Genomic and metabolomic diversity within a familial population of Aspergillus flavus.

Aspergillus flavus is an agriculturally significant micro-fungus having potential to contaminate food and feed crops with toxic secondary metabolites such as aflatoxin (AF) and cyclopiazonic acid (CPA). Research has shown A. flavus strains can overcome heterokaryon incompatibility and undergo meiotic recombination as teleomorphs. Although evidence of recombination in the AF gene cluster has been reported, the impacts of recombination on genotype and metabolomic phenotype in a single generation are lacking. In previous studies, we paired an aflatoxigenic MAT1-1 A. flavus strain with a non-aflatoxigenic MAT1-2 A. flavus strain that had been tagged with green fluorescent protein and then 10 F1 progenies (a mix of fluorescent and non-fluorescent) were randomly selected from single-ascospore colonies and broadly examined for evidence of recombination. In this study, we determined four of those 10 F1 progenies were recombinants because they were not vegetatively compatible with either parent or their siblings, and they exhibited other distinctive traits that could only result from meiotic recombination. The other six progenies examined shared genomic identity with the non-aflatoxigenic, fluorescent, and MAT1-2 parent, but were metabolically distinct. This study highlights phenotypic and genomic changes that may occur in a single generation from the outcrossing of sexually compatible strains of A. flavus.

Assessing Microbial Metabolic and Biological Diversity to Inform Natural Product Library Assembly.

The pressing need for novel chemical matter to support bioactive compound discovery has led natural product researchers to explore a wide range of source organisms and environments. One of the implicit guiding principles behind those efforts is the notion that sampling different environments is critical to accessing unique natural products. This idea was tested by comparing fungi from disparate biomes: aquatic sediments from Lake Michigan (USA) and terrestrial samples taken from the surrounding soils. Matched sets of Penicillium brevicompactum, Penicillium expansum, and Penicillium oxalicum from the two source environments were compared, revealing modest differences in physiological performance and chemical output. Analysis of LC-MS/MS-derived molecular feature data showed no source-dependent differences in chemical richness. High levels of scaffold homogeneity were also observed with 78-83% of scaffolds shared among the terrestrial and aquatic Penicillium spp. isolates. A comparison of the culturable fungi from the two biomes indicated that certain genera were more strongly associated with aquatic sediments (e.g., Trichoderma, Pseudeurotium, Cladosporium, and Preussia) versus the surrounding terrestrial environment (e.g., Fusarium, Pseudogymnoascus, Humicola, and Acremonium). Taken together, these results suggest that focusing efforts on sampling the microbial resources that are unique to an environment may have a more pronounced effect on enhancing the sought-after natural product diversity needed for chemical discovery and screening collections.

Altertoxin II, a Highly Effective and Specific Compound against Ewing Sarcoma.

A screening program designed to identify natural products with selective cytotoxic effects against cell lines representing different types of pediatric solid tumors led to the identification of altertoxin II as a highly potent and selective cytotoxin against Ewing sarcoma cell lines. Altertoxin II, but not the related compounds altertoxin I and alteichin, was highly effective against every Ewing sarcoma cell line tested, with an average 25-fold selectivity for these cells as compared to cells representing other pediatric and adult cancers. Mechanism of action studies revealed that altertoxin II causes DNA double-strand breaks, a rapid DNA damage response, and cell cycle accumulation in the S phase. Our studies also demonstrate that the potent effects of altertoxin II are partially dependent on the progression through the cell cycle, because the G1 arrest initiated by a CDK4/6 inhibitor decreased antiproliferative potency more than 10 times. Importantly, the cell-type-selective DNA-damaging effects of altertoxin II in Ewing sarcoma cells occur independently of its ability to bind directly to DNA. Ultimately, we found that altertoxin II has a dose-dependent in vivo antitumor efficacy against a Ewing sarcoma xenograft, suggesting that it has potential as a therapeutic drug lead and will be useful to identify novel targets for Ewing-sarcoma-specific therapies.

Building Natural Product Libraries Using Quantitative Clade-Based and Chemical Clustering Strategies.

The success of natural product-based drug discovery is predicated on having chemical collections that offer broad coverage of metabolite diversity. We propose a simple set of tools combining genetic barcoding and metabolomics to help investigators build natural product libraries aimed at achieving predetermined levels of chemical coverage. It was found that such tools aided in identifying overlooked pockets of chemical diversity within taxa, which could be useful for refocusing collection strategies. We have used fungal isolates identified as Alternaria from a citizen-science-based soil collection to demonstrate the application of these tools for assessing and carrying out predictive measurements of chemical diversity in a natural product collection. Within Alternaria, different subclades were found to contain nonequivalent levels of chemical diversity. It was also determined that a surprisingly modest number of isolates (195 isolates) was sufficient to afford nearly 99% of Alternaria chemical features in the data set. However, this result must be considered in the context that 17.9% of chemical features appeared in single isolates, suggesting that fungi like Alternaria might be engaged in an ongoing process of actively exploring nature's metabolic landscape. Our results demonstrate that combining modest investments in securing internal transcribed spacer (ITS)-based sequence information (i.e., establishing gene-based clades) with data from liquid chromatography-mass spectrometry (i.e., generating feature accumulation curves) offers a useful route to obtaining actionable insights into chemical diversity coverage trends in a natural product library. It is anticipated that these outcomes could be used to improve opportunities for accessing bioactive molecules that serve as the cornerstone of natural product-based drug discovery. IMPORTANCE Natural product drug discovery efforts rely on libraries of organisms to provide access to diverse pools of compounds. Actionable strategies to rationally maximize chemical diversity, rather than relying on serendipity, can add value to such efforts. Readily implementable biological (i.e., ITS sequence analysis) and chemical (i.e., mass spectrometry-based feature and scaffold measurements) diversity assessment tools can be employed to monitor and adjust library development tactics in real time. In summary, metabolomics-driven technologies and simple gene-based specimen barcoding approaches have broad applicability to building chemically diverse natural product libraries.