Characterization of Peptaibols Produced by a Marine Strain of the Fungus Trichoderma endophyticum via Mass Spectrometry, Genome Mining and Phylogeny-Based Prediction.

Trichoderma is recognized as a prolific producer of nonribosomal peptides (NRPs) known as peptaibols, which have remarkable biological properties, such as antimicrobial and anticancer activities, as well as the ability to promote systemic resistance in plants against pathogens. In this study, the sequencing of 11-, 14- and 15-res peptaibols produced by a marine strain of Trichoderma isolated from the ascidian Botrylloides giganteus was performed via liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/MS). Identification, based on multilocus phylogeny, revealed that our isolate belongs to the species T. endophyticum, which has never been reported in marine environments. Through genome sequencing and genome mining, 53 biosynthetic gene clusters (BGCs) were identified as being related to bioactive natural products, including two NRP-synthetases: one responsible for the biosynthesis of 11- and 14-res peptaibols, and another for the biosynthesis of 15-res. Substrate prediction, based on phylogeny of the adenylation domains in combination with molecular networking, permitted extensive annotation of the mass spectra related to two new series of 15-res peptaibols, which are referred to herein as "endophytins". The analyses of synteny revealed that the origin of the 15-module peptaibol synthetase is related to 18, 19 and 20-module peptaibol synthetases, and suggests that the loss of modules may be a mechanism used by Trichoderma species for peptaibol diversification. This study demonstrates the importance of combining genome mining techniques, mass spectrometry analysis and molecular networks for the discovery of new natural products.

Discovery of dual PKS involved in sclerotiorin biosynthesis in Penicillium meliponae using genome mining and gene knockout.

Fungi of the genus Penicillium section Sclerotiora have as their main characteristic the presence of orange-pigmented mycelium, which is associated with sclerotiorin, a chlorinated secondary metabolite of the azaphilone subclass of polyketides. Sclerotiorin presents anti-diabetes, antioxidant, anti-inflammatory, anti-Alzheimer, antiviral, and antimicrobial activities, which has always attracted the attention of researchers worldwide. During our ongoing search for azaphilone-producing Amazonian fungi, the strain of Penicillium MMSRG-058 was isolated as an endophyte from the roots of Duguetia stelechantha and showed great capacity for producing sclerotiorin-like metabolites. Using multilocus phylogeny, this strain was identified as Penicillium meliponae. Moreover, based on the genome mining of this strain through the reverse approach, a cluster of putative biosynthetic genes (BGC) responsible for the biosynthesis of sclerotiorin-like metabolites (scl cluster) was identified. The knockout of the sclA (highly reducing PKS) and sclI (non-reducing PKS) genes resulted in mutants with loss of mycelial pigmentation and terminated the biosynthesis of sclerotiorin-like metabolites: geumsanol B, chlorogeumsanol B, 7-deacetylisochromophilone VI, isochromophilone VI, ochrephilone, isorotiorin, and sclerotiorin. Based on these results, a biosynthetic pathway was proposed considering the homology of BGC scl genes with the azaphilone BGCs that have already been functionally characterized.

Population Genetic Analysis of Fusarium decemcellulare, a Guaraná Pathogen, Reveals High Genetic Diversity in the Amazonas State, Brazil.

Guaraná is indigenous to the Brazilian Amazon where it has cultural and agroeconomic significance. However, its cultivation is constrained by a disease termed oversprouting of guaraná caused by Fusarium decemcellulare, with yield losses reaching as high as 100%. The disease can affect different parts of the plant, causing floral hypertrophy and hyperplasia, stem galls, and oversprouting of vegetative buds. To date, no study has been conducted characterizing the genetic diversity and population structure of this pathogen. Here, we report genetic diversity and genetic structure among 224 isolates from eight guaraná production areas of Amazonas State, Brazil, that were genotyped using a set of 10 inter-simple-sequence repeat (ISSR) markers. Despite moderate gene diversity (Hexp = 0.21 to 0.32), genotypic diversity was at or near maximum (223 multilocus genotypes among 224 isolates). Population genetic analysis of the 10 ISSR marker fragments with STRUCTURE software identified two populations designated C1 and C2 within the F. decemcellulare collection from the eight sites. Likewise, UPGMA hierarchical clustering and discriminant analysis of principal components of the strains from guaraná resolved these same two groups. Analysis of molecular variance demonstrated that 71% of genetic diversity occurred within the C1 and C2 populations. A pairwise comparison of sampling sites for both genetic populations revealed that 59 of 66 were differentiated from one another (P < 0.05), and high and significant gene flow was detected only between sampling sites assigned to the same genetic population. The presence of MAT1-1 and MAT1-2 strains, in conjunction with the high genotypic diversity and no significant linkage disequilibrium, suggests that each population of F. decemcellulare might be undergoing sexual reproduction. Isolation by distance was not observed (R2 = 0.02885, P > 0.05), which suggests that human-mediated movement of seedlings may have played a role in shaping the F. decemcellulare genetic structure in Amazonas State, Brazil.

Abundance, distribution and potential impact of transposable elements in the genome of Mycosphaerella fijiensis.

BACKGROUND: Mycosphaerella fijiensis is a ascomycete that causes Black Sigatoka in bananas. Recently, the M. fijiensis genome was sequenced. Repetitive sequences are ubiquitous components of fungal genomes. In most genomic analyses, repetitive sequences are associated with transposable elements (TEs). TEs are dispersed repetitive DNA sequences found in a host genome. These elements have the ability to move from one location to another within the genome, and their insertion can cause a wide spectrum of mutations in their hosts. Some of the deleterious effects of TEs may be due to ectopic recombination among TEs of the same family. In addition, some transposons are physically linked to genes and can control their expression. To prevent possible damage caused by the presence of TEs in the genome, some fungi possess TE-silencing mechanisms, such as RIP (Repeat Induced Point mutation). In this study, the abundance, distribution and potential impact of TEs in the genome of M. fijiensis were investigated. RESULTS: A total of 613 LTR-Gypsy and 27 LTR-Copia complete elements of the class I were detected. Among the class II elements, a total of 28 Mariner, five Mutator and one Harbinger complete elements were identified. The results of this study indicate that transposons were and are important ectopic recombination sites. A distribution analysis of a transposable element from each class of the M. fijiensis isolates revealed variable hybridization profiles, indicating the activity of these elements. Several genes encoding proteins involved in important metabolic pathways and with potential correlation to pathogenicity systems were identified upstream and downstream of transposable elements. A comparison of the sequences from different transposon groups suggested the action of the RIP silencing mechanism in the genome of this microorganism. CONCLUSIONS: The analysis of TEs in M. fijiensis suggests that TEs play an important role in the evolution of this organism because the activity of these elements, as well as the rearrangements caused by ectopic recombination, can result in deletion, duplication, inversion and translocation. Some of these changes can potentially modify gene structure or expression and, thus, facilitate the emergence of new strains of this pathogen.