Gene Activation Strategies in Filamentous Fungi for Natural Products Discovery

Fungi have long been a prolific source of clinically approved drugs. The antibiotic penicillin, the cholesterol lowering statins, and the immunosuppressant cyclosporine have changed the treatment of human diseases in the last century. Recently, genome analysis of filamentous fungi has revealed that the biosynthetic potential for natural product expression is far greater than what is traditionally observed in laboratory culture. To this end, strategies to induce the expression of these otherwise transcriptionally silent natural product gene clusters are key to the continued discovery of chemical diversity. The research presented includes four manuscripts on gene activation strategies in fungi, with foci including ecological, chemical, and genetic approaches. Knockout of an epigenetic regulator in Fusarium graminearum resulted in the identification of two novel terpenes (tricinolonoic acid and tricinolone) and one novel polyketide (protofusarin). Furthermore, bioinformatics analysis allowed for the identification of the gene clusters responsible for the production of tricin terpenes and gibepyrones. In Chalara sp. 6661, treatment with the HDAC inhibitor vorinostat resulted in the production of four novel xanthones (chalanilines A-B and two xanthone-adenosine derivatives). Synthesis of isotopically labeled vorinostat and consequent feeding studies confirmed that chalanilines A and B were biotransformation products of vorinostat. A nutrient manipulation study in Aspergillius terreus showed selective production of metabolites via LCMS based metabolomics and enabled the discovery of new compounds including 7-desmethyl citreoviridin. Co-cultivation of two developmental forms of Aspergillius alliaceus resulted in the expression of novel, cytotoxic, bianthrone polyketides (allianthrones A-C). In each instance presented, application of a gene activation technique elicited the production of previously undescribed natural products

Development of a Antibacterial Disc Diffusion Assay to Differentiate Fusarin C from Non-Fusarin C activity in Early Screening of Mutant Fusarium graminearum Extracts

The discovery of novel compounds with antibacterial properties continues to be critically important.3 One potential source of such compounds is the cryptic genome of fungi known to produce biologically active molecules. A kmt6 mutant of the cereal pathogen Fusarium graminearum was previously developed through a histone H3 lysine 27 methyltransferase gene knockout, and has been shown to express hundreds of additional genes compared to wild type under the same growth conditions.1 Both the kmt6 mutant and the wild type F. graminearum produce the biologically active metabolite fusarin C, shown in Figure 1, and its analogs. Activity due to fusarin C can mask any activity of non-fusarin C compounds during early screening of extracts. Using a broad panel of bacteria, two samples were tested for differentiating activity profiles. These included an ethyl acetate extract of kmt6 growth media (i.e., the “160001E” fraction), and a more purified flash column fraction containing fusarin C and its analogs (i.e., the “fusarin C isolate”). Intriguingly, both Bacillus cereus and Staphylococcus aureus were inhibited by the 160001E extract, but were not inhibited by the fusarin C isolate. This indicates that activity from the 160001E fraction is not due to fusarin C or its analogs, but rather due to non-fusarin C compounds. With these results, a panel of 5 bacteria, including both gram negative and gram positive strains, has been developed as an early screening tool to differentiate activity between fusarin C isolates and non-fusarin C compounds from kmt6 mutant F. graminearum. This will aid in the bioassay-guided fractionation of extracts exhibiting antibacterial activity due to non-fusarin C compounds and toward the important aim of identifying novel compounds with antibacterial activity

Precursor-Directed Biosynthesis of Aminofulvenes: New Chalanilines from Endophytic Fungus Chalara sp.

The plant endophyte Chalara sp. is able to biotransform the epigenetic modifier vorinostat to form unique, aniline-containing polyketides named chalanilines. Here, we sought to expand the chemical diversity of chalaniline A-type molecules by changing the aniline moiety in the precursor vorinostat. In total, twenty-three different vorinostat analogs were prepared via two-step synthesis, and nineteen were incorporated by the fungus into polyketides. The highest yielding substrates were selected for large-scale precursor-directed biosynthesis and five novel compounds, including two fluorinated chalanilines, were isolated, purified, and structurally characterized. Structure elucidation relied on 1D and 2D NMR techniques and was supported by low- and high-resolution mass spectrometry. All compounds were tested for their bioactivity but were not active in antimicrobial or cell viability assays. Aminofulvene-containing natural products are rare, and this high-yielding, precursor-directed process allows for the diversification of this class of compounds.</jats:p

Precursor-Directed Biosynthesis of Aminofulvenes: New Chalanilines from Endophytic Fungus Chalara sp.

The plant endophyte Chalara sp. is able to biotransform the epigenetic modifier vorinostat to form unique, aniline-containing polyketides named chalanilines. Here, we sought to expand the chemical diversity of chalaniline A-type molecules by changing the aniline moiety in the precursor vorinostat. In total, twenty-three different vorinostat analogs were prepared via two-step synthesis, and nineteen were incorporated by the fungus into polyketides. The highest yielding substrates were selected for large-scale precursor-directed biosynthesis and five novel compounds, including two fluorinated chalanilines, were isolated, purified, and structurally characterized. Structure elucidation relied on 1D and 2D NMR techniques and was supported by low- and high-resolution mass spectrometry. All compounds were tested for their bioactivity but were not active in antimicrobial or cell viability assays. Aminofulvene-containing natural products are rare, and this high-yielding, precursor-directed process allows for the diversification of this class of compounds

Discovery and Biosynthesis of a Structurally Dynamic Antibacterial Diterpenoid

A new bicyclic diterpenoid, benditerpenoic acid, was isolated from soil-dwelling Streptomyces sp. (CL12-4). We sequenced the bacterial genome, identified the responsible biosynthetic gene cluster, verified the function of the terpene synthase, and heterologously produced the core diterpene. Comparative bioinformatics indicated this Streptomyces strain is phylogenetically unique and possesses nine terpene synthases. The absolute configurations of the new trans-fused bicyclo[8.4.0]tetradecanes were achieved by extensive spectroscopic analyses, including Mosher’s analysis, J-based coupling analysis, and computations based on sparse NMR-derived experimental restraints. Interestingly, benditerpenoic acid exists in two distinct ring-flipped bicyclic conformations with a rotational barrier of ~16 kcal mol(−1) in solution. The diterpenes exhibit moderate antibacterial activity against Gram-positive bacteria including methicillin and multi-drug resistant Staphylococcus aureus. This is the first isolation of an eunicellane-type diterpenoid from bacteria and the first identification of a diterpene synthase and biosynthetic gene cluster responsible for the construction of the eunicellane scaffold

Interrogation of solution conformation of complex macrocyclic peptides utilizing a combined SEC-HDX-MS, circular dichroism, and NMR workflow

CD/SEC-HDX-MS/NMR workflow for accelerated determination of the 3D conformation of pharmaceutically relevant peptides in a free solution state.</jats:p