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Major Goal 1: The purpose of this project is to characterize secondary metabolite-producing biosynthetic gene clusters (BGCs) of Sarocladium zeae due to its production of antibacterial compounds that inhibit aflatoxins and fumonisins, two major microbiological threats to corn. As the selective action of S. zeae against mycotoxins has incurred an interest in its use as a targeted biocontrol agent, all other possible metabolites must be characterized to ensure no toxins can be produced under any conditions. As only two of the 29 bioinformatically detected BGCs have been studied, the intention of the proposed work is to characterize metabolites produced by 17 of the remaining BGCs.Project objectives to attain this goal:Perform heterologous expression (HEx) with non-native host Aspergillus oryzae on the six terpene synthase and one non-ribosomal peptide synthase-polyketide synthase (NRPS-PKS) hybrid megasynthase-containing BGCs.Develop a transformation method for S. zeae using the translation elongation factor (TEF) promoter, which has been reported to be successful with S. zeae.Perform transcription factor (TF) overexpression on the 12 BGCs containing TFs.Elucidate the structures of all discovered compounds through HEx and TF overexpression work.Determine potential toxicity of elucidated structures.Report this new knowledge to the scientific and agricultural communities through conference presentations and peer-reviewed publications.Major Goal 2: Another major goal to be realized over the duration of this project is the enrichment of knowledge and professional development of the graduate student acting as the project director (PD).Project objectives to attain this goal:Teaching and training of any undergraduate student mentees involved in the completion of the proposed work and ensuring subsequent completion of mentee responsibilities. Undergraduate students Ian Sartor, Tram Nguyen, and Timothy Lim are going to be involved in the heterologous expression of terpene synthase genes in the coming months.Communication of project progress and findings through at least two conference presentations per year.Weekly meetings with the primary mentor and research group for the evaluation of project successes, possible improved methods, and practice for professional presentation of progress made for the PD.Biannual meetings with the doctoral committee for the purpose of obtaining expert advice and effective accountability on all listed objectives. As it stands, the doctoral committee is composed of the primary mentor and fungal natural products chemist, Dr. Elizabeth Skellam an analytical chemist, Dr. Jeffry Kelber; and a bioanalytical chemist with a specialty in mass spectrometry, Dr. Guido Verbeck IV. The addition of a biologist is planned before the end of 2023 to ensure an expert biological perspective is able to be given as well.Completion and submission of annual project updates to the USDA-NIFA-EWD program through REEport as an additional level of accountability.Completion of the doctoral dissertation and subsequent graduation.Major Goal 3: Lastly, the training of an earlier-stage graduate student by the PD will be completed in order to ensure the remaining BGCs that are not characterized through the course of this project will begin immediately upon award termination.Project objectives to attain this goal:Selection of a graduate student interested in continuing the work of identifying remaining secondary metabolites from S. zeae 1.5-2 years into the progress of this work.Training of the selected graduate student on relevant methods and project background already completed by the PD.Training of the selected graduate student on HEx with a specific focus on uncharacterized BGCs that will be the responsibility of the new student after the completion of the proposed work by the PD.Establishment and commitment to regular meetings with the PD and primary mentor to assess progress.

Schoellhorn, S. M.
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