biosensors, biofuel cells and chemical syntheses. Escalating attention in this area is driven by remarkable progress in designing efficient interfaces for transferring electrons between electrode surfaces and redox proteins. Research in interface design is slowly shifting from modifying electrode surfaces towards the engineering of redox proteins. Protein engineering, which encompasses rational design, directed evolution and combined methods, offers many powerful methods and strategies for improving the electron transfer properties of redox proteins.

Catalysis↗