Understanding evolutionary similarity and diversification of complex pathways of soil fungi to synthesize compounds with diverse ecological, biological and pharmaceutical functions

真菌通过复杂的生物合成途径产生生物活性分子，如抗生素和免疫系统抑制剂。本研究调查了在这些途径多样化中运作的进化过程，以及它们产生的代谢物，使用土壤栖息真菌toolypocladium。众所周知，环孢菌的种类特别富含生物活性代谢物，最著名的例子是环孢菌素A，它在医学上用于抑制器官移植患者的免疫系统。基因组学和计算生物学的最新进展表明，与传统的化学分析相比，代谢途径的多样性显著增加。因此，科学只观察到真菌生物化学多样性的“冰山一角”，而大多数真菌正在等待发现，作为有希望的药物来源、控制害虫的替代品和污染土壤的生物修复，以及生物燃料发展的关键。对导致真菌基因组多样化的模式和过程以及随后的生物活性分子的更强有力的理解，将增强利用真菌改善社会的能力。这项研究建立了一种案例研究方法，通过这种方法可以研究和利用其他真菌来促进科学进步。为了实现这些进步，学生和年轻研究人员接受跨学科方式的培训，并配备研究和开发应对现代世界挑战的生物解决方案。将对20个真菌分离株的基因组进行测序，鉴定和表征次生代谢物基因簇。这些基因组分析将与操纵生长实验和化学分析相结合，将遗传因素、环境和生态与产生的代谢物联系起来，并对这类真菌进行代谢物普查。进化过程和多样化模式（例如，基因复制-分化、水平基因转移、基因融合、多样化选择）将使用系统基因组学方法进行测试。分析将结合液相色谱-串联质谱代谢物分析、选择性生长实验和RNA测序，将次级代谢物基因簇与其代谢物联系起来。具体的目标和实验计划是为了比较两种主要的假设。首先，次生代谢物的多样化是与系统发育不一致的复杂过程的结果。第二，同源结构域产生化学上不同的代谢物，以前没有怀疑有共同的进化起源。该研究确定了遗传上不同的非核糖体肽合成酶和多酮合成酶所显示的复杂进化模式是否分别代表同源结构域和基因，这些进化过程是由复制-分化、水平基因转移、基因融合和多样化选择等进化过程产生的。结合液相色谱-串联质谱代谢物分析、选择性生长实验、RNA测序和系统基因组分析，将二级代谢物基因簇与其产生的代谢物联系起来，显示基因型与化学表型之间的进化联系。

Fungi produce biologically active molecules such as antibiotics and immune system suppressants through complex biosynthetic pathways. This research investigates the evolutionary processes that operate in the diversification of these pathways, and thus the metabolites they produce, using the soil dwelling fungus Tolypocladium. Species of Tolypocladium are known to be particularly rich in biologically active metabolites with the best-known example being Cyclosporin A, which is used medicinally to suppress the immune system in organ transplant patients. Recent advancements in genomics and computational biology show significantly greater diversity of metabolic pathways than previously detected by traditional chemical analyses. As such, science has only observed the "tip of the iceberg" of fungal biochemical diversity, while the majority await discovery as promising sources of pharmaceuticals, alternatives for the control of insect pests and bioremediation of contaminated soils, and essential in the development of biofuels. A more robust understanding of patterns and processes that have led to the diversification of fungal genomes, and subsequently their biologically active molecules, will enhance the ability to exploit fungi for the betterment of society. This research establishes a case study methodology by which other fungi can be studied and exploited for scientific advancements. In achieving these advancements, students and young researchers are trained in an interdisciplinary manner and equipped to study and develop biological solutions to the challenges of the modern World.Genomes of 20 fungal isolates will be sequenced, identifying and characterizing secondary metabolite gene clusters. These genomic analyses will be coupled with manipulative growth experiments and chemical analyses, linking the genetic factors, environment and ecology, with the metabolites produced, and resulting in a metabolite census of this group of fungi. Evolutionary processes and patterns of diversification (e.g., gene duplication-divergence, horizontal gene transfer, gene fusion, diversifying selection) will be tested using phylogenomic methodologies. The analyses will be coupled with liquid chromatography-tandem mass spectrometry metabolite profiling, selective growth experiments, and RNA sequencing to link secondary metabolite gene clusters with their metabolites. The specific aims and experimental plan are designed to compare two major hypotheses. The first is that diversification of secondary metabolites results from complex processes incongruent with phylogeny. The second is that homologous domains produce chemically distinct metabolites not previously suspected of having common evolutionary origins. The research determines if the complex patterns of evolution shown by genetically distinct non-ribosomal peptide synthetases and polyketide synthases represent homologous domains and genes, respectively, produced by evolutionary processes such as duplication-divergence, horizontal genet transfer, gene fusion, and diversifying selection. The integration of liquid chromatography-tandem mass spectrometry metabolite profiling, selective growth experiments, RNA sequencing and phylogenomic analyses will link secondary metabolite gene clusters and the metabolites they produce, showing the evolutionary connection between genotypes and chemical phenotypes.