scholarly journals Evolution-guided engineering of small-molecule biosensors

2019 ◽  
Author(s):  
Tim Snoek ◽  
Evan K. Chaberski ◽  
Francesca Ambri ◽  
Stefan Kol ◽  
Sara P. Bjørn ◽  
...  
Keyword(s):  
Small Molecule ◽  
Transfer Functions ◽  
Binding Affinities ◽  
Ligand Specificity ◽  
Muconic Acid ◽  
New Host ◽  
High Throughput Method ◽  
Operational Range ◽  
Effector Binding ◽  
Complete Inversion

AbstractAllosteric transcription factors (aTFs) have proven widely applicable for biotechnology and synthetic biology as ligand-specific biosensors enabling real-time monitoring, selection and regulation of cellular metabolism. However, both the biosensor specificity and the correlation between ligand concentration and biosensor output signal, also known as the transfer function, often needs to be optimized before meeting application needs. Here, we present a versatile and high-throughput method to evolve and functionalize prokaryotic aTF specificity and transfer functions in a eukaryote chassis, namely baker’s yeastSaccharomyces cerevisiae. From a single round of directed evolution of the effector-binding domain (EBD) coupled with various toggled selection regimes, we robustly select aTF variants of thecis, cis-muconic acid-inducible transcription factor BenM evolved for change in ligand specificity, increased dynamic output range, shifts in operational range, and a complete inversion of function from activation to repression. Importantly, by targeting only the EBD, the evolved biosensors display DNA-binding affinities similar to BenM, and are functional when ported back into a non-native prokaryote chassis. The developed platform technology thus leverages aTF evolvability for the development of new host-agnostic biosensors with user-defined small-molecule specificities and transfer functions.

scholarly journals Evolution-guided engineering of small-molecule biosensors

2019 ◽  
Vol 48 (1) ◽  
pp. e3-e3 ◽  
Author(s):  
Tim Snoek ◽  
Evan K Chaberski ◽  
Francesca Ambri ◽  
Stefan Kol ◽  
Sara P Bjørn ◽  
...  
Keyword(s):  
Small Molecule ◽  
Transfer Functions ◽  
Binding Affinities ◽  
Ligand Specificity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Muconic Acid ◽  
New Host ◽  
High Throughput Method ◽  
Operational Range ◽  
Effector Binding

Abstract Allosteric transcription factors (aTFs) have proven widely applicable for biotechnology and synthetic biology as ligand-specific biosensors enabling real-time monitoring, selection and regulation of cellular metabolism. However, both the biosensor specificity and the correlation between ligand concentration and biosensor output signal, also known as the transfer function, often needs to be optimized before meeting application needs. Here, we present a versatile and high-throughput method to evolve prokaryotic aTF specificity and transfer functions in a eukaryote chassis, namely baker's yeast Saccharomyces cerevisiae. From a single round of mutagenesis of the effector-binding domain (EBD) coupled with various toggled selection regimes, we robustly select aTF variants of the cis,cis-muconic acid-inducible transcription factor BenM evolved for change in ligand specificity, increased dynamic output range, shifts in operational range, and a complete inversion-of-function from activation to repression. Importantly, by targeting only the EBD, the evolved biosensors display DNA-binding affinities similar to BenM, and are functional when ported back into a prokaryotic chassis. The developed platform technology thus leverages aTF evolvability for the development of new host-agnostic biosensors with user-defined small-molecule specificities and transfer functions.

scholarly journals Evolving small-molecule biosensors with improved performance and reprogrammed ligand specificity using OrthoRep

2021 ◽  
Author(s):  
Alex A Javanpour ◽  
Chang C Liu
Keyword(s):  
Small Molecule ◽  
High Throughput Screening ◽  
High Performance ◽  
Dynamic Range ◽  
Yeast Cells ◽  
Pathway Engineering ◽  
Muconic Acid ◽  
Metabolic Pathway Engineering ◽  
Operational Range

Genetically-encoded biosensors are valuable for the optimization of small-molecule biosynthesis pathways, because they transduce the production of small-molecule ligands into a readout compatible with high-throughput screening or selection in vivo. However, engineering biosensors with appropriate response functions and ligand specificities remains challenging. Here, we show that the continuous hypermutation system, OrthoRep, can be effectively applied to evolve biosensors with high dynamic range, reprogrammed activity towards desired non-cognate ligands, and proper operational range for coupling to biosynthetic pathways. In particular, we encoded the allosteric transcriptional factor, BenM, on OrthoRep such that propagation of host yeast cells resulted in BenM's rapid and continuous diversification. When these cells were subjected to cycles of culturing and sorting on BenM activity in the presence and absence of its cognate ligand, muconic acid, or the non-cognate ligand, adipic acid, we obtained multiple BenM variants that respond to their corresponding ligands. These biosensors outperform previously-engineered BenM-based biosensors by achieving substantially greater dynamic range (up to ~180-fold-induction) and broadened operational range. Expression of select BenM variants in the presence of a muconic acid biosynthetic pathway demonstrated sensitive biosensor activation without saturating response, which should enable pathway and host engineering for higher production of muconic and adipic acids. Given the streamlined manner in which high-performance and versatile biosensors were evolved using OrthoRep, this study provides a template for generating custom biosensors for metabolic pathway engineering and other biotechnology goals.

scholarly journals Reconcilingin vitroandin vivoactivities of engineered, LacI-based repressor proteins: Contributions of DNA looping and operator sequence variation

2018 ◽  
Author(s):  
Sudheer Tungtur ◽  
Kristen M. Schwingen ◽  
Joshua J. Riepe ◽  
Chamitha J. Weeramange ◽  
Liskin Swint-Kruse
Keyword(s):  
Amino Acid ◽  
Dna Looping ◽  
Published Data ◽  
Binding Affinities ◽  
Ligand Specificity ◽  
Primary Operator ◽  
Repressor Proteins ◽  
Operator Sequence

AbstractOne way to create new components for synthetic transcription circuits is to re-purpose naturally occurring transcription factor proteins and their cognate DNA operators. For the proteins, re-engineering can be accomplished via domain recombination (to create chimeric regulators) and/or amino acid substitutions. The resulting activities of new protein regulators are often assessedin vitrousing a representative operator. However, when functioningin vivo, transcription factors can interact with multiple operators. We comparedin vivoandin vitroresults for two LacI-based transcription repressor proteins, their mutational variants, and four operator sequences. The two sets of repressor variants differed in their overallin vivorepression, even though theirin vitrobinding affinities for the primary operator spanned the same range. Here, we show that the offset can be explained by different abilities to simultaneously bind and “loop” two DNA operators. Furtherin vitrostudies of the looping-competent repressors were carried out to measure binding to a secondary operator sequence. Surprisingly, binding to this operator was largely insensitive to amino acid changes in the repressor protein.In vitroexperiments with additional operators and analyses of published data indicates that amino acid changes in these repressor proteins leads to complicated changes in ligand specificity. These results raise new considerations for engineering components of synthetic transcription circuits and – more broadly – illustrate difficulties encountered when trying to extrapolate information about specificity determinant positions among protein homologs.

scholarly journals Determination of Effector Binding Affinities Using Photoacoustic Calorimetry

2018 ◽  
Vol 114 (3) ◽  
pp. 416a
Author(s):  
Jovany J. Betancourt ◽  
Jaroslava Miksovska
Keyword(s):  
Binding Affinities ◽  
Photoacoustic Calorimetry ◽  
Effector Binding

scholarly journals Optimization of Fluorescently Labeled Nrf2 Peptide Probes and the Development of a Fluorescence Polarization Assay for the Discovery of Inhibitors of Keap1-Nrf2 Interaction

2011 ◽  
Vol 17 (4) ◽  
pp. 435-447 ◽  
Author(s):  
Daigo Inoyama ◽  
Yu Chen ◽  
Xinyi Huang ◽  
Lesa J. Beamer ◽  
Ah-Ng Tony Kong ◽  
...  
Keyword(s):  
Small Molecule ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Dynamic Range ◽  
Antioxidant Response ◽  
Binding Affinities ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Peptide Amide

Activation of the antioxidant response element (ARE) upregulates enzymes involved in detoxification of electrophiles and reactive oxygen species. The induction of ARE genes is regulated by the interaction between redox sensor protein Keap1 and the transcription factor Nrf2. Fluorescently labeled Nrf2 peptides containing the ETGE motif were synthesized and optimized as tracers in the development of a fluorescence polarization (FP) assay to identify small-molecule inhibitors of the Keap1-Nrf2 interaction. The tracers were optimized to increase the dynamic range of the assay and their binding affinities to the Keap1 Kelch domain. The binding affinities of Nrf2 peptide inhibitors obtained in our FP assay using FITC-9mer Nrf2 peptide amide as the probe were in good agreement with those obtained previously by a surface plasmon resonance assay. The FP assay exhibits considerable tolerance toward DMSO and produced a Z′ factor greater than 0.6 in a 384-well format. Further optimization of the probe led to cyanine-labeled 9mer Nrf2 peptide amide, which can be used along with the FITC-9mer Nrf2 peptide amide in a high-throughput screening assay to discover small-molecule inhibitors of Keap1-Nrf2 interaction.

scholarly journals SAMPL7 TrimerTrip Host-Guest Binding Affinities from Extensive Alchemical and End-Point Free Energy Calculations

2020 ◽  
Author(s):  
Zhe Huai ◽  
Huaiyu Yang ◽  
Xiao Li ◽  
Zhaoxi Sun
Keyword(s):  
Free Energy ◽  
Computational Modelling ◽  
Free Energy Calculations ◽  
Binding Affinities ◽  
Enhanced Sampling ◽  
New Host ◽  
Energy Calculations ◽  
Configurational Space ◽  
End Point ◽  
Guest Binding

<p>The prediction of host-guest binding affinities with computational modelling is still a challenging task. In the 7<sup>th</sup> statistical assessment of the modeling of proteins and ligands (SAMPL) challenge, a new host named TrimerTrip is synthesized and the thermodynamic parameters of 16 structurally diverse guests binding to the host are characterized. The challenge provides only structures of the host and the guests, which indicates that the predictions of both the binding poses and the binding affinities are under assessment. In this work, starting from the binding poses obtained from our previous enhanced sampling simulations in the configurational space, we perform extensive alchemical and end-point free energy calculations to calculate the host-guest binding affinities. The alchemical predictions with two widely accepted charge schemes (i.e. AM1-BCC and RESP) are in good agreement with the experimental reference, while the end-point estimates show significant deviations. Surprisingly, the end-point MM/PBSA method seems very powerful in reproducing the experimental rank of binding affinities. Although the length of our simulations is already very long and the intermediate spacing is very dense, the convergence behavior is not very good, which may arise from the flexibility of the host molecule. Enhanced sampling techniques in the configurational space may be required to obtain fully converged sampling. Further, as the length of sampling in alchemical free energy calculations already achieves several hundred ns, performing direct simulations of the binding/unbinding event in the physical space could be more useful and insightful. More details about the binding pathway and mechanism could be obtained in this way. </p>

A New Host for Polymer and Small-Molecule Clathration

1999 ◽  
Vol 11 (9) ◽  
pp. 2478-2485 ◽  
Author(s):  
Harry R. Allcock ◽  
Nicolas J. Sunderland ◽  
A. Paul Primrose ◽  
Arnold L. Rheingold ◽  
Ilia A. Guzei ◽  
...  
Keyword(s):  
Small Molecule ◽  
New Host

scholarly journals Perception of structurally distinct effectors by the integrated WRKY domain of a plant immune receptor

2021 ◽  
Vol 118 (50) ◽  
pp. e2113996118
Author(s):  
Nitika Mukhi ◽  
Hannah Brown ◽  
Danylo Gorenkin ◽  
Pingtao Ding ◽  
Adam R. Bentham ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Binding Domain ◽  
Effector Proteins ◽  
Binding Affinities ◽  
Wrky Domain ◽  
Nucleotide Binding Domain ◽  
In Planta ◽  
Integrated Domains ◽  
Effector Binding

Plants use intracellular nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)–containing immune receptors (NLRs) to detect pathogen-derived effector proteins. The Arabidopsis NLR pair RRS1-R/RPS4 confers disease resistance to different bacterial pathogens by perceiving the structurally distinct effectors AvrRps4 from Pseudomonas syringae pv. pisi and PopP2 from Ralstonia solanacearum via an integrated WRKY domain in RRS1-R. How the WRKY domain of RRS1 (RRS1WRKY) perceives distinct classes of effector to initiate an immune response is unknown. Here, we report the crystal structure of the in planta processed C-terminal domain of AvrRps4 (AvrRps4C) in complex with RRS1WRKY. Perception of AvrRps4C by RRS1WRKY is mediated by the β2-β3 segment of RRS1WRKY that binds an electronegative patch on the surface of AvrRps4C. Structure-based mutations that disrupt AvrRps4C–RRS1WRKY interactions in vitro compromise RRS1/RPS4-dependent immune responses. We also show that AvrRps4C can associate with the WRKY domain of the related but distinct RRS1B/RPS4B NLR pair, and the DNA-binding domain of AtWRKY41, with similar binding affinities and how effector binding interferes with WRKY–W-box DNA interactions. This work demonstrates how integrated domains in plant NLRs can directly bind structurally distinct effectors to initiate immunity.

scholarly journals SAMPL7 TrimerTrip Host-Guest Binding Affinities from Extensive Alchemical and End-Point Free Energy Calculations

2020 ◽  
Author(s):  
Zhe Huai ◽  
Huaiyu Yang ◽  
Xiao Li ◽  
Zhaoxi Sun
Keyword(s):  
Free Energy ◽  
Computational Modelling ◽  
Free Energy Calculations ◽  
Binding Affinities ◽  
Enhanced Sampling ◽  
New Host ◽  
Energy Calculations ◽  
Configurational Space ◽  
End Point ◽  
Guest Binding

<p></p><p> The prediction of host-guest binding affinities with computational modelling is still a challenging task. In the 7<sup>th</sup> statistical assessment of the modeling of proteins and ligands (SAMPL) challenge, a new host named TrimerTrip was synthesized and the thermodynamic parameters of 16 structurally diverse guests binding to the host were characterized. In the TrimerTrip-guest challenge, only structures of the host and the guests are provided, which indicates that the predictions of both the binding poses and the binding affinities are under assessment. In this work, starting from the binding poses obtained from our previous enhanced sampling simulations in the configurational space, we perform extensive alchemical and end-point free energy calculations to calculate the host-guest binding affinities retrospectively. The alchemical predictions with two widely accepted charge schemes (i.e. AM1-BCC and RESP) are in good agreement with the experimental reference, while the end-point estimates perform poorly in reproducing the experimental binding affinities. Aside from the absolute value of the binding affinity, the rank of binding free energies is also crucial in drug design. Surprisingly, the end-point MM/PBSA method seems very powerful in reproducing the experimental rank of binding affinities. Although the length of our simulations is long and the intermediate spacing is dense, the convergence behavior is not very good, which may arise from the flexibility of the host molecule. Enhanced sampling techniques in the configurational space may be required to obtain fully converged sampling. Further, as the length of sampling in alchemical free energy calculations already achieves several hundred ns, performing direct simulations of the binding/unbinding event in the physical space could be more useful and insightful. More details about the binding pathway and mechanism could be obtained in this way. The nonequilibrium method could also be a nice choice if one insists to use the alchemical method, as the intermediate sampling is avoided to some extent. </p><p></p>

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