7. Project Summary Ergot alkaloids improve human health as powerful and versatile pharmaceuticals for treatment of multiple conditions including senile dementia, Alzheimer's disease, Parkinson's disease, migraines, hyperprolactinemia, and type 2 diabetes. Tremendous diversity in structure and activity can be found among the natural and semi-synthetic ergot alkaloids. Small changes in structure may lead to a great changes in activity. The long-term goal of this research program is to understand and ultimately control the biosynthesis of diverse ergot alkaloids by determining the functions of genes and gene products that produce and diversify ergot alkaloids. One goal of this particular project is to understand and control the biosynthesis of lysergic acid amides. Many of the more important pharmaceutical ergot alkaloids are lysergic acid amides or could be derived from lysergic acid amides. The synthesis of these compounds involves alternate and competitive mechanisms for off-loading amides from a non-ribosomal peptide synthetase. An understanding of the genes involved in making these amide ergot alkaloids is now more accessible because of their recent discovery in an experimentally tractable fungi, including three species of Aspergillus as well as Metarhizium brunneum. A second goal of this project is to test investigate recently discovered genes hypothesized to control regulation and secretion of lysergic acid amides; both of these processes have translational significance. Specific aims of the proposed project are to: 1) Understand and control competing non-ribosomal peptide synthetase offloading mechanisms for lysergic acid amides; and, 2) Determine roles of novel ergot alkaloid synthesis genes in accumulation and secretion of lysergic acid amides. Aim 1 will be pursued through two reciprocal but independent approaches with alternate schemes for producing mutants of the reductase domain of a lysergyl peptide synthetase to pair with other ergot alkaloid synthesis genes to understand steps in liberating lysergic acid amides. The experimental approach to Aim 2 will involve application of transformation and CRISPR/Cas9- based technologies we have developed to assess functions of a transcriptional regulator and a major facilitator family transporter. The fungal strains produced by these genetic manipulations will be analyzed by molecular and biochemical methods to determine how expression or alteration of the candidate genes has affected the fungus's ergot alkaloid profile. Results of the proposed project will reveal roles of specific genes and provide strains of fungi that produce molecules with pharmaceutical significance. Additional benefits include meaningful experiences for graduate and undergraduate students and further development of platforms for modification and improvement of additional or novel ergot alkaloids.