Browse
Submit Data
Databases
API
Help

Dataset Information

18 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Disulfide-compatible phage-assisted continuous evolution in the periplasmic space.

ABSTRACT: The directed evolution of antibodies has yielded important research tools and human therapeutics. The dependence of many antibodies on disulfide bonds for stability has limited the application of continuous evolution technologies to antibodies and other disulfide-containing proteins. Here we describe periplasmic phage-assisted continuous evolution (pPACE), a system for continuous evolution of protein-protein interactions in the disulfide-compatible environment of the E. coli periplasm. We first apply pPACE to rapidly evolve novel noncovalent and covalent interactions between subunits of homodimeric YibK protein and to correct a binding-defective mutant of the anti-GCN4 Ω-graft antibody. We develop an intein-mediated system to select for soluble periplasmic expression in pPACE, leading to an eight-fold increase in soluble expression of the Ω-graft antibody. Finally, we evolve disulfide-containing trastuzumab antibody variants with improved binding to a Her2-like peptide and improved soluble expression. Together, these results demonstrate that pPACE can rapidly optimize proteins containing disulfide bonds, broadening the applicability of continuous evolution.

SUBMITTER: Morrison MS

PROVIDER: S-EPMC8514426 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Phage-assisted continuous and non-continuous evolution.

Project description:Directed evolution, which applies the principles of Darwinian evolution to a laboratory setting, is a powerful strategy for generating biomolecules with diverse and tailored properties. This technique can be implemented in a highly efficient manner using continuous evolution, which enables the steps of directed evolution to proceed seamlessly over many successive generations with minimal researcher intervention. Phage-assisted continuous evolution (PACE) enables continuous directed evolution in bacteria by mapping the steps of Darwinian evolution onto the bacteriophage life cycle and allows directed evolution to occur on much faster timescales compared to conventional methods. This protocol provides detailed instructions on evolving proteins using PACE and phage-assisted non-continuous evolution (PANCE) and includes information on the preparation of selection phage and host cells, the assembly of a continuous flow apparatus and the performance and analysis of evolution experiments. This protocol can be performed in as little as 2 weeks to complete more than 100 rounds of evolution (complete cycles of mutation, selection and replication) in a single PACE experiment.

| S-EPMC7865204 | biostudies-literature

Phage-assisted continuous evolution of proteases with altered substrate specificity.

Project description:Here we perform phage-assisted continuous evolution (PACE) of TEV protease, which canonically cleaves ENLYFQS, to cleave a very different target sequence, HPLVGHM, that is present in human IL-23. A protease emerging from ∼2500 generations of PACE contains 20 non-silent mutations, cleaves human IL-23 at the target peptide bond, and when pre-mixed with IL-23 in primary cultures of murine splenocytes inhibits IL-23-mediated immune signaling. We characterize the substrate specificity of this evolved enzyme, revealing shifted and broadened specificity changes at the six positions in which the target amino acid sequence differed. Mutational dissection and additional protease specificity profiling reveal the molecular basis of some of these changes. This work establishes the capability of changing the substrate specificity of a protease at many positions in a practical time scale and provides a foundation for the development of custom proteases that catalytically alter or destroy target proteins for biotechnological and therapeutic applications.Proteases are promising therapeutics to treat diseases such as hemophilia which are due to endogenous protease deficiency. Here the authors use phage-assisted continuous evolution to evolve a variant TEV protease with altered target peptide sequence specificities.

| S-EPMC5643515 | biostudies-literature

Negative selection and stringency modulation in phage-assisted continuous evolution.

Project description:Phage-assisted continuous evolution (PACE) uses a modified filamentous bacteriophage life cycle to substantially accelerate laboratory evolution experiments. In this work, we expand the scope and capabilities of the PACE method with two key advances that enable the evolution of biomolecules with radically altered or highly specific new activities. First, we implemented small molecule-controlled modulation of selection stringency that enables otherwise inaccessible activities to be evolved directly from inactive starting libraries through a period of evolutionary drift. Second, we developed a general negative selection that enables continuous counterselection against undesired activities. We integrated these developments to continuously evolve mutant T7 RNA polymerase enzymes with ∼10,000-fold altered, rather than merely broadened, substrate specificities during a single three-day PACE experiment. The evolved enzymes exhibit specificity for their target substrate that exceeds that of wild-type RNA polymerases for their cognate substrates while maintaining wild type-like levels of activity.

| S-EPMC3977541 | biostudies-literature

Phage-Assisted Continuous Evolution and Selection of Enzymes for Chemical Synthesis.

Project description:Ligand-dependent biosensors are valuable tools for coupling the intracellular concentrations of small molecules to easily detectable readouts such as absorbance, fluorescence, or cell growth. While ligand-dependent biosensors are widely used for monitoring the production of small molecules in engineered cells and for controlling or optimizing biosynthetic pathways, their application to directed evolution for biocatalysts remains underexplored. As a consequence, emerging continuous evolution technologies are rarely applied to biocatalyst evolution. Here, we develop a panel of ligand-dependent biosensors that can detect a range of small molecules. We demonstrate that these biosensors can link enzymatic activity to the production of an essential phage protein to enable biocatalyst-dependent phage-assisted continuous evolution (PACE) and phage-assisted continuous selection (PACS). By combining these phage-based evolution and library selection technologies, we demonstrate that we can evolve enzyme variants with improved and expanded catalytic properties. Finally, we show that the genetic diversity resulting from a highly mutated PACS library is enriched for active enzyme variants with altered substrate scope. These results lay the foundation for using phage-based continuous evolution and selection technologies to engineer biocatalysts with novel substrate scope and reactivity.

| S-EPMC8461764 | biostudies-literature

Phage-Assisted Continuous Evolution (PACE): A Guide Focused on Evolving Protein-DNA Interactions.

Project description:The uptake of directed evolution methods is increasing, as these powerful systems can be utilized to develop new biomolecules with altered/novel activities, for example, proteins with new catalytic functions or substrate specificities and nucleic acids that recognize an intended target. Especially useful are systems that incorporate continuous evolution, where the protein under selective pressure undergoes continuous mutagenesis with little-to-no input from the researcher once the system is started. However, continuous evolution methods can be challenging to implement and a daunting investment of time and resources. Our intent is to provide basic information and helpful suggestions that we have gained from our experience with bacterial phage-assisted continuous evolution (PACE) toward the evolution of proteins that bind to a specific DNA target. We discuss factors to consider before adopting PACE for a given evolution scheme with focus on the PACE bacterial one-hybrid selection system and what optimization of a PACE selection circuit may look like using the evolution of the DNA-binding protein ME47 as a case study. We outline different types of selection circuits and techniques that may be added onto a basic PACE setup. With this information, researchers will be better equipped to determine whether PACE is a valid strategy to adopt for their research program and how to set up a valid selection circuit.

| S-EPMC7593997 | biostudies-literature

Experimental interrogation of the path dependence and stochasticity of protein evolution using phage-assisted continuous evolution.

Project description:To what extent are evolutionary outcomes determined by a population's recent environment, and to what extent do they depend on historical contingency and random chance? Here we apply a unique experimental system to investigate evolutionary reproducibility and path dependence at the protein level. We combined phage-assisted continuous evolution with high-throughput sequencing to analyze evolving protein populations as they adapted to divergent and then convergent selection pressures over hundreds of generations. Independent populations of T7 RNA polymerase genes were subjected to one of two selection histories ("pathways") demanding recognition of distinct intermediate promoters followed by a common final promoter. We observed distinct classes of solutions with unequal phenotypic activity and evolutionary potential evolve from the two pathways, as well as from replicate populations exposed to identical selection conditions. Mutational analysis revealed specific epistatic interactions that explained the observed path dependence and irreproducibility. Our results reveal in molecular detail how protein adaptation to different environments, as well as stochasticity among populations evolved in the same environment, can both generate evolutionary outcomes that preclude subsequent convergence.

| S-EPMC3670371 | biostudies-literature

Improved pyrrolysine biosynthesis through phage assisted non-continuous directed evolution of the complete pathway.

Project description:Pyrrolysine (Pyl, O) exists in nature as the 22nd proteinogenic amino acid. Despite being a fundamental building block of proteins, studies of Pyl have been hindered by the difficulty and inefficiency of both its chemical and biological syntheses. Here, we improve Pyl biosynthesis via rational engineering and directed evolution of the entire biosynthetic pathway. To accommodate toxicity of Pyl biosynthetic genes in Escherichia coli, we also develop Alternating Phage Assisted Non-Continuous Evolution (Alt-PANCE) that alternates mutagenic and selective phage growths. The evolved pathway provides 32-fold improved yield of Pyl-containing reporter protein compared to the rationally engineered ancestor. Evolved PylB mutants are present at up to 4.5-fold elevated levels inside cells, and show up to 2.2-fold increased protease resistance. This study demonstrates that Alt-PANCE provides a general approach for evolving proteins exhibiting toxic side effects, and further provides an improved pathway capable of producing substantially greater quantities of Pyl-proteins in E. coli.

| S-EPMC8225853 | biostudies-literature

Continuous evolution of SpCas9 variants compatible with non-G PAMs.

Project description:The targeting scope of Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants is largely restricted to protospacer-adjacent motif (PAM) sequences containing G bases. Here we report the evolution of three new SpCas9 variants that collectively recognize NRNH PAMs (where R is A or G and H is A, C or T) using phage-assisted non-continuous evolution, three new phage-assisted continuous evolution strategies for DNA binding and a secondary selection for DNA cleavage. The targeting capabilities of these evolved variants and SpCas9-NG were characterized in HEK293T cells using a library of 11,776 genomically integrated protospacer-sgRNA pairs containing all possible NNNN PAMs. The evolved variants mediated indel formation and base editing in human cells and enabled A•T-to-G•C base editing of a sickle cell anemia mutation using a previously inaccessible CACC PAM. These new evolved SpCas9 variants, together with previously reported variants, in principle enable targeting of most NR PAM sequences and substantially reduce the fraction of genomic sites that are inaccessible by Cas9-based methods.

| S-EPMC7145744 | biostudies-literature

Phage-Assisted Evolution of Bacillus methanolicus Methanol Dehydrogenase 2.

Project description:Synthetic methylotrophy, the modification of organisms such as E. coli to grow on methanol, is a longstanding goal of metabolic engineering and synthetic biology. The poor kinetic properties of NAD-dependent methanol dehydrogenase, the first enzyme in most methanol assimilation pathways, limit pathway flux and present a formidable challenge to synthetic methylotrophy. To address this bottleneck, we used a formaldehyde biosensor to develop a phage-assisted noncontinuous evolution (PANCE) selection for variants of Bacillus methanolicus methanol dehydrogenase 2 (Bm Mdh2). Using this selection, we evolved Mdh2 variants with up to 3.5-fold improved Vmax. The mutations responsible for enhanced activity map to the predicted active site region homologous to that of type III iron-dependent alcohol dehydrogenases, suggesting a new critical region for future methanol dehydrogenase engineering strategies. Evolved Mdh2 variants enable twice as much 13C-methanol assimilation into central metabolites than previously reported state-of-the-art methanol dehydrogenases. This work provides improved Mdh2 variants and establishes a laboratory evolution approach for metabolic pathways in bacterial cells.

| S-EPMC6479731 | biostudies-literature

Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity.

Project description:Although bespoke, sequence-specific proteases have the potential to advance biotechnology and medicine, generation of proteases with tailor-made cleavage specificities remains a major challenge. We developed a phage-assisted protease evolution system with simultaneous positive and negative selection and applied it to three botulinum neurotoxin (BoNT) light-chain proteases. We evolved BoNT/X protease into separate variants that preferentially cleave vesicle-associated membrane protein 4 (VAMP4) and Ykt6, evolved BoNT/F protease to selectively cleave the non-native substrate VAMP7, and evolved BoNT/E protease to cleave phosphatase and tensin homolog (PTEN) but not any natural BoNT protease substrate in neurons. The evolved proteases display large changes in specificity (218- to >11,000,000-fold) and can retain their ability to form holotoxins that self-deliver into primary neurons. These findings establish a versatile platform for reprogramming proteases to selectively cleave new targets of therapeutic interest.

| S-EPMC8175023 | 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