Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A Photoclick-Based High-Throughput Screening for the Directed Evolution of Decarboxylase OleT.

ABSTRACT: Enzymatic oxidative decarboxylation is an up-and-coming reaction yet lacking efficient screening methods for the directed evolution of decarboxylases. Here, we describe a simple photoclick assay for the detection of decarboxylation products and its application in a proof-of-principle directed evolution study on the decarboxylase OleT. The assay was compatible with two frequently used OleT operation modes (directly using hydrogen peroxide as the enzyme's co-substrate or using a reductase partner) and the screening of saturation mutagenesis libraries identified two enzyme variants shifting the enzyme's substrate preference from long chain fatty acids toward styrene derivatives. Overall, this photoclick assay holds promise to speed-up the directed evolution of OleT and other decarboxylases.

SUBMITTER: Markel U

PROVIDER: S-EPMC7839715 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

A Photoclick-Based High-Throughput Screening for the Directed Evolution of Decarboxylase OleT.

Markel Ulrich U Lanvers Pia P Sauer Daniel F DF Wittwer Malte M Dhoke Gaurao V GV Davari Mehdi D MD Schiffels Johannes J Schwaneberg Ulrich U

Chemistry (Weinheim an der Bergstrasse, Germany) 20201209 3

Enzymatic oxidative decarboxylation is an up-and-coming reaction yet lacking efficient screening methods for the directed evolution of decarboxylases. Here, we describe a simple photoclick assay for the detection of decarboxylation products and its application in a proof-of-principle directed evolution study on the decarboxylase OleT. The assay was compatible with two frequently used OleT operation modes (directly using hydrogen peroxide as the enzyme's co-substrate or using a reductase partner) ...[more]

PMID: 32955127

Similar Datasets

High Throughput Screening and Selection Methods for Directed Enzyme Evolution.

Project description:Successful evolutionary enzyme engineering requires a high throughput screening or selection method, which considerably increases the chance of obtaining desired properties and reduces the time and cost. In this review, a series of high throughput screening and selection methods are illustrated with significant and recent examples. These high throughput strategies are also discussed with an emphasis on compatibility with phenotypic analysis during directed enzyme evolution. Lastly, certain limitations of current methods, as well as future developments, are briefly summarized.

| S-EPMC4461044 | biostudies-other

Ultrahigh-throughput screening in drop-based microfluidics for directed evolution.

Project description:The explosive growth in our knowledge of genomes, proteomes, and metabolomes is driving ever-increasing fundamental understanding of the biochemistry of life, enabling qualitatively new studies of complex biological systems and their evolution. This knowledge also drives modern biotechnologies, such as molecular engineering and synthetic biology, which have enormous potential to address urgent problems, including developing potent new drugs and providing environmentally friendly energy. Many of these studies, however, are ultimately limited by their need for even-higher-throughput measurements of biochemical reactions. We present a general ultrahigh-throughput screening platform using drop-based microfluidics that overcomes these limitations and revolutionizes both the scale and speed of screening. We use aqueous drops dispersed in oil as picoliter-volume reaction vessels and screen them at rates of thousands per second. To demonstrate its power, we apply the system to directed evolution, identifying new mutants of the enzyme horseradish peroxidase exhibiting catalytic rates more than 10 times faster than their parent, which is already a very efficient enzyme. We exploit the ultrahigh throughput to use an initial purifying selection that removes inactive mutants; we identify approximately 100 variants comparable in activity to the parent from an initial population of approximately 10(7). After a second generation of mutagenesis and high-stringency screening, we identify several significantly improved mutants, some approaching diffusion-limited efficiency. In total, we screen approximately 10(8) individual enzyme reactions in only 10 h, using < 150 microL of total reagent volume; compared to state-of-the-art robotic screening systems, we perform the entire assay with a 1,000-fold increase in speed and a 1-million-fold reduction in cost.

| S-EPMC2840095 | biostudies-literature

A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A.

Project description:Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N',N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2-8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.

| S-EPMC8002391 | biostudies-literature

Origin and evolution of high throughput screening.

Project description:This article reviews the origin and evolution of high throughput screening (HTS) through the experience of an individual pharmaceutical company, revealing some of the mysteries of the early stages of drug discovery to the wider pharmacology audience. HTS in this company (Pfizer, Groton, USA) had its origin in natural products screening in 1986, by substituting fermentation broths with dimethyl sulphoxide solutions of synthetic compounds, using 96-well plates and reduced assay volumes of 50-100 microl. A nominal 30 mM source compound concentration provided high microM assay concentrations. Starting at 800 compounds each week, the process reached a steady state of 7200 compounds per week by 1989. Screening in the Applied Biotechnology and Screening Group was centralized with screens operating in lock-step to maximize efficiency. Initial screens were full files run in triplicate. Autoradiography and image analysis were introduced for (125)I receptor ligand screens. Reverse transcriptase (RT) coupled with quantitative PCR and multiplexing addressed several targets in a single assay. By 1992 HTS produced 'hits' as starting matter for approximately 40% of the Discovery portfolio. In 1995, the HTS methodology was expanded to include ADMET targets. ADME targets required each compound to be physically detected leading to the development of automated high throughput LC-MS. In 1996, 90 compounds/week were screened in microsomal, protein binding and serum stability assays. Subsequently, the mutagenic Ames assay was adapted to a 96-well plate liquid assay and novel algorithms permitted automated image analysis of the micronucleus assay. By 1999 ADME HTS was fully integrated into the discovery cycle.

| S-EPMC1978279 | biostudies-other

DMSO-tolerant ornithine decarboxylase (ODC) tandem assay optimised for high-throughput screening.

Project description:Ornithine decarboxylase (ODC), the first rate-limiting enzyme in polyamine synthesis, has emerged as a therapeutic target for cancer and Alzheimer's disease (AD). To inhibit ODC, α-difluoromethylornithine (DFMO), an irreversible ODC inhibitor, has been widely used. However, due to its poor pharmacokinetics, the need for discovery of better ODC inhibitors is inevitable. For high-throughput screening (HTS) of ODC inhibitors, an ODC enzyme assay using supramolecular tandem assay has been introduced. Nevertheless, there has been no study utilising the ODC tandem assay for HTS, possibly due to its intolerability to dimethyl sulfoxide (DMSO), a common amphipathic solvent used for drug libraries. Here we report a DMSO-tolerant ODC tandem assay in which DMSO-dependent fluorescence quenching becomes negligible by separating enzyme reaction and putrescine detection. Furthermore, we optimised human cell-line-based mass production of ODC for HTS. Our newly developed assay can be a crucial first step in discovering more effective ODC modulators than DFMO.

| S-EPMC9721421 | biostudies-literature

High-throughput screening for phosphatidylserine decarboxylase inhibitors using a distyrylbenzene-bis-aldehyde (DSB-3)-based fluorescence assay.

Project description:Phosphatidylserine decarboxylases (PSDs) catalyze the decarboxylation of phosphatidylserine to generate phosphatidylethanolamine, a critical step in phospholipid metabolism in both prokaryotes and eukaryotes. Most PSDs are membrane-bound, and classical radioisotope-based assays for determining their activity in vitro are not suitable for high-throughput drug screening. The finding that the PkPSD from Plasmodium knowlesi can be purified in a soluble and active form and the recent development of a fluorescence-based distyrylbenzene-bis-aldehyde (DSB-3) assay to measure PSD activity in vitro have laid the groundwork for screening chemical libraries for PSD inhibitors. Using this assay, here we conducted a high-throughput screen of a structurally diverse 130,858-compound library against PkPSD. Further characterization of the hits identified in this screening yielded five PkPSD inhibitors with IC50 values ranging from 3.1 to 42.3 μm Lead compounds were evaluated against the pathogenic yeast Candida albicans in the absence or presence of exogenous ethanolamine, and YU253467 and YU254403 were identified as inhibiting both native C. albicans PSD mitochondrial activity and C. albicans growth, with an MIC50 of 22.5 and 15 μg/ml without ethanolamine and an MIC50 of 75 and 60 μg/ml with ethanolamine, respectively. Together, these results provide the first proof of principle for the application of DSB-3-based fluorescent readouts in high-throughput screening for PSD inhibitors. The data set the stage for future analyses to identify more selective and potent PSD inhibitors with antimicrobial or antitumor activities.

| S-EPMC6690695 | biostudies-literature

Yeast-based directed-evolution for high-throughput structural stabilization of G protein-coupled receptors (GPCRs).

Project description:The immense potential of G protein-coupled receptors (GPCRs) as targets for drug discovery is not fully realized due to the enormous difficulties associated with structure elucidation of these profoundly unstable membrane proteins. The existing methods of GPCR stability-engineering are cumbersome and low-throughput; in addition, the scope of GPCRs that could benefit from these techniques is limited. Here, we present a yeast-based screening platform for a single-step isolation of GRCR variants stable in the presence of short-chain detergents, a feature essential for their successful crystallization using vapor diffusion method. The yeast detergent-resistant cell wall presents a unique opportunity for compartmentalization, to physically link the receptor's phenotype to its encoding DNA, and thus enable discovery of stable GPCR variants with unprecedent efficiency. The scope of mutations identified by the method reveals a surprising amenability of the GPCR scaffold to stabilization, and suggests an intriguing possibility of amending the stability properties of GPCR by varying the structural status of the C-terminus.

| S-EPMC9126886 | biostudies-literature

Directed evolution of the substrate specificity of dialkylglycine decarboxylase.

Project description:Dialkylglycine decarboxylase (DGD) is an unusual pyridoxal phosphate dependent enzyme that catalyzes decarboxylation in the first and transamination in the second half-reaction of its ping-pong catalytic cycle. Directed evolution was employed to alter the substrate specificity of DGD from 2-aminoisobutyrate (AIB) to 1-aminocyclohexane-1-carboxylate (AC6C). Four rounds of directed evolution led to the identification of several mutants, with clones in the final rounds containing five persistent mutations. The best clones show ~2.5-fold decrease in KM and ~2-fold increase in kcat, giving a modest ~5-fold increase in catalytic efficiency for AC6C. Additional rounds of directed evolution did not improve catalytic activity toward AC6C. Only one (S306F) of the five persistent mutations is close to the active site. S306F was observed in all 33 clones except one, and the mutation is shown to stabilize the enzyme toward denaturation. The other four persistent mutations are near the surface of the enzyme. The S306F mutation and the distal mutations all have significant effects on the kinetic parameters for AIB and AC6C. Molecular dynamics simulations suggest that the mutations alter the conformational landscape of the enzyme, favoring a more open active site conformation that facilitates the reactivity of the larger substrate. We speculate that the small increases in kcat/KM for AC6C are due to two constraints. The first is the mechanistic requirement for catalyzing oxidative decarboxylation via a concerted decarboxylation/proton transfer transition state. The second is that DGD must catalyze transamination at the same active site in the second half-reaction of the ping-pong catalytic cycle.

| S-EPMC4334570 | biostudies-literature

Directed evolution of an ?1,3-fucosyltransferase using a single-cell ultrahigh-throughput screening method.

Project description:Fucosylated glycoconjugates are involved in a variety of physiological and pathological processes. However, economical production of fucosylated drugs and prebiotic supplements has been hampered by the poor catalytic efficiency of fucosyltransferases. Here, we developed a fluorescence-activated cell sorting system that enables the ultrahigh-throughput screening (>107 mutants/hour) of such enzymes and designed a companion strategy to assess the screening performance of the system. After three rounds of directed evolution, a mutant M32 of the ?1,3-FucT from Helicobacter pylori was identified with 6- and 14-fold increases in catalytic efficiency (k cat/K m) for the synthesis of Lewis x and 3'-fucosyllactose, respectively. The structure of the M32 mutant revealed that the S45F mutation generates a clamp-like structure that appears to improve binding of the galactopyranose ring of the acceptor substrate. Moreover, molecular dynamic simulations reveal that helix ?5, is more mobile in the M32 mutant, possibly explaining its high fucosylation activity.

| S-EPMC6785251 | biostudies-literature

Identification of Small-Molecule Inhibitors of Brucella Diaminopimelate Decarboxylase by Using a High-Throughput Screening Assay.

Project description:Brucellosis, caused by intracellular gram-negative pathogens of the genus Brucella, continues to be one of the most pandemic zoonotic diseases in most countries. At present, the therapeutic treatment of brucellosis relies on a combination of multiple antibiotics that involves a long course of treatment, easy relapse, and high side effects from the use of certain antibiotics (such as streptomycin). Thus, the need to identify novel drugs or targets to control this disease is urgent. Diaminopimelate decarboxylase (DAPDC), a key enzyme involved in the bacterial diaminopimelate (DAP) biosynthetic pathway, was suggested to be a promising anti-Brucella target in our previous study. In this work, the biological activity of Brucella melitensis DAPDC was characterized, and a library of 1,591 compounds was screened for inhibitors of DAPDC. The results of a high-throughput screening (HTS) assay showed that 24 compounds inhibited DAPDC activity. In a further in vitro bacterial inhibition experiment, five compounds exhibited anti-Brucella activity (SID3, SID4, SID14, SID15, and SID20). These results suggested that the identified compounds can be used as potent molecules against brucellosis and that the application ranges of these approved drugs can be expanded in the future.

| S-EPMC6986272 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data