Comprehensive untargeted lipidomic profiling of third generation lentiviral vectors and packaging cells.

Lentiviral vectors (LV) are emerging tools for genetic therapies and novel cancer treatments. While effective, LV-based therapies have extremely large costs associated with their manufacturing and delivery. LV technology descends from human immunodeficiency virus (HIV), whose lipid envelope has been previously measured and shown to have a direct impact on its transduction efficiency.

Thioesterases as tools for chemoenzymatic synthesis of macrolactones.

Macrocycles are a key functional group that can impart unique properties into molecules. Their synthesis has led to the development of many outstanding chemical methodologies and yet still remains challenging. Thioesterase (TE) domains are frequently responsible for macrocyclization in natural product biosynthesis and provide unique strengths for the enzymatic synthesis of macrocycles.

Total and chemoenzymatic synthesis of the lipodepsipeptide rhizomide A.

Rhizomides are a family of depsipeptide macrolactones synthesized by a non-ribosomal peptide synthetase (NRPS) encoded in the genome of Paraburkholderia rhizoxinica str. HKI 454. In this study, the total and chemoenzymatic synthesis of the depsipeptide rhizomide A is described.

Double Stranded DNA Binding Stapled Peptides: An Emerging Tool for Transcriptional Regulation.

Stapled peptides have rapidly established themselves as a powerful technique to mimic α-helical interactions with a short peptide sequence. There are many examples of stapled peptides that successfully disrupt α-helix-mediated protein-protein interactions, with an example currently in clinical trials. DNA-protein interactions are also often mediated by α-helices and are involved in all transcriptional regulation processes.

Macrocyclization strategies for the total synthesis of cyclic depsipeptides.

Cyclic depsipeptides are an important class of peptide natural products that are defined by the presence of ester and amide bonds within the macrocycle. The structural diversity of depsipeptides has required the development of a broad range of synthetic strategies to access these biologically active compounds. Solid phase peptide synthesis (SPPS) strategies have been an invaluable tool in their synthesis.

CRISPR-Cas9 Gene Editing and Secondary Metabolite Screening Confirm C16 Biosynthetic Gene Cluster Products as Decalin-Containing Diterpenoid Pyrones.

is a causal organism of Fusarium head blight in cereals and maize. Although a few secondary metabolites produced by are considered disease virulence factors, many molecular products of biosynthetic gene clusters expressed by during infection and their associated role in the disease are unknown. In particular, the predicted meroterpenoid products of the biosynthetic gene cluster historically designated as "C16" are likely associated with pathogenicity.

Characterization of Arthropeptide B, an Antifungal Cyclic Tetrapeptide from Arthrobacter humicola.

Disease suppressive composts are known, yet little information on the potential role of specific microbial antagonist within are available. Arthrobacter humicola isolate M9-1A has been obtained from a compost prepared from marine residues and peat moss. The bacterium is a non-filamentous actinomycete with antagonistic activity against plant pathogenic fungi and oomycetes sharing its ecological niche in agri-food microecosystems.

In Vitro Biochemical Characterization of Excised Macrocyclizing Thioesterase Domains from Non-ribosomal Peptide Synthetases.

Characterization of thioesterases (TEs) is an important step in understanding natural product biosynthesis. Studying non-ribosomal peptide synthetase (NRPS) TEs presents a unique set of challenges with specific cloning and expression issues as well as the challenging synthesis of the thioester peptides substrate required for characterization of the TE. In this method, we describe the cloning and expression of NRPS TEs, the synthesis of thioester peptides, and the in vitro biochemical characterization of the enzyme.

Armeniaspirol analogues disrupt the electrical potential (ΔΨ) of the proton motive force.

The armeniaspirol family of natural product antibiotics have been shown to inhibit the ATP-dependent proteases ClpXP and ClpYQ and disrupt membrane potential through shuttling of protons across the membrane. Herein we investigate their ability to disrupt the proton motive force (PMF). We show, using a voltage sensitive, that armeniaspiols disrupt the electrical membrane potential (ΔΨ) component of the PMF and not the transmembrane proton gradient (ΔpH).

Untargeted metabolomics screening reveals unique secondary metabolite production from section .

section is comprised of many species that infect a broad diversity of important crop plants and cause post-harvest spoilage. section species, such as and , are prolific producers of secondary metabolites that act as virulence factors of disease and are mycotoxins that accumulate in infected tissues-metabolites that can vary in their spectrum of production between individuals from the same fungal species. Untargeted metabolomics profiling of secondary metabolite production using mass spectrometry is an effective means to detect phenotypic anomalies in secondary metabolism within a species.

Synthesis of a Constitutional Isomer of Armeniaspirol A, Pseudoarmeniaspirol A, via Lewis Acid-Mediated Rearrangement.

The natural product armeniaspirol possesses a unique spirocyclic N,O-ketal in an α,β-dichloro-α,β-unsaturated lactam scaffold that has proved challenging to synthesize. Herein, we characterize the oxidative chlorination of pyrrole-2-carboxylate derivatives that rapidly generates this scaffold. The scope of this oxidation was extended to a series of esters and amides.

Total and Chemoenzymatic Synthesis of Seongsanamide E.

The total and chemoenzymatic synthesis of the depsipeptide natural product seongsanamide E, , is described. The synthetic C-terminal -acetylcysteamine thioester of linear natural product was macrolactonized by the excised recombinant purified seongsanamide thioesterase (Sgd-TE) domain, generating . Sgd-TE also effects the ring opening of .

Biomineralization in Cave Bacteria-Popcorn and Soda Straw Crystal Formations, Morphologies, and Potential Metabolic Pathways.

Caves are extreme, often oligotrophic, environments that house diverse groups of microorganisms. Many of these microbes can perform microbiologically induced carbonate precipitation (MICP) to form crystalline secondary cave deposits known as speleothems. The urease family is a group of enzymes involved in MICP that catalyze the breakdown of urea, which is a source of energy, into ammonia and carbonate.

RpoN-Based stapled peptides with improved DNA binding suppress virulence.

Stapled peptides have the ability to mimic α-helices involved in protein binding and have proved to be effective pharmacological agents for disrupting protein-protein interactions. DNA-binding proteins such as transcription factors bind their cognate DNA sequences an α-helix interacting with the major groove of DNA. We previously developed a stapled peptide based on the bacterial alternative sigma factor RpoN capable of binding the RpoN DNA promoter sequence and inhibiting RpoN-mediated expression in .

Armeniaspirol analogues with more potent Gram-positive antibiotic activity show enhanced inhibition of the ATP-dependent proteases ClpXP and ClpYQ.

Antibiotics with fundamentally new mechanisms of action such as the armeniaspirols, which target the ATP-dependent proteases ClpXP and ClpYQ, must be developed to combat antimicrobial resistance. While the mechanism of action of armeniaspirol against Gram-positive bacteria is understood, little is known about the structure-activity relationship for its antibiotic activity. Based on the preliminary data showing that modifications of armeniaspirol's -methyl group increased antibiotic potency, we probed the structure-activity relationship of -alkyl armeniaspirol derivatives.

Evolution of the Ergot Alkaloid Biosynthetic Gene Cluster Results in Divergent Mycotoxin Profiles in Sclerotia.

Research into ergot alkaloid production in major cereal cash crops is crucial for furthering our understanding of the potential toxicological impacts of upon Canadian agriculture and to ensure consumer safety. An untargeted metabolomics approach profiling extracts of sclerotia from four different grain crops separated the strains into two distinct metabolomic classes based on ergot alkaloid content. Variances in alkaloid profiles were correlated to genetic differences within the gene of the ergot alkaloid biosynthetic gene cluster from previously published genomes and from newly sequenced, long-read genome assemblies of Canadian strains.

A metabolomic study of vegetative incompatibility in Cryphonectria parasitica.

Vegetative incompatibility (VI) is a form of non-self allorecognition in filamentous fungi that restricts conspecific hyphal fusion and the formation of heterokaryons. In the chestnut pathogenic fungus, Cryphonectria parasitica, VI is controlled by six vic loci and has been of particular interest because it impedes the spread of hypoviruses and thus biocontrol strategies. We use nuclear magnetic resonance and high-resolution mass spectrometry to characterize alterations in the metabolome of C.

Apicidin biosynthesis is linked to accessory chromosomes in Fusarium poae isolates.

Background: Fusarium head blight is a disease of global concern that reduces crop yields and renders grains unfit for consumption due to mycotoxin contamination. Fusarium poae is frequently associated with cereal crops showing symptoms of Fusarium head blight. While previous studies have shown F.

Armeniaspirols inhibit the AAA+ proteases ClpXP and ClpYQ leading to cell division arrest in Gram-positive bacteria.

Multi-drug-resistant bacteria present an urgent threat to modern medicine, creating a desperate need for antibiotics with new modes of action. As natural products remain an unsurpassed source for clinically viable antibiotic compounds, we investigate the mechanism of action of armeniaspirol. The armeniaspirols are a structurally unique class of Gram-positive antibiotic discovered from Streptomyces armeniacus for which resistance cannot be readily obtained.

Accessory Chromosome-Acquired Secondary Metabolism in Plant Pathogenic Fungi: The Evolution of Biotrophs Into Host-Specific Pathogens.

Accessory chromosomes are strain- or pathotype-specific chromosomes that exist in addition to the core chromosomes of a species and are generally not considered essential to the survival of the organism. Among pathogenic fungal species, accessory chromosomes harbor pathogenicity or virulence factor genes, several of which are known to encode for secondary metabolites that are involved in plant tissue invasion. Accessory chromosomes are of particular interest due to their capacity for horizontal transfer between strains and their dynamic "crosstalk" with core chromosomes.

Thioesterase from Cereulide Biosynthesis Is Responsible for Oligomerization and Macrocyclization of a Linear Tetradepsipeptide.

Cereulide is a toxic cyclic depsidodecapeptide produced in by two nonribosomal peptide synthetases, CesA and CesB. While highly similar in structure to valinomycin and with a homologous biosynthetic gene cluster, recent work suggests that cereulide is produced via a different mechanism that relies on a noncanonical coupling of two didepsipeptide-peptidyl carrier protein (PCP) bound intermediates. Ultimately this alternative mechanism generates a tetradepsipeptide-PCP bound intermediate that differs from the tetradepsipeptide-PCP intermediate predicted from canonical activity of CesA and CesB.

Whole Genome Sequencing and Metabolomic Study of Cave Isolates ICC1 and ICC4.

The terrestrial subsurface microbiome has gained considerable amount of interests in the recent years because of its rich potential resource for biomining novel genes coding for metabolites possessing antimicrobial activities. In our previous study, we identified two isolates, designated as ICC1 and ICC4, from the Iron Curtain Cave, Chilliwack, Canada that exhibited antagonistic activities against the multidrug resistant strains of . In this study, the genomes of these two isolates were sequenced by Illumina MiSeq, assembled and annotated.

Trapping biosynthetic acyl-enzyme intermediates with encoded 2,3-diaminopropionic acid.

Many enzymes catalyse reactions that proceed through covalent acyl-enzyme (ester or thioester) intermediates. These enzymes include serine hydrolases (encoded by one per cent of human genes, and including serine proteases and thioesterases), cysteine proteases (including caspases), and many components of the ubiquitination machinery. Their important acyl-enzyme intermediates are unstable, commonly having half-lives of minutes to hours.

Draft Genome Sequence of the Type Strain Streptomyces armeniacus ATCC 15676.

The Streptomyces genus represents a prolific and significant source for secondary metabolite discovery. Here, we report a assembly and draft genome sequence for the type strain Streptomyces armeniacus ATCC 15676.

Chemoenzymatic macrocycle synthesis using resorcylic acid lactone thioesterase domains.

A key missing tool in the chemist's toolbox is an effective biocatalyst for macrocyclization. Macrocycles limit the conformational flexibility of small molecules, often improving their ability to bind selectively and with high affinity to a target, making them a privileged structure in drug discovery. Macrocyclic natural product biosynthesis offers an obvious starting point for biocatalyst discovery via the native macrocycle forming biosynthetic mechanism.