Bacterial growth-mediated systems remodelling of Nicotiana benthamiana defines unique signatures of target protein production in molecular pharming.

The need for therapeutics to treat a plethora of medical conditions and diseases is on the rise and the demand for alternative approaches to mammalian-based production systems is increasing. Plant-based strategies provide a safe and effective alternative to produce biological drugs but have yet to enter mainstream manufacturing at a competitive level. Limitations associated with batch consistency and target protein production levels are present; however, strategies to overcome these challenges are underway. In this study, we apply state-of-the-art mass spectrometry-based proteomics to define proteome remodelling of the plant following agroinfiltration with bacteria grown under shake flask or bioreactor conditions. We observed distinct signatures of bacterial protein production corresponding to the different growth conditions that directly influence the plant defence responses and target protein production on a temporal axis. Our integration of proteomic profiling with small molecule detection and quantification reveals the fluctuation of secondary metabolite production over time to provide new insight into the complexities of dual system modulation in molecular pharming. Our findings suggest that bioreactor bacterial growth may promote evasion of early plant defence responses towards Agrobacterium tumefaciens (updated nomenclature to Rhizobium radiobacter). Furthermore, we uncover and explore specific targets for genetic manipulation to suppress host defences and increase recombinant protein production in molecular pharming.

Epistatic Relationship between MGV1 and TRI6 in the Regulation of Biosynthetic Gene Clusters in Fusarium graminearum.

Genetic studies have shown that the MAP kinase MGV1 and the transcriptional regulator TRI6 regulate many of the same biosynthetic gene clusters (BGCs) in Fusarium graminearum. This study sought to investigate the relationship between MGV1 and TRI6 in the regulatory hierarchy. Transgenic F. graminearum strains constitutively expressing MGV1 and TRI6 were generated to address both independent and epistatic regulation of BGCs by MGV1 and TRI6. We performed a comparative transcriptome analysis between axenic cultures grown in nutrient-rich and secondary metabolite-inducing conditions. The results indicated that BGCs regulated independently by Mgv1 included genes of BGC52, whereas genes uniquely regulated by TRI6 included the gene cluster (BGC49) that produces gramillin. To understand the epistatic relationship between MGV1 and TRI6, CRISPR/Cas9 was used to insert a constitutive promoter to drive TRI6 expression in the Δmgv1 strain. The results indicate that BGCs that produce deoxynivalenol and fusaoctaxin are co-regulated, with TRI6 being partially regulated by MGV1. Overall, the findings from this study indicate that MGV1 provides an articulation point to differentially regulate various BGCs. Moreover, TRI6, embedded in one of the BGCs provides specificity to regulate the expression of the genes in the BGC.

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

Fusarium graminearum is a causal organism of Fusarium head blight in cereals and maize. Although a few secondary metabolites produced by F. graminearum are considered disease virulence factors, many molecular products of biosynthetic gene clusters expressed by F. graminearum 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. Presented here are the results of CRISPR-Cas9 gene-editing experiments disrupting the polyketide synthase and terpene synthase genes associated with the C16 biosynthetic gene cluster in F. graminearum. Culture medium screening experiments using transformant strains were profiled by UHPLC-HRMS and targeted MS2 experiments to confirm the associated secondary metabolite products of the C16 biosynthetic gene cluster as the decalin-containing diterpenoid pyrones, FDDP-D and FDDP-E. Both decalin-containing diterpenoid pyrones were confirmed to be produced in wheat heads challenged with F. graminearum in growth chamber trials. The extent to which the F. graminearum C16 biosynthetic gene cluster is dispersed within the genus Fusarium is discussed along with a proposed role of the FDDPs as pathogen virulence factors.

Multiple Clonostachys rosea UDP-Glycosyltransferases Contribute to the Production of 15-Acetyl-Deoxynivalenol-3-O-Glycoside When Confronted with Fusarium graminearum.

Mycotoxins, derived from toxigenic fungi such as Fusarium, Aspergillus, and Penicillium species have impacted the human food chain for thousands of years. Deoxynivalenol (DON), is a tetracyclic sesquiterpenoid type B trichothecene mycotoxin predominantly produced by F. culmorum and F. graminearum during the infection of corn, wheat, oats, barley, and rice. Glycosylation of DON is a protective detoxification mechanism employed by plants. More recently, DON glycosylating activity has also been detected in fungal microparasitic (biocontrol) fungal organisms. Here we follow up on the reported conversion of 15-acetyl-DON (15-ADON) into 15-ADON-3-O-glycoside (15-ADON-3G) in Clonostachys rosea. Based on the hypothesis that the reaction is likely being carried out by a uridine diphosphate glycosyl transferase (UDP-GTase), we applied a protein structural comparison strategy, leveraging the availability of the crystal structure of rice Os70 to identify a subset of potential C. rosea UDP-GTases that might have activity against 15-ADON. Using CRISPR/Cas9 technology, we knocked out several of the selected UDP-GTases in the C. rosea strain ACM941. Evaluation of the impact of knockouts on the production of 15-ADON-3G in confrontation assays with F. graminearum revealed multiple UDP-GTase enzymes, each contributing partial activities. The relationship between these positive hits and other UDP-GTases in fungal and plant species is discussed.

Untargeted metabolomics screening reveals unique secondary metabolite production from Alternaria section Alternaria.

Alternaria section Alternaria is comprised of many species that infect a broad diversity of important crop plants and cause post-harvest spoilage. Alternaria section Alternaria species, such as A. alternata and A. arborescens, 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. Secondary metabolite phenotypes from 36 Alternaria section Alternaria isolates were constructed to observe frequency of production patterns. A clear and unique mass feature pattern was observed for three of the strains that were linked with the production of the dehydrocurvularin family of toxins and associated detoxification products. Examination of corresponding genomes revealed the presence of the dehydrocurvularin biosynthesis gene cluster associated with a sub-telomeric accessory region. A comparison of sequence similarity and occurrences of the dehydrocurvularin biosynthetic gene cluster within Pleosporalean fungi is presented and discussed.

CRISPR/Cas9 disruption of UGT71L1 in poplar connects salicinoid and salicylic acid metabolism and alters growth and morphology.

Salicinoids are salicyl alcohol-containing phenolic glycosides with strong antiherbivore effects found only in poplars and willows. Their biosynthesis is poorly understood, but recently a UDP-dependent glycosyltransferase, UGT71L1, was shown to be required for salicinoid biosynthesis in poplar tissue cultures. UGT71L1 specifically glycosylates salicyl benzoate, a proposed salicinoid intermediate. Here, we analyzed transgenic CRISPR/Cas9-generated UGT71L1 knockout plants. Metabolomic analyses revealed substantial reductions in the major salicinoids, confirming the central role of the enzyme in salicinoid biosynthesis. Correspondingly, UGT71L1 knockouts were preferred to wild-type by white-marked tussock moth (Orgyia leucostigma) larvae in bioassays. Greenhouse-grown knockout plants showed substantial growth alterations, with decreased internode length and smaller serrated leaves. Reinserting a functional UGT71L1 gene in a transgenic rescue experiment demonstrated that these effects were due only to the loss of UGT71L1. The knockouts contained elevated salicylate (SA) and jasmonate (JA) concentrations, and also had enhanced expression of SA- and JA-related genes. SA is predicted to be released by UGT71L1 disruption, if salicyl salicylate is a pathway intermediate and UGT71L1 substrate. This idea was supported by showing that salicyl salicylate can be glucosylated by recombinant UGT71L1, providing a potential link of salicinoid metabolism to SA and growth impacts. Connecting this pathway with growth could imply that salicinoids are under additional evolutionary constraints beyond selective pressure by herbivores.

Extraction methods for untargeted metabolomics influence enzymatic activity in diverse soils.

Soil organic matter (SOM) is the largest carbon pool in terrestrial ecosystems and underpins the health and productivity of soil. Accurate characterization of its chemical composition will improve our understanding of biotic and abiotic processes regulating its stabilization. Our purpose in this study was to estimate the loss of SOM by microbial and exoenzymatic activity that might occur when soil is extracted for analysis of representative low molecular weight mass features using untargeted metabolomics. Two mined clays (kaolinite, montmorillonite) and three diverse soils (varying in texture, specific surface area and cation exchange capacity) were used to assess the extraction efficiency and loss of three enzymatic activity indicators (2,6-dichloroindophenol sodium salt hydrate [DCIP], 4-methylumbelliferyl phosphate [MUBph] and 3,4-dihydroxy-L-phenylalanine [LDOPA]) during extraction with two different solvents (water and methanol). Losses of the indicators were attributed to extraction method (ultrasonication, shaking, or shaking following chloroform fumigation), physical properties associated with the soil/clay type, and microbial activity. Soil/clay type strongly influenced indicator recovery and hence, SOM recovery. Choice of extraction method strongly influenced the composition and recovery of representative SOM mass features, while the choice of solvent determined whether the soil type or extraction method had a greater influence of compositional differences in the SOM mass features extracted. Extraction following chloroform fumigation had the greatest loss of the indicators, due to enzymatic activity and/or adsorption onto the soil matrix. Minimal variation in composition and loss of SOM mass features occurred during extraction by shaking for the soils tested; we therefore recommend it as the method of choice for untargeted SOM extraction studies.

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

Research into ergot alkaloid production in major cereal cash crops is crucial for furthering our understanding of the potential toxicological impacts of Claviceps purpurea upon Canadian agriculture and to ensure consumer safety. An untargeted metabolomics approach profiling extracts of C. purpurea sclerotia from four different grain crops separated the C. purpurea strains into two distinct metabolomic classes based on ergot alkaloid content. Variances in C. purpurea alkaloid profiles were correlated to genetic differences within the lpsA gene of the ergot alkaloid biosynthetic gene cluster from previously published genomes and from newly sequenced, long-read genome assemblies of Canadian strains. Based on gene cluster composition and unique polymorphisms, we hypothesize that the alkaloid content of C. purpurea sclerotia is currently undergoing adaptation. The patterns of lpsA gene diversity described in this small subset of Canadian strains provides a remarkable framework for understanding accelerated evolution of ergot alkaloid production in Claviceps purpurea.

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. parasitica over an eight-day time course of vic3 incompatibility. Our findings support transcriptomic data that indicated remodeling of secondary metabolite profiles occurs during vic3 -associated VI. VI-associated secondary metabolites include novel forms of calbistrin, decumbenone B, a sulfoxygenated farnesyl S-cysteine analog, lysophosphatidylcholines, and an as-yet unidentified group of lipid disaccharides. The farnesyl S-cysteine analog is structurally similar to pheromones predicted to be produced during VI and is here named 'crypheromonin'. Mass features associated with C. parasitica secondary metabolites skyrin, rugulosin and cryphonectric acid were also detected but were not VI specific. Partitioning of VI-associated secondary metabolites was observed, with crypheromonins and most calbistrins accumulating in the growth medium over time, whereas lysophosphatidylcholines, lipid disaccharide-associated mass features and other calbistrin-associated mass features peaked at distinct time points in the mycelium. Secondary metabolite biosynthetic gene clusters and potential biological roles associated with the detected secondary metabolites are discussed.

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. poae isolates produce a range of known mycotoxins, including type A and B trichothecenes, fusarins and beauvericin, genomic analysis suggests that this species may have lineage-specific accessory chromosomes with secondary metabolite biosynthetic gene clusters awaiting description. METHODS: We examined the biosynthetic potential of 38 F. poae isolates from Eastern Canada using a combination of long-read and short-read genome sequencing and untargeted, high resolution mass spectrometry metabolome analysis of extracts from isolates cultured in multiple media conditions. RESULTS: A high-quality assembly of isolate DAOMC 252244 (Fp157) contained four core chromosomes as well as seven additional contigs with traits associated with accessory chromosomes. One of the predicted accessory contigs harbours a functional biosynthetic gene cluster containing homologs of all genes associated with the production of apicidins. Metabolomic and genomic analyses confirm apicidins are produced in 4 of the 38 isolates investigated and genomic PCR screening detected the apicidin synthetase gene APS1 in approximately 7% of Eastern Canadian isolates surveyed. CONCLUSIONS: Apicidin biosynthesis is linked to isolate-specific putative accessory chromosomes in F. poae. The data produced here are an important resource for furthering our understanding of accessory chromosome evolution and the biosynthetic potential of F. poae.

Four phylogenetic species of ergot from Canada and their characteristics in morphology, alkaloid production, and pathogenicity.

Four ergot species (Claviceps ripicola, C. quebecensis, C. perihumidiphila, and C. occidentalis) were recognized based on analyses of DNA sequences from multiple loci, including two housekeeping genes, RNA polymerase II second largest subunit (RPB2), and translation elongation factor 1-α (TEF1-α), and a single-copy ergot alkaloid synthesis gene (easE) encoding chanoclavine I synthase oxidoreductase. Morphological features, ergot alkaloid production, and pathogenicity on five common cereal crops of each species were evaluated and presented in taxonomic descriptions. A synoptic key was also provided for identification.

Activation of biosynthetic gene clusters by the global transcriptional regulator TRI6 in Fusarium graminearum.

In F. graminearum, the transcription factor TRI6 positively regulates the trichothecene biosynthetic gene cluster (BGC) leading to the production of the secondary metabolite 15-acetyl deoxynivalenol. Secondary metabolites are not essential for survival, instead, they enable the pathogen to successfully infect its host. F. graminearum has the potential to produce a diverse array of secondary metabolites (SMs). However, given high functional specificity and energetic cost, most of these clusters remain silent, unless the organism is subjected to an environment conducive to SM production. Alternatively, secondary metabolite gene clusters (SMCs) can be activated by genetically manipulating their activators or repressors. In this study, a combination of transcriptomic and metabolomics analyses with a deletion and overexpressor mutants of TRI6 was used to establish the role of TRI6 in the regulation of several BGCs in F. graminearum. Evidence for direct and indirect regulation of BGCs by TRI6 was obtained by chromatin immunoprecipitation and yeast two-hybrid experiments. The results showed that the trichothecene genes are under direct control, while the gramillin gene cluster is indirectly controlled by TRI6 through its interaction with the pathway-specific transcription factor GRA2.

Transcriptomic and Exometabolomic Profiling Reveals Antagonistic and Defensive Modes of Clonostachys rosea Action Against Fusarium graminearum.

The mycoparasite Clonostachys rosea ACM941 is under development as a biocontrol organism against Fusarium graminearum, the causative agent of Fusarium head blight in cereals. To identify molecular factors associated with this interaction, the transcriptomic and exometabolomic profiles of C. rosea and F. graminearum GZ3639 were compared during coculture. Prior to physical contact, the antagonistic activity of C. rosea correlated with a response heavily dominated by upregulation of polyketide synthase gene clusters, consistent with the detected accumulation of corresponding secondary metabolite products. Similarly, prior to contact, trichothecene gene clusters were upregulated in F. graminearum, while those responsible for fusarielin and fusarin biosynthesis were downregulated, correlating with an accumulation of trichothecene products in the interaction zone over time. A concomitant increase in 15-acetyl deoxynivalenol-3-glucoside in the interaction zone was also detected, with C. rosea established as the source of this detoxified mycotoxin. After hyphal contact, C. rosea was found to predominantly transcribe genes encoding cell wall-degradation enzymes, major facilitator superfamily sugar transporters, anion:cation symporters, as well as alternative carbon source utilization pathways, together indicative of a transition to necrotropism at this stage. F. graminearum notably activated the transcription of phosphate starvation pathway signature genes at this time. Overall, a number of signature molecular mechanisms likely contributing to antagonistic activity by C. rosea against F. graminearum, as well as its mycotoxin tolerance, are identified in this report, yielding several new testable hypotheses toward understanding the basis of C. rosea as a biocontrol agent for continued agronomic development and application.

Enniatin Production Influences Fusarium avenaceum Virulence on Potato Tubers, but not on Durum Wheat or Peas.

Fusarium avenaceum is a generalist pathogen responsible for diseases in numerous crop species. The fungus produces a series of mycotoxins including the cyclohexadepsipeptide enniatins. Mycotoxins can be pathogenicity and virulence factors in various plant-pathogen interactions, and enniatins have been shown to influence aggressiveness on potato tubers. To determine the role of these mycotoxins in other F. avenaceum-host interactions, enniatin synthase 1 (ESYN1) disruption and overexpression mutants were generated and their ability to infect wheat and peas investigated. As a preliminary study, the transformants were screened for their ability to cause potato tuber necrosis and, consistent with a previous report, enniatin production increased necrotic lesion size on the tubers. By contrast, when the same mutants were assessed in their ability to cause disease in pea roots or durum wheat spikes, no changes in disease symptoms or virulence were observed. While it is known that, at least in the case of wheat, exogenously applied enniatins can cause tissue necrosis, this group of mycotoxins does not appear to be a key factor on its own in disease development on peas or durum wheat.

DNA Methylation Is Responsive to the Environment and Regulates the Expression of Biosynthetic Gene Clusters, Metabolite Production, and Virulence in Fusarium graminearum.

Histone modifications play a significant role in the regulation of biosynthetic gene clusters (BGCs) in the phytopathogen Fusarium graminearum, by contrast, epigenetic regulation by DNA methyltransferases (DNMTs) is less documented. In this study, we characterized two DNMTs (FgDIM-2 and FgRID) in F. graminearum, with homologies to "Deficient in methylation" (DIM-2) and "Repeat-induced point (RIP) deficient" (RID) from Neurospora. The loss of DNMTs resulted in not only a decrease in average methylation density in the nutrient-poor, compared to nutrient-rich conditions, but also differences in the genes expressed between the WT and the DNMT mutant strains, implicating the external environment as an important trigger in altering DNA methylation patterns. Consequently, we observed significant changes in the regulation of multiple BGCs and alterations in the pathogenicity of the fungus.

Terrosamycins A and B, Bioactive Polyether Ionophores from Streptomyces sp. RKND004 from Prince Edward Island Sediment.

Terrosamycins A (1) and B (2), two polycyclic polyether natural products, were purified from the fermentation broth of Streptomyces sp. RKND004 isolated from Prince Edward Island sediment. The one strain-many compounds (OSMAC) approach coupled with UPLC-HRMS-based metabolomics screening led to the identification of these compounds. The structure of 1 was determined from analysis of NMR, HRMS, and X-ray diffraction data. NMR experiments performed on 2 revealed the presence of two methoxy groups replacing two hydroxy groups in 1. Like other polyether ionophores, 1 and 2 exhibited excellent antibiotic activity against Gram-positive pathogens. Interestingly, the terrosamycins also exhibited activity against two breast cancer cell lines.

Whole Genome Sequencing and Metabolomic Study of Cave Streptomyces 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 Streptomyces isolates, designated as ICC1 and ICC4, from the Iron Curtain Cave, Chilliwack, Canada that exhibited antagonistic activities against the multidrug resistant strains of Escherichia coli. In this study, the genomes of these two isolates were sequenced by Illumina MiSeq, assembled and annotated. The genes associated with secondary metabolite production were identified and annotated using the bioinformatics platforms antiSMASH and BAGEL. ICC1 and ICC4 were then cultivated and ICC1 metabolome characterized by UHPLC-ESI-HRMS. The Global Natural Products Social Molecular Networking was used to identify metabolites based on the MS/MS spectral data. ICC1 and ICC4 showed a high level of sequence identity with the terrestrial bacteria Streptomyces lavendulae; however, they possess a greater secondary metabolite potential as estimated by the total number of identified biosynthetic gene clusters (BGCs). In particular, ICC1 and ICC4 had a greater number of polyketide and non-ribosomal peptide BGCs. The most frequently detected BGCs were those predicted to generate terpenes, small and low complexity dipeptides and lipids. Spectral analysis clearly identified a number of diketopiperazine products through matched reference spectra for cyclo (Leu-Pro), cyclo (Pro-Val) and cyclo [(4-hydroxyPro)-Leu]. One of the terpenes gene clusters predicted by antiSMASH possesses a seven-gene pathway consistent with diazepinomicin biosynthesis. This molecule contains a very rare core structure and its BGC, to date, has only been identified from a single bacterial genome. The tetrapeptide siderophore coelichelin BGC was unambiguously identified in the genome, however, the metabolite could not be identified from the culture extracts. Two type III polyketides, 2', 5' - dimethoxyflavone and nordentatin, were identified from the UHPLC-HRMS data of the aqueous and n-butanolic fractions of Streptomyces sp. ICC1, respectively. A BGC likely encoding these metabolites was predicted in both genomes. The predicted similarities in molecule production and genome shared by these two strains could be an indicative of a cooperative mode of living in extreme habitats instead of a competitive one. This secondary metabolite potential may contribute to the fitness of ICC1 and ICC4 in the Iron Curtain Cave.

Discovery of a New Natural Product and a Deactivation of a Quorum Sensing System by Culturing a "Producer" Bacterium With a Heat-Killed "Inducer" Culture.

Herein we describe a modified bacterial culture methodology as a tool to discover new natural products via supplementing actinomycete fermentation media with autoclaved cultures of "inducer" microbes. Using seven actinomycetes and four inducer microbes, we detected 28 metabolites that were induced in UHPLC-HRESIMS-based analysis of bacterial fermentations. Metabolomic analysis indicated that each inducer elicited a unique response from the actinomycetes and that some chemical responses were specific to each inducer-producer combination. Among these 28 metabolites, hydrazidomycin D, a new hydrazide-containing natural product was isolated from the pair Streptomyces sp. RKBH-B178 and Mycobacterium smegmatis. This result validated the effectiveness of the strategy in discovering new natural products. From the same set of induced metabolites, an in-depth investigation of a fermentation of Streptomyces sp. RKBH-B178 and autoclaved Pseudomonas aeruginosa led to the discovery of a glucuronidated analog of the pseudomonas quinolone signal (PQS). We demonstrated that RKBH-B178 is able to biotransform the P. aeruginosa quorum sensing molecules, 2-heptyl-4-quinolone (HHQ), and PQS to form PQS-GlcA. Further, PQS-GlcA was shown to have poor binding affinity to PqsR, the innate receptor of HHQ and PQS.