Microevolution in the Pan-Secondary Metabolome of Aspergillus flavus and its Potential Macroevolutionary Implications for Filamentous Fungi

Milton T. Drott1*, Tomás A. Rush2, Tatum R. Satterlee3, Richard J. Giannone2, Paul E. Abraham2, Claudio Greco4, Nandhitha Venkatesh5,6, Jeffrey M. Skerker7, N. Louise Glass7,8,9, Jesse L. Labbé2, Michael G. Milgroom10, Nancy P. Keller1,5*

1Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53703, USA, 2Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830, USA, 3Department of Genetics, University of Wisconsin-Madison, Madison, WI 53703, USA, 4Department of Molecular Microbiology, John Innes Centre, Norwich, United Kingdom, 5Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI 53703, USA, 6Department of Plant Pathology, University of Wisconsin-Madison, Madison, WI 53706, USA, 7Innovative Genomics Institute, The University of California, Berkeley, CA 94720 USA, 8Department of Plant and Microbial Biology, The University of California, Berkeley, CA 94720, USA, 9Environmental Genomics and Systems Biology, The Lawrence Berkeley National Laboratory, Berkeley, CA 94720 USA,10School of Integrative Plant Science, Plant Pathology and Plant-Microbe Biology Section, Cornell University, Ithaca, NY 14853, USA

Fungi produce a wealth of pharmaceutically bioactive secondary metabolites (SMs) from biosynthetic gene clusters (BGCs). It is common practice for drug-discovery efforts to treat species’ secondary metabolomes as being well represented by a single or a small number of representative genomes. However, this approach misses the possibility that intraspecific population dynamics, such as adaptation to environmental conditions or local microbiomes, may harbor novel BGCs that contribute to the overall niche breadth of species. Using 94 isolates of Aspergillus flavus, a cosmopolitan model fungus, sampled from seven states in the United States, we dereplicate 7,821 BGCs into 92 unique BGCs. We find that more than 25% of pan-genomic BGCs show population-specific patterns of presence/absence or protein divergence. Population-specific BGCs make up most of the accessory-genome BGCs, suggesting that different ecological forces that maintain accessory genomes may be partially mediated by population-specific differences in secondary metabolism. We use ultra-high-performance high resolution mass spectrometry to confirm that these genetic differences in BGCs also result in chemotypic differences in SM production in different populations that could mediate ecological interactions and be acted on by selection. Thus, our results suggest a paradigm shift that previously unrealized population-level reservoirs of SM diversity may be of significant evolutionary, ecological, and pharmaceutical importance. Lastly, we find that several population-specific BGCs from A. flavus are present in Aspergillus parasiticus and Aspergillus minisclerotigenes and discuss how the importance of microevolutionary patterns we uncover inform macroevolutionary inferences and help to align fungal secondary metabolism with existing evolutionary theory.


Transcription Factor Repurposing Offers Insights into Evolution of Biosynthetic Gene Cluster Regulation

Wenjie Wang1,2, Grant R. Nickles1, Milton Drott1, Lindsay K. Caesar3, PinMei Wang2, Nancy P. Keller1,4

1Department of Medical Microbiology and Immunology, University of Wisconsin – Madison, Madison, Wisconsin, USA; 2Ocean College, Zhejiang University, Zhoushan, Zhejiang, China; 3Department of Chemistry, Northwestern University, Evanston, Illinois, IL; 4Department of Bacteriology, University of Wisconsin – Madison, Madison, Wisconsin, USA

The fungal kingdom has provided advances in our ability to identify biosynthetic gene clusters (BGCs) and to examine how gene composition of BGCs evolves across species and genera. However, little is known about the evolution of specific BGC regulators that mediate how BGCs produce secondary metabolites (SMs). A bioinformatic search for conservation of the Aspergillus fumigatus xanthocillin BGC revealed a xan-like BGC in Pencillium expansum. However, in contrast to the role of the xan transcription factor (e.g. AfXanC) in A. fumigatus, overexpression (OE) of the conserved xan BGC TF gene, PexanC, did not activate xan BGC expression or xanthocillin production in P. expansum. Surprisingly, OE::PexanC was instead found to promote citrinin synthesis in P. expansum via trans induction of the cit pathway specific TF, ctnA, as determined by cit BGC expression and chemical profiling of ctnA deletion and OE::PexanC single and double mutants. Further, Gliovictin and other Glitoxin derivatives were found to be increased in OE::PexanC strain, suggesting a global role for PeXanC in SM regulation. Bioinformatic and promoter mutation analysis led to the identification of an AfXanC binding site, 5’-AGTCAGCA-3’, in promoter regions of the A. fumigatus xan BGC genes. This motif was not in the ctnA promoter suggesting a different binding site of PeXanC.

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Discovery of Fungal-Specific Targets and Inhibitors Using Chemical Phenotyping of Pathogenic Spore Germination

Sébastien C. Ortiz.1 Mingwei Huang1, Christina M. Hull1,2

1Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin–Madison, Madison, Wisconsin, USA, 2Department of Medical Microbiology and Immunology, School of Medicine and Public Health, University of Wisconsin–Madison, Madison, Wisconsin, USA

There is a critical need for new antifungal drugs; however, the lack of available fungal-specific targets is a major hurdle in the development of antifungal therapeutics. Spore germination is a differentiation process absent in humans that could harbor uncharacterized fungal-specific targets. To capitalize on this possibility, we developed novel phenotypic assays to identify and characterize inhibitors of spore germination of the human fungal pathogen Cryptococcus. Using these assays, we carried out a high throughput screen of ~75,000 drug-like small molecules and identified and characterized 191 novel inhibitors of spore germination, many of which also inhibited yeast replication and demonstrated low cytotoxicity against mammalian cells. Using an automated, microscopy-based, quantitative germination assay (QGA), we discovered that germinating spore populations can exhibit unique phenotypes in response to chemical inhibitors. Through the characterization of these spore population dynamics in the presence of the newly identified inhibitors, we classified 6 distinct phenotypes based on differences in germination synchronicity, germination rates, and overall population behavior. Similar chemical phenotypes were induced by inhibitors that targeted the same cellular function or had shared substructures. Leveraging these features, we used QGAs to identify outliers among compounds that fell into similar structural groups and thus refined relevant structural moieties, facilitating target identification. This approach led to the identification of complex II of the electron transport chain as the putative target of a promising structural cluster of germination inhibitory compounds. These inhibitors showed high potency against Cryptococcus spore germination, while maintaining low cytotoxicity against mammalian cells, making them prime candidates for development into novel antifungal therapeutics.


New Tools For Targeted Cloning and Over Expression Of Biosynthetic Gene Clusters

Robb Stankey1, Don Johnson1, Katherine Wozniak1, Rana Montaser2, Neil L. Kelleher2, Alinne Pereira3, Megan Sandoval-Powers3, Joyanne MacDonald1, Phil Brumm1, Håvard Sletta4, Trond Ellingsen4, Alexander Wentzel4, Mark Liles3, and David Mead1

1Varigen Biosciences, Madison, WI, USA, 2Northwestern University, Evanston, IL, USA, 3Auburn University, Auburn, AL, USA, 4SINTEF Industry, Trondheim, Norway

105 biosynthetic gene clusters (BGCs) ranging 12 to 130 kb from 95 diverse bacterial and fungal strains were successfully captured and cloned using CRISPR-Cas9 to precisely excise the pathway of interest. To improve the success of heterologous expression, we developed a new Streptomyces BGC expression vector (pDualP) which uniquely includes two inducible promoter elements, one flanking each side of the cloning site. As a proof of concept, we cloned the ACT and RED BGCs from S. coelicolor in both orientations of the pDualP vector, integrated them into S. lividans ΔredΔact, and observed inducible production of the blue ACT cluster product and the red RED cluster product but not from the native promoters. Second, we observed a substantial enhancement of the antimicrobial activity of heterologously-expressed, soil-derived metagenomic BGCs through induction with pDualP. Finally, we de-orphaned the stravidin BGC from Streptomyces sp. NRRL S-98 using the same approach. These results indicate that virtually any sequenced BGC can be cloned intact from complex genomes, and that direct cloning to a dual-inducible expression vector can greatly accelerate downstream small molecule characterization.

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