De novo design of ribosomally synthesized and post-translationally modified peptides.

In nature, peptides are enzymatically modified to constrain their structure and introduce functional moieties. De novo peptide structures could be built by combining enzymes from different pathways, but determining the rules of their use is difficult. We present a biophysical model to combine enzymes sourced from bacterial ribosomally synthesized and post-translationally modified peptide (RiPP) gene clusters.

Systematic mining of the human microbiome identifies antimicrobial peptides with diverse activity spectra.

Human-associated bacteria secrete modified peptides to control host physiology and remodel the microbiota species composition. Here we scanned 2,229 Human Microbiome Project genomes of species colonizing skin, gastrointestinal tract, urogenital tract, mouth and trachea for gene clusters encoding RiPPs (ribosomally synthesized and post-translationally modified peptides). We found 218 lanthipeptides and 25 lasso peptides, 70 of which were synthesized and expressed in E.

Functional expression of diverse post-translational peptide-modifying enzymes in Escherichia coli under uniform expression and purification conditions.

RiPPs (ribosomally-synthesized and post-translationally modified peptides) are a class of pharmaceutically-relevant natural products expressed as precursor peptides before being enzymatically processed into their final functional forms. Bioinformatic methods have illuminated hundreds of thousands of RiPP enzymes in sequence databases and the number of characterized chemical modifications is growing rapidly; however, it remains difficult to functionally express them in a heterologous host. One challenge is peptide stability, which we addressed by designing a RiPP stabilization tag (RST) based on a small ubiquitin-like modifier (SUMO) domain that can be fused to the N- or C-terminus of the precursor peptide and proteolytically removed after modification.

Selection for constrained peptides that bind to a single target protein.

Peptide secondary metabolites are common in nature and have diverse pharmacologically-relevant functions, from antibiotics to cross-kingdom signaling. Here, we present a method to design large libraries of modified peptides in Escherichia coli and screen them in vivo to identify those that bind to a single target-of-interest. Constrained peptide scaffolds were produced using modified enzymes gleaned from microbial RiPP (ribosomally synthesized and post-translationally modified peptide) pathways and diversified to build large libraries.

Genetically modifying skin microbe to produce violacein and augmenting microbiome did not defend Panamanian golden frogs from disease.

We designed two probiotic treatments to control chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) on infected Panamanian golden frogs (Atelopus zeteki), a species that is thought to be extinct in the wild due to Bd. The first approach disrupted the existing skin microbe community with antibiotics then exposed the frogs to a core golden frog skin microbe (Diaphorobacter sp.) that we genetically modified to produce high titers of violacein, a known antifungal compound.

Gut-inhabiting Clostridia build human GPCR ligands by conjugating neurotransmitters with diet- and human-derived fatty acids.

Human physiology is regulated by endogenous signalling compounds, including fatty acid amides (FAAs), chemical mimics of which are made by bacteria. The molecules produced by human-associated microbes are difficult to identify because they may only be made in a local niche or they require a substrate sourced from the host, diet or other microbes. We identified a set of uncharacterized gene clusters in metagenomics data from the human gut microbiome.

Escherichia coli "Marionette" strains with 12 highly optimized small-molecule sensors.

Cellular processes are carried out by many genes, and their study and optimization requires multiple levers by which they can be independently controlled. The most common method is via a genetically encoded sensor that responds to a small molecule. However, these sensors are often suboptimal, exhibiting high background expression and low dynamic range.

DNAplotlib: Programmable Visualization of Genetic Designs and Associated Data.

DNAplotlib ( www.dnaplotlib.org ) is a computational toolkit for the programmable visualization of highly customizable, standards-compliant genetic designs.

Integration of high-content screening and untargeted metabolomics for comprehensive functional annotation of natural product libraries.

Traditional natural products discovery using a combination of live/dead screening followed by iterative bioassay-guided fractionation affords no information about compound structure or mode of action until late in the discovery process. This leads to high rates of rediscovery and low probabilities of finding compounds with unique biological and/or chemical properties. By integrating image-based phenotypic screening in HeLa cells with high-resolution untargeted metabolomics analysis, we have developed a new platform, termed Compound Activity Mapping, that is capable of directly predicting the identities and modes of action of bioactive constituents for any complex natural product extract library.

Probing the Physicochemical Boundaries of Cell Permeability and Oral Bioavailability in Lipophilic Macrocycles Inspired by Natural Products.

Cyclic peptide natural products contain a variety of conserved, nonproteinogenic structural elements such as d-amino acids and amide N-methylation. In addition, many cyclic peptides incorporate γ-amino acids and other elements derived from polyketide synthases. We hypothesized that the position and orientation of these extended backbone elements impact the ADME properties of these hybrid molecules, especially their ability to cross cell membranes and avoid metabolic degradation.

Large-scale cytological profiling for functional analysis of bioactive compounds.

Cytological profiling (CP) is an unbiased image-based screening technique that uses automated microscopy and image analysis to profile compounds based on numerous quantifiable phenotypic features. We used CP to evaluate a library of nearly 500 compounds with documented mechanisms of action (MOAs) spanning a wide range of biological pathways. We developed informatics techniques for generating dosage-independent phenotypic "fingerprints" for each compound, and for quantifying the likelihood that a compound's CP fingerprint corresponds to its annotated MOA.

Conformational dynamics accompanying the proteolytic degradation of trimeric collagen I by collagenases.

Collagenases are the principal enzymes responsible for the degradation of collagens during embryonic development, wound healing, and cancer metastasis. However, the mechanism by which these enzymes disrupt the highly chemically and structurally stable collagen triple helix remains incompletely understood. We used a single-molecule magnetic tweezers assay to characterize the cleavage of heterotrimeric collagen I by both the human collagenase matrix metalloproteinase-1 (MMP-1) and collagenase from Clostridium histolyticum.