Diversity and Biosynthetic Potential of Fungi Isolated from St. John's Island, Singapore.

Adaptation to a wide variety of habitats allows fungi to develop unique abilities to produce diverse secondary metabolites with diverse bioactivities. In this study, 30 Ascomycetes fungi isolated from St. John's Island, Singapore were investigated for their general biosynthetic potential and their ability to produce antimicrobial secondary metabolites (SMs). All the 30 fungal isolates belong to the Phylum Ascomycota and are distributed into 6 orders and 18 genera with Order Hypocreales having the highest number of representative (37%). Screening for polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes using degenerate PCR led to the identification of 23 polyketide synthases (PKSs) and 5 nonribosomal peptide synthetases (NRPSs) grouped into nine distinct clades based on their reduction capabilities. Some of the identified PKSs genes share high similarities between species and known reference genes, suggesting the possibility of conserved biosynthesis of closely related compounds from different fungi. Fungal extracts were tested for their antimicrobial activity against S. aureus, Methicillin-resistant S. aureus (MRSA), and Candida albicans. Bioassay-guided fractionation of the active constituents from two promising isolates resulted in the isolation of seven compounds: Penilumamides A, D, and E from strain F4335 and xanthomegnin, viomellein, pretrichodermamide C and vioxanthin from strain F7180. Vioxanthin exhibited the best antibacterial activity with IC50 values of 3.0 µM and 1.6 µM against S. aureus and MRSA respectively. Viomellein revealed weak antiproliferative activity against A549 cells with an IC50 of 42 µM. The results from this study give valuable insights into the diversity and biosynthetic potential of fungi from this unique habitat and forms a background for an in-depth analysis of the biosynthetic capability of selected strains of interest with the aim of discovering novel fungal natural products.

Chemical elicitation as an avenue for discovery of bioactive compounds from fungal endophytes.

The present study investigated the molecular phylogeny, antimicrobial and cytotoxic activities of fungal endophytes obtained from the A*STAR Natural Organism Library (NOL) and previously isolated from Sungei Buloh Wetland Reserve, Singapore. Phylogenetic analysis based on ITS2 gene suggests that these isolates belong to 46 morphotypes and are affiliated to 23 different taxa in 17 genera of the Ascomycota phylum. Colletotrichum was the most dominant fungal genus accounting for 37% of all the isolates, followed by Diaporthe (13%), Phyllosticta (10.9%) and Diplodia (8.7%). Chemical elicitation using 5-azacytidine, a DNA methyltransferase inhibitor and suberoylanilide hydroxamic acid, a histone deacetylase inhibitor resulted in an increase in the number of active strains. Bioassay-guided isolation and structural elucidation yielded pestahivin and two new analogues from Bartalinia sp. F9447. Pestahivin and its related analogues did not exhibit antibacterial activity against Staphylococcus aureus but displayed strong antifungal activities against Candida albicans and Aspergillus brasiliensis, with IC50 values ranging from 0.46 ± 0.06 to 144 ± 18 µM. Pestahivin and its two analogues furthermore exhibited cytotoxic activity against A549 and MIA PACA-2 cancer cell lines with IC50 values in the range of 0.65 ± 0.12 to 42 ± 5.2 µM. The finding from this study reinforces that chemical epigenetic induction is a promising approach for the discovery of bioactive fungal secondary metabolites encoded by cryptic gene clusters.

CRISPR/Cas9 RNP-assisted validation of palmarumycin biosynthetic gene cluster in Lophiotrema sp. F6932.

Lophiotrema is a genus of ascomycetous fungi within the family Lophiotremataceae. Members of this genus have been isolated as endophytes from a wide range of host plants and also from plant debris within terrestrial and marine habitats, where they are thought to function as saprobes. Lophiotrema sp. F6932 was isolated from white mangrove (Avicennia officinalis) in Pulau Ubin Island, Singapore. Crude extracts from the fungus exhibited strong antibacterial activity, and bioassay-guided isolation and structure elucidation of bioactive constituents led to the isolation of palmarumycin C8 and a new analog palmarumycin CP30. Whole-genome sequencing analysis resulted in the identification of a putative type 1 iterative PKS (iPKS) predicated to be involved in the biosynthesis of palmarumycins. To verify the involvement of palmarumycin (PAL) gene cluster in the biosynthesis of these compounds, we employed ribonucleoprotein (RNP)-mediated CRISPR-Cas9 to induce targeted deletion of the ketosynthase (KS) domain in PAL. Double-strand breaks (DSBs) upstream and downstream of the KS domain was followed by homology-directed repair (HDR) with a hygromycin resistance cassette flanked by a 50 bp of homology on both sides of the DSBs. The resultant deletion mutants displayed completely different phenotypes compared to the wild-type strain, as they had different colony morphology and were no longer able to produce palmarumycins or melanin. This study, therefore, confirms the involvement of PAL in the biosynthesis of palmarumycins, and paves the way for implementing a similar approach in the characterization of other gene clusters of interest in this largely understudied fungal strain.

Enhancing the Discovery of Bioactive Secondary Metabolites From Fungal Endophytes Using Chemical Elicitation and Variation of Fermentation Media.

Endophytic microorganisms are an important source of bioactive secondary metabolites. In this study, fungal endophytes obtained from A*STAR's Natural Product Library (NPL) and previously isolated from different habitats of Singapore were investigated for their diversity, antimicrobial, and cytotoxic activities. A total of 222 fungal strains were identified on the basis of sequence analysis of ITS region of the rDNA gene. The identified fungal strains belong to 59 genera distributed in 20 orders. Majority of the identified strains (99%; 219 strains) belong to the phylum Ascomycota, while two strains belonged to the phylum Basidiomycota, and only one strain was from Mucoromycota phylum. The most dominant genus was Colletotrichum accounting for 27% of all the identified strains. Chemical elicitation using 5-azacytidine and suberoylanilide hydroxamic acid (SAHA) and variation of fermentation media resulted in the discovery of more bioactive strains. Bioassay-guided isolation and structure elucidation of active constituents from three prioritized fungal strains: Lophiotrema sp. F6932, Muyocopron laterale F5912, and Colletotrichum tropicicola F10154, led to the isolation of a known compound; palmarumycin C8 and five novel compounds; palmarumycin CP30, muyocopronol A-C and tropicicolide. Tropicicolide displayed the strongest antifungal activity against Aspergillus fumigatus with an IC50 value of 1.8 µg/ml but with a weaker activity against the Candida albicans presenting an IC50 of 7.1 µg/ml. Palmarumycin C8 revealed the best antiproliferative activity with IC50 values of 1.1 and 2.1 µg/ml against MIA PaCa-2 and PANC-1 cells, respectively.

Fungal Endophytes: A Promising Frontier for Discovery of Novel Bioactive Compounds.

For years, fungi have served as repositories of bioactive secondary metabolites that form the backbone of many existing drugs. With the global rise in infections associated with antimicrobial resistance, in addition to the growing burden of non-communicable disease, such as cancer, diabetes and cardiovascular ailments, the demand for new drugs that can provide an improved therapeutic outcome has become the utmost priority. The exploration of microbes from understudied and specialized niches is one of the promising ways of discovering promising lead molecules for drug discovery. In recent years, a special class of plant-associated fungi, namely, fungal endophytes, have emerged as an important source of bioactive compounds with unique chemistry and interesting biological activities. The present review focuses on endophytic fungi and their classification, rationale for selection and prioritization of host plants for fungal isolation and examples of strategies that have been adopted to induce the activation of cryptic biosynthetic gene clusters to enhance the biosynthetic potential of fungal endophytes.

Proposal of Carbonactinosporaceae fam. nov. within the class Actinomycetia. Reclassification of Streptomyces thermoautotrophicus as Carbonactinospora thermoautotrophica gen. nov., comb. nov.

Streptomyces thermoautotrophicus UBT1T has been suggested to merit generic status due to its phylogenetic placement and distinctive phenotypes among Actinomycetia. To evaluate whether 'S. thermoautotrophicus' represents a higher taxonomic rank, 'S. thermoautotrophicus' strains UBT1T and H1 were compared to Actinomycetia using 16S rRNA gene sequences and comparative genome analyses. The UBT1T and H1 genomes each contain at least two different 16S rRNA sequences, which are closely related to those of Acidothermus cellulolyticus (order Acidothermales). In multigene-based phylogenomic trees, UBT1T and H1 typically formed a sister group to the Streptosporangiales-Acidothermales clade. The Average Amino Acid Identity, Percentage of Conserved Proteins, and whole-genome Average Nucleotide Identity (Alignment Fraction) values were ≤58.5%, ≤48%, ≤75.5% (0.3) between 'S. thermoautotrophicus' and Streptosporangiales members, all below the respective thresholds for delineating genera. The values for genomics comparisons between strains UBT1T and H1 with Acidothermales, as well as members of the genus Streptomyces, were even lower. A review of the 'S. thermoautotrophicus' proteomic profiles and KEGG orthology demonstrated that UBT1T and H1 present pronounced differences, both tested and predicted, in phenotypic and chemotaxonomic characteristics compared to its sister clades and Streptomyces. The distinct phylogenetic position and the combination of genotypic and phenotypic characteristics justify the proposal of Carbonactinospora gen. nov., with the type species Carbonactinospora thermoautotrophica comb. nov. (type strain UBT1T, = DSM 100163T = KCTC 49540T) belonging to Carbonactinosporaceae fam. nov. within Actinomycetia.

Hypoculoside, a sphingoid base-like compound from Acremonium disrupts the membrane integrity of yeast cells.

We have isolated Hypoculoside, a new glycosidic amino alcohol lipid from the fungus Acremonium sp. F2434 belonging to the order Hypocreales and determined its structure by 2D-NMR (Nuclear Magnetic Resonance) spectroscopy. Hypoculoside has antifungal, antibacterial and cytotoxic activities. Homozygous profiling (HOP) of hypoculoside in Saccharomyces cerevisiae (budding yeast) revealed that several mutants defective in vesicular trafficking and vacuolar protein transport are sensitive to hypoculoside. Staining of budding yeast cells with the styryl dye FM4-64 indicated that hypoculoside damaged the vacuolar structure. Furthermore, the propidium iodide (PI) uptake assay showed that hypoculoside disrupted the plasma membrane integrity of budding yeast cells. Interestingly, the glycosidic moiety of hypoculoside is required for its deleterious effect on growth, vacuoles and plasma membrane of budding yeast cells.