Morgan A. Wyatt

Genomes to natural products PRediction Informatics for Secondary Metabolomes (PRISM)

Microbial natural products are an invaluable source of evolved bioactive small molecules and pharmaceutical agents. Next-generation and metagenomic sequencing indicates untapped genomic potential, yet high rediscovery rates of known metabolites increasingly frustrate conventional natural product screening programs. New methods to connect biosynthetic gene clusters to novel chemical scaffolds are therefore critical to enable the targeted discovery of genetically encoded natural products. Here, we present PRISM, a computational resource for the identification of biosynthetic gene clusters, prediction of genetically encoded nonribosomal peptides and type I and II polyketides, and bio- and cheminformatic dereplication of known natural products. PRISM implements novel algorithms which render it uniquely capable of predicting type II polyketides, deoxygenated sugars, and starter units, making it a comprehensive genome-guided chemical structure prediction engine. A library of 57 tailoring reactions is leveraged for combinatorial scaffold library generation when multiple potential substrates are consistent with biosynthetic logic. We compare the accuracy of PRISM to existing genomic analysis platforms. PRISM is an open-source, user-friendly web application available at http://magarveylab.ca/prism/.

Gold biomineralization by a metallophore from a gold-associated microbe

Microorganisms produce and secrete secondary metabolites to assist in their survival. We report that the gold resident bacterium Delftia acidovorans produces a secondary metabolite that protects from soluble gold through the generation of solid gold forms. This finding is the first demonstration that a secreted metabolite can protect against toxic gold and cause gold biomineralization.

Staphylococcus aureus Nonribosomal Peptide Secondary Metabolites Regulate Virulence

Golden Regulator Staphylococcus aureus is a common cause of intractable infections that are exacerbated by an array of toxins and virulence factors. The agr pheromone has been thought to represent the master regulator of virulence in this pathogen, but it is not always expressed and is also found in many nonpathogenic cocci. A strictly conserved, nonribosomal peptide synthetase has now been found by Wyatt et al. (p. 294, published online 3 June) by genome mining. The enzyme assembles valine and tyrosine into cyclic dipeptides called aureusimines that are expressed by all sequenced strains of S. aureus, including the “superbug” MRSA (Methicillin-resistant Staphylococcus aureus). Microarray analysis showed a striking effect of mutation in the synthetase locus on the production of immunomodulators, hemolysins, and other exotoxins by the pathogen. Indeed, mice infected systemically with the mutant strain showed a restricted spread of infection compared with the wild type. Dipeptides produced by a major bacterial pathogen are essential for successful infection. Staphylococcus aureus is a major human pathogen that is resistant to numerous antibiotics in clinical use. We found two nonribosomal peptide secondary metabolites—the aureusimines, made by S. aureus—that are not antibiotics, but function as regulators of virulence factor expression and are necessary for productive infections. In vivo mouse models of bacteremia showed that strains of S. aureus unable to produce aureusimines were attenuated and/or cleared from major organs, including the spleen, liver, and heart. Targeting aureusimine synthesis may offer novel leads for anti-infective drugs.

An automated Genomes-to-Natural Products platform (GNP) for the discovery of modular natural products.

Bacterial natural products are a diverse and valuable group of small molecules, and genome sequencing indicates that the vast majority remain undiscovered. The prediction of natural product structures from biosynthetic assembly lines can facilitate their discovery, but highly automated, accurate, and integrated systems are required to mine the broad spectrum of sequenced bacterial genomes. Here we present a genome-guided natural products discovery tool to automatically predict, combinatorialize and identify polyketides and nonribosomal peptides from biosynthetic assembly lines using LC–MS/MS data of crude extracts in a high-throughput manner. We detail the directed identification and isolation of six genetically predicted polyketides and nonribosomal peptides using our Genome-to-Natural Products platform. This highly automated, user-friendly programme provides a means of realizing the potential of genetically encoded natural products.

Heterologous Expression and Structural Characterisation of a Pyrazinone Natural Product Assembly Line

Through a number of strategies nonribosomal peptide assembly lines give rise to a metabolic diversity not possible by ribosomal synthesis. One distinction within nonribosomal assembly is that products are elaborated on an enzyme‐tethered substrate, and their release is enzyme catalysed. Reductive release by NAD(P)H‐dependent catalysts is one observed nonribosomal termination and release strategy. Here we probed the selectivity of a terminal reductase domain by using a full‐length heterologously expressed nonribosomal peptide synthetase for the dipeptide aureusimine and were able to generate 17 new analogues. Further, we generated an X‐ray structure of aureusimine terminal reductase to gain insight into the structural details associated with this enzymatic domain.

Biosynthesis of ebelactone A: isotopic tracer, advanced precursor and genetic studies reveal a thioesterase-independent cyclization to give a polyketide β-lactone

Macrocyclization of polyketides generates arrays of molecular architectures that are directly linked to biological activities. The four-membered ring in oxetanones (β-lactones) is found in a variety of bioactive polyketides (for example, lipstatin, hymeglusin and ebelactone), yet details of its molecular assembly have not been extensively elucidated. Using ebelactone as a model system, and its producer Streptomyces aburaviensis ATCC 31860, labeling with sodium [1-13C,18O2]propionate afforded ebelactone A that contains 18O at all oxygen sites. The pattern of 13C–18O bond retention defines the steps for ebelactone biosynthesis, and demonstrates that β-lactone ring formation occurs by attack of a β-hydroxy group onto the carbonyl moiety of an acyclic precursor. Reaction of ebelactone A with N-acetylcysteamine (NAC) gives the β-hydroxyacyl thioester, which cyclizes quantitatively to give ebelactone A in aqueous ethanol. The putative gene cluster encoding the polyketide synthase (PKS) for biosynthesis of 1 was also identified; notably the ebelactone PKS lacks a terminal thioesterase (TE) domain and no stand alone TE was found. Thus the formation of ebelactone is not TE dependent, supporting the hypothesis that cyclization occurs on the PKS surface in a process that is modeled by the chemical cyclization of the NAC thioester.

Gold nanoparticle formation via microbial metallophore chemistries

Microbes are evolved to live in almost every environment and often produce small molecules to protect themselves, communicate, and sequester nutrients and minerals from their surroundings. Our recent discovery that the gold-associated microbe, Delftia acidovorans, overcomes Au3+ toxicity through complexation and precipitation of soluble gold by the nonribosomal peptide metallophore, delftibactin, revealed a new mechanism for gold biomineralization and a possible industrial application for gold sequestration. In this work, we have sought to define more the scope of nanoparticle formation by other microbial metallophores and the physical properties of gold nanoparticles created by microbial natural products (hydroxamates, catechols, citrates, mixed ligands) and synthetics.

Informatic search strategies to discover analogues and variants of natural product archetypes

Natural products are a crucial source of antimicrobial agents, but reliance on low-resolution bioactivity-guided approaches has led to diminishing interest in discovery programmes. Here, we demonstrate that two in-house automated informatic platforms can be used to target classes of biologically active natural products, specifically, peptaibols. We demonstrate that mass spectrometry-based informatic approaches can be used to detect natural products with high sensitivity, identifying desired agents present in complex microbial extracts. Using our specialised software packages, we could elaborate specific branches of chemical space, uncovering new variants of trichopolyn and demonstrating a way forward in mining natural products as a valuable source of potential pharmaceutical agents.

Bioinformatic evaluation of the secondary metabolism of antistaphylococcal environmental bacterial isolates.

The increasing occurrence of drug-resistant Staphylococcus aureus is exacerbated with a declining rate of antibiotic discovery, particularly those with new mechanisms of action. The decline in antibiotic discovery from traditional sources, such as soil actinobacteria, necessitates examination of lesser studied microbes. Here, we present a strategy to select for organisms that may have a propensity to result in new antistaphylococcal agents by using S. aureus as a bait organism, and selecting organisms that have a natural lytic activity towards it. We have isolated over 80 environmental isolates and typed these organisms using 16S rDNA sequence comparison and deployed bioinformatics to assess the secondary metabolic potential of the isolated antistaphylococcal bacteria using genomic sequences. Bioinformatic analysis highlights the enriched and unique suite of potential antibiotic polyketides and nonribosomal peptides and lantibiotic gene clusters from these organisms. Profiling organic microbial extracts further showed that many of the organisms from the 10 staphylolytic genera secrete agents with antistaphylococcal activity and may serve as new sources for future antistaphylococcal drug discovery.

Optimizing dimodular nonribosomal peptide synthetases and natural dipeptides in an Escherichia coli heterologous host.

Nonribosomal peptides are an important class of natural products that have a broad range of biological activities. Their structural complexity often prevents simple chemical synthesis, and production from the natural producer is often low, which deters pharmaceutical development. Expression of biosynthetic machinery in heterologous host organisms like Escherichia coli is one way to access these structures, and subsequent optimization of these systems is critical for future development. We utilized the aureusimine biosynthetic gene cluster as a model system to identify the optimal conditions to produce nonribosomal peptides in the isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible T7 promoter system of pET28. Single reaction monitoring of nonribosomal products was used to find the optimal concentration of IPTG, postinduction temperature, and the effect of amino acid precursor supplementation. In addition, principle component analysis of these extracts identified 3 previously undiscovered pyrazine products of the aureusimine biosynthetic locus, highlighting the utility of heterologously expressing nonribosomal peptide synthetases to find new products.