Depside and Depsidone Synthesis in Lichenized Fungi Comes into Focus through a Genome-Wide Comparison of the Olivetoric Acid and Physodic Acid Chemotypes of Pseudevernia furfuracea

Primary biosynthetic enzymes involved in the synthesis of lichen polyphenolic compounds depsides and depsidones are non-reducing polyketide synthases (NR-PKSs), and cytochrome P450s. However, for most depsides and depsidones the corresponding PKSs are unknown. Additionally, in non-lichenized fungi specific fatty acid synthases (FASs) provide starters to the PKSs. Yet, the presence of such FASs in lichenized fungi remains to be investigated. Here we implement comparative genomics and metatranscriptomics to identify the most likely PKS and FASs for olivetoric acid and physodic acid biosynthesis, the primary depside and depsidone defining the two chemotypes of the lichen Pseudevernia furfuracea. We propose that the gene cluster PF33-1_006185, found in both chemotypes, is the most likely candidate for the olivetoric acid and physodic acid biosynthesis. This is the first study to identify the gene cluster and the FAS likely responsible for olivetoric acid and physodic acid biosynthesis in a lichenized fungus. Our findings suggest that gene regulation and other epigenetic factors determine whether the mycobiont produces the depside or the depsidone, providing the first direct indication that chemotype diversity in lichens can arise through regulatory and not only through genetic diversity. Combining these results and existing literature, we propose a detailed scheme for depside/depsidone synthesis.

Depside and depsidone synthesis in lichenized fungi comes into focus through a genome-wide comparison of the olivetoric and physodic acid chemotype of Pseudevernia furfuracea

ABSTRACTPrimary biosynthetic enzymes involved in the synthesis of lichen polyphenolic compounds depsides and depsidones are Non-Reducing Polyketide Synthases (NR-PKSs), and cytochrome P450s (CytP450). However, for most depsides and depsidones the corresponding PKSs are unknown. Additionally, in non-lichenized fungi specific fatty acyl synthases (FASs) provide starters to the PKSs. Yet, the presence of such FASs in lichenized fungi remains to be investigated. Here we implement comparative genomics and metatranscriptomics to identify the most likely PKS and FASs for the synthesis of olivetoric and physodic acid, the primary depside and depsidone defining the two chemotypes of the lichen Pseudevernia furfuracea. We propose that the gene cluster PF33-1_006185, found in both chemotypes, is the most likely candidate for olivetoric and physodic acid biosynthesis. This is the first study to identify the gene cluster and the FAS likely responsible for physodic and olivetoric acid biosynthesis in a lichenized fungus. Our findings suggest that gene regulation and other epigenetic factors determine whether the mycobiont produces the depside or the depsidone, providing the first direct indication that chemotype diversity in lichens can arise through regulatory and not only through genetic diversity. Combining these results and existing literature, we propose a detailed scheme for depside/depsidone synthesis.

Population Genetic Structure and Chemotype Diversity of Fusarium graminearum Populations from Wheat in Canada and North Eastern United States

Fusarium head blight (FHB) is a major disease in wheat causing severe economic losses globally by reducing yield and contaminating grain with mycotoxins. In Canada, Fusarium graminearum is the principal etiological agent of FHB in wheat, producing mainly the trichothecene mycotoxin, deoxynivalenol (DON) and its acetyl derivatives (15-acetyl deoxynivalenol (15ADON) and 3-acetyl deoxynivalenol (3ADON)). Understanding the population biology of F. graminearum such as the genetic variability, as well as mycotoxin chemotype diversity among isolates is important in developing sustainable disease management tools. In this study, 570 F. graminearum isolates collected from commercial wheat crops in five geographic regions in three provinces in Canada in 2018 and 2019 were analyzed for population diversity and structure using 10 variable number of tandem repeats (VNTR) markers. A subset of isolates collected from the north-eastern United States was also included for comparative analysis. About 75% of the isolates collected in the Canadian provinces of Saskatchewan and Manitoba were 3ADON indicating a 6-fold increase in Saskatchewan and a 2.5-fold increase in Manitoba within the past 15 years. All isolates from Ontario and those collected from the United States were 15ADON and isolates had a similar population structure. There was high gene diversity (H = 0.803–0.893) in the F. graminearum populations in all regions. Gene flow was high between Saskatchewan and Manitoba (Nm = 4.971–21.750), indicating no genetic differentiation between these regions. In contrast, less gene flow was observed among the western provinces and Ontario (Nm = 3.829–9.756) and USA isolates ((Nm = 2.803–6.150). However, Bayesian clustering model analyses of trichothecene chemotype subpopulations divided the populations into two clusters, which was correlated with trichothecene types. Additionally, population cluster analysis revealed there was more admixture of isolates among isolates of the 3ADON chemotypes than among the 15ADON chemotype, an observation that could play a role in the increased virulence of F. graminearum. Understanding the population genetic structure and mycotoxin chemotype variations of the pathogen will assist in developing FHB resistant wheat cultivars and in mycotoxin risk assessment in Canada.

Molecular Diversity Assessment using Chemotypes

Background: Many techniques to design chemical libraries for screening have been put forward over time. General use libraries are still important when screening against novel targets and their design has relied on the use of molecular descriptors, while chemotype or scaffold analysis has been used less often. Objective: We describe a simple method to assess chemical diversity based on counts of the chemotypes that offers an alternative to model chemical diversity based on computed molecular properties. We show how chemotype counts can be used to evaluate the diversity of a library and compare diversity selection algorithms. We demonstrate an efficient compound selection algorithm based on chemotype analysis. Methods: We use automated chemotype perception algorithms and compare them to traditional techniques for diversity analysis to check their effectiveness in designing diverse libraries for screening. Results: The best type of molecular fingerprints for diversity selection in our analysis are extended circular fingerprints, but they can be outperformed by the use of a chemotype diversity algorithm, which can be more intuitive than traditional techniques based on molecular descriptors. Chemotype based algorithms retrieve a larger share of the chemotypes contained in a library when picking a subset of the chemicals in a collection. Conclusions: chemotype analysis offers an alternative for the generation of a general-purpose screening library as it maximizes the number of chemotypes present in a subset with the smallest number of compounds. The application of methods based on chemotype analysis that do not resort to the use of molecular descriptors are a very promising but seldom explored area of chemoinformatics.

Microcystis Chemotype Diversity in the Alimentary Tract of Bigheaded Carp

Most cyanobacterial organisms included in the genus Microcystis can produce a wide repertoire of secondary metabolites. In the mid-2010s, summer cyanobacterial blooms of Microcystis sp. occurred regularly in Lake Balaton. During this period, we investigated how the alimentary tract of filter-feeding bigheaded carps could deliver different chemotypes of viable cyanobacteria with specific peptide patterns. Twenty-five Microcystis strains were isolated from pelagic plankton samples (14 samples) and the hindguts of bigheaded carp (11 samples), and three bloom samples were collected from the scums of cyanobacterial blooms. An LC-MS/MS-based untargeted approach was used to analyze peptide patterns, which identified 36 anabaenopeptin, 17 microginin, and 13 microcystin variants. Heat map clustering visualization was used to compare the identified chemotypes. A lack of separation was observed in peptide patterns of Microcystis that originated from hindguts, water samples, and bloom-samples. Except for 13 peptides, all other congeners were detected from the viable and cultivated chemotypes of bigheaded carp. This finding suggests that the alimentary tract of bigheaded carps is not simply an extreme habitat, but may also supply the cyanobacterial strains that represent the pelagic chemotypes.

Molecular Phylogenetic Relationships, Trichothecene Chemotype Diversity and Aggressiveness of Strains in a Global Collection of Fusarium graminearum Species

Fusarium head blight (FHB), caused principally by the species belonging to the Fusarium graminearum species complex (FGSC), is an important disease in wheat, barley, and other small grain crops worldwide. Grain infected with species in the FGSC may be contaminated with trichothecene mycotoxins such as deoxynivalenol (DON) and nivalenol (NIV). In this study, we characterized the phylogenetic relationships, chemotype diversity, phenotypic characters, and aggressiveness of 150 strains in FGSC collected from eight different countries. Phylogenetic analysis based on portions of translation elongation factor 1-α (EF-1α) gene from 150 strains revealed six species in the FGSC, F. graminearum s.s, F. asiaticum, F. meridionale, F. cortaderiae, F. boothii, and F. austroamericanum. In this collection, 50% of the strains were 15-acetyldeoxynivalenol (15-ADON), 35% were 3-acetyldeoxynivalenol (3-ADON) and 15% were NIV. Evaluation of strains on moderately resistant (MR) wheat cultivar Carberry indicated that there is no significant difference among the species for FHB disease severity (DS), fusarium damaged kernel percentage (FDK%) and DON production. However, significant differences were observed among the chemotypes. Results showed significantly higher FHB DS, FDK%, DON production, growth rates, and macroconidia production for the 3-ADON strains than the 15-ADON and NIV strains. In addition, significant differences for FHB response variables were observed among the strains from different countries. Our results demonstrate that type and amount of trichothecene produced by a strain play a key role in determining the level of aggressiveness of that particular strain, regardless of the species.