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1. Computer-aided, resistance gene-guided genome mining for proteasome and HMG-CoA reductase inhibitors

   https://doi.org/10.1093/jimb/kuad045  

   Jenkinson, Cory B; Podgorny, Adam R; Zhong, Cuncong; Oakley, Berl R (January 2023, Journal of Industrial Microbiology and Biotechnology)

   Abstract  Secondary metabolites (SMs) are biologically active small molecules, many of which are medically valuable. Fungal genomes contain vast numbers of SM biosynthetic gene clusters (BGCs) with unknown products, suggesting that huge numbers of valuable SMs remain to be discovered. It is challenging, however, to identify SM BGCs, among the millions present in fungi, that produce useful compounds. One solution is resistance gene-guided genome mining, which takes advantage of the fact that some BGCs contain a gene encoding a resistant version of the protein targeted by the compound produced by the BGC. The bioinformatic signature of such BGCs is that they contain an allele of an essential gene with no SM biosynthetic function, and there is a second allele elsewhere in the genome. We have developed a computer-assisted approach to resistance gene-guided genome mining that allows users to query large databases for BGCs that putatively make compounds that have targets of therapeutic interest. Working with the MycoCosm genome database, we have applied this approach to look for SM BGCs that target the proteasome β6 subunit, the target of the proteasome inhibitor fellutamide B, or HMG-CoA reductase, the target of cholesterol reducing therapeutics such as lovastatin. Our approach proved effective, finding known fellutamide and lovastatin BGCs as well as fellutamide- and lovastatin-related BGCs with variations in the SM genes that suggest they may produce structural variants of fellutamides and lovastatin. Gratifyingly, we also found BGCs that are not closely related to lovastatin BGCs but putatively produce novel HMG-CoA reductase inhibitors. One-Sentence SummaryA new computer-assisted approach to resistance gene-directed genome mining is reported along with its use to identify fungal biosynthetic gene clusters that putatively produce proteasome and HMG-CoA reductase inhibitors. 

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2. Beyond the Biosynthetic Gene Cluster Paradigm: Genome-Wide Coexpression Networks Connect Clustered and Unclustered Transcription Factors to Secondary Metabolic Pathways

   https://doi.org/10.1128/Spectrum.00898-21  

   Kwon, Min Jin; Steiniger, Charlotte; Cairns, Timothy C.; Wisecaver, Jennifer H.; Lind, Abigail L.; Pohl, Carsten; Regner, Carmen; Rokas, Antonis; Meyer, Vera (October 2021, Microbiology Spectrum)

   Goldman, Gustavo H. (Ed.)

   ABSTRACT Fungal secondary metabolites are widely used as therapeutics and are vital components of drug discovery programs. A major challenge hindering discovery of novel secondary metabolites is that the underlying pathways involved in their biosynthesis are transcriptionally silent under typical laboratory growth conditions, making it difficult to identify the transcriptional networks that they are embedded in. Furthermore, while the genes participating in secondary metabolic pathways are typically found in contiguous clusters on the genome, known as biosynthetic gene clusters (BGCs), this is not always the case, especially for global and pathway-specific regulators of pathways’ activities. To address these challenges, we used 283 genome-wide gene expression data sets of the ascomycete cell factory Aspergillus niger generated during growth under 155 different conditions to construct two gene coexpression networks based on Spearman’s correlation coefficients (SCCs) and on mutual rank-transformed Pearson’s correlation coefficients (MR-PCCs). By mining these networks, we predicted six transcription factors, named MjkA to MjkF, to regulate secondary metabolism in A. niger . Overexpression of each transcription factor using the Tet-On cassette modulated the production of multiple secondary metabolites. We found that the SCC and MR-PCC approaches complemented each other, enabling the delineation of putative global (SCC) and pathway-specific (MR-PCC) transcription factors. These results highlight the potential of coexpression network approaches to identify and activate fungal secondary metabolic pathways and their products. More broadly, we argue that drug discovery programs in fungi should move beyond the BGC paradigm and focus on understanding the global regulatory networks in which secondary metabolic pathways are embedded. IMPORTANCE There is an urgent need for novel bioactive molecules in both agriculture and medicine. The genomes of fungi are thought to contain vast numbers of metabolic pathways involved in the biosynthesis of secondary metabolites with diverse bioactivities. Because these metabolites are biosynthesized only under specific conditions, the vast majority of the fungal pharmacopeia awaits discovery. To discover the genetic networks that regulate the activity of secondary metabolites, we examined the genome-wide profiles of gene activity of the cell factory Aspergillus niger across hundreds of conditions. By constructing global networks that link genes with similar activities across conditions, we identified six putative global and pathway-specific regulators of secondary metabolite biosynthesis. Our study shows that elucidating the behavior of the genetic networks of fungi under diverse conditions harbors enormous promise for understanding fungal secondary metabolism, which ultimately may lead to novel drug candidates. 

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3. Uncovering a miltiradiene biosynthetic gene cluster in the Lamiaceae reveals a dynamic evolutionary trajectory

   https://doi.org/10.1038/s41467-023-35845-1  

   Bryson, Abigail E.; Lanier, Emily R.; Lau, Kin H.; Hamilton, John P.; Vaillancourt, Brieanne; Mathieu, Davis; Yocca, Alan E.; Miller, Garret P.; Edger, Patrick P.; Buell, C. Robin; et al (December 2023, Nature Communications)

   Abstract The spatial organization of genes within plant genomes can drive evolution of specialized metabolic pathways. Terpenoids are important specialized metabolites in plants with diverse adaptive functions that enable environmental interactions. Here, we report the genome assemblies of Prunella vulgaris , Plectranthus barbatus , and Leonotis leonurus . We investigate the origin and subsequent evolution of a diterpenoid biosynthetic gene cluster (BGC) together with other seven species within the Lamiaceae (mint) family. Based on core genes found in the BGCs of all species examined across the Lamiaceae, we predict a simplified version of this cluster evolved in an early Lamiaceae ancestor. The current composition of the extant BGCs highlights the dynamic nature of its evolution. We elucidate the terpene backbones generated by the Callicarpa americana BGC enzymes, including miltiradiene and the terpene (+)-kaurene, and show oxidization activities of BGC cytochrome P450s. Our work reveals the fluid nature of BGC assembly and the importance of genome structure in contributing to the origin of metabolites. 

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4. Tomato root specialized metabolites evolved through gene duplication and regulatory divergence within a biosynthetic gene cluster

   https://doi.org/10.1126/sciadv.adn3991  

   Kerwin, Rachel E; Hart, Jaynee E; Fiesel, Paul D; Lou, Yann-Ru; Fan, Pengxiang; Jones, A Daniel; Last, Robert L (April 2024, Science Advances)

   Tremendous plant metabolic diversity arises from phylogenetically restricted specialized metabolic pathways. Specialized metabolites are synthesized in dedicated cells or tissues, with pathway genes sometimes colocalizing in biosynthetic gene clusters (BGCs). However, the mechanisms by which spatial expression patterns arise and the role of BGCs in pathway evolution remain underappreciated. In this study, we investigated the mechanisms driving acylsugar evolution in the Solanaceae. Previously thought to be restricted to glandular trichomes, acylsugars were recently found in cultivated tomato roots. We demonstrated that acylsugars in cultivated tomato roots and trichomes have different sugar cores, identified root-enriched paralogs of trichome acylsugar pathway genes, and characterized a key paralog required for root acylsugar biosynthesis,SlASAT1-LIKE(SlASAT1-L), which is nested within a previously reported trichome acylsugar BGC. Last, we provided evidence thatASAT1-Larose through duplication of its paralog,ASAT1, and was trichome-expressed before acquiring root-specific expression in theSolanumgenus. Our results illuminate the genomic context and molecular mechanisms underpinning metabolic diversity in plants. 

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5. Secondary Metabolism Gene Clusters Exhibit Increasingly Dynamic and Differential Expression during Asexual Growth, Conidiation, and Sexual Development in Neurospora crassa

   https://doi.org/10.1128/msystems.00232-22  

   Wang, Zheng; Lopez-Giraldez, Francesc; Slot, Jason; Yarden, Oded; Trail, Frances; Townsend, Jeffrey P. (June 2022, mSystems)

   Hug, Laura A. (Ed.)

   ABSTRACT Secondary metabolite clusters (SMCs) encode the machinery for fungal toxin production. However, understanding their function and analyzing their products requires investigation of the developmental and environmental conditions in which they are expressed. Gene expression is often restricted to specific and unexamined stages of the life cycle. Therefore, we applied comparative genomics analyses to identify SMCs in Neurospora crassa and analyzed extensive transcriptomic data spanning nine independent experiments from diverse developmental and environmental conditions to reveal their life cycle-specific gene expression patterns. We reported 20 SMCs comprising 177 genes—a manageable set for investigation of the roles of SMCs across the life cycle of the fungal model N. crassa —as well as gene sets coordinately expressed in 18 predicted SMCs during asexual and sexual growth under three nutritional and two temperature conditions. Divergent activity of SMCs between asexual and sexual development was reported. Of 126 SMC genes that we examined for knockout phenotypes, al-2 and al-3 exhibited phenotypes in asexual growth and conidiation, whereas os-5 , poi-2 , and pmd-1 exhibited phenotypes in sexual development. SMCs with annotated function in mating and crossing were actively regulated during the switch between asexual and sexual growth. Our discoveries call for attention to roles that SMCs may play in the regulatory switches controlling mode of development, as well as the ecological associations of those developmental stages that may influence expression of SMCs. IMPORTANCE Secondary metabolites (SMs) are low-molecular-weight compounds that often mediate interactions between fungi and their environments. Fungi enriched with SMs are of significant research interest to agriculture and medicine, especially from the aspects of pathogen ecology and environmental epidemiology. However, SM clusters (SMCs) that have been predicted by comparative genomics alone have typically been poorly defined and insufficiently functionally annotated. Therefore, we have investigated coordinate expression in SMCs in the model system N. crassa , and our results suggest that SMCs respond to environmental signals and to stress that are associated with development. This study examined SMC regulation at the level of RNA to integrate observations and knowledge of these genes in various growth and development conditions, supporting combining comparative genomics and inclusive transcriptomics to improve computational annotation of SMCs. Our findings call for detailed study of the function of SMCs during the asexual-sexual switch, a key, often-overlooked developmental stage. 

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