Recipient Organization
UNIVERSITY OF NORTH TEXAS
1155 UNION CIR #305250
DENTON,TX 76203-5017
Performing Department
(N/A)
Non Technical Summary
One third of the global corn supply is produced in the United States, making maize the largest American agricultural product. Fungal pathogens are a major threat to the production of this crop by causing rot and producing toxic compounds. Corn showing signs of fungal pathogen growth are discarded, but levels of these compounds above the accepted safe threshold have been detected in recent studies. This implies that not only can there be a decreased crop yield from these toxic fungi, but that there are still traces of toxins that make it into the food supply. Additionally, lower crop yield may result in higher costs of corn products, including animal feed. This may cause an economic ripple effect as higher animal feed costs may lead to increased costs of meat and animal products. These are significant consequences for the general consumer.The fungus Sarocladium zeae grows within corn and produces antibacterial compounds, pyrrocidines, that naturally inhibit the production of the toxic compounds. Besides pyrrocidines, most compounds propagated by S. zeae are not known. The natural inhibition of the harmful fungi makes S. zeae an attractive candidate for a biocontrol agent. However, it must first be established that there are no toxins produced by S. zeae that simply have not been discovered yet. As it is impossible to test every single possible set of growing conditions, analysis of natural product-producing subsets of its fungal DNA will be done instead. These otherwise inactive DNA subsets will be activated with genetic modification in a fungal host, the modified fungus will be grown, and the new compounds will be identified. Once identified, the potential toxicity of the compounds can be determined, beginning to answer the question of whether S. zeae can be applied as a targeted biocontrol agent in the growth of corn.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
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.
Project Methods
The methods to be employed in this work are as follows:Bioinformatic analyses to be performed periodically to stay up to date with advances in literature methods as they are developed and published.In vitro work will begin with plasmid building for a projected 21 expression plasmids as described in the literature.1Performance of fungal PEG-mediated protoplast transformation via heterologous expression in Aspergillus oryzae as described in the literature.1Application and/or possible development of a transformation method based on those described in the literature2,3 with the use of a previously successful promoter4 in place of previous promoters attempted.Transcription factor (TF) overexpression of TF-containing biosynthetic gene clusters (BGCs) using the described transformation method.Solid phase microextraction (SPME) and Liquid-liquid extraction (LLE) of secondary metabolites from the media and cells of transformed fungus as described in the literature.5,6Instrumental analysis. These methods must be designed at the appropriate stage of the research i.e., when peaks have been isolated for compound identification. Instruments projected for use include HS-SPME-GC-MS, LCMS, HR-LCMS, MS/MS, NMR, and 2D NMR.Efforts to be performed under this work:Laboratory instruction for undergraduates and graduates involved in the procession of this work.In-lab workshops hosted monthly as opportunities for peer-to-peer instruction.Applications to workshops and conferences, such as those submitted already to the NPLinker bioinformatics workshop (not selected) and Gordon Research Symposium and Conference on Mycotoxins and Phycotoxins (selected for attendance in June 2023)Publications submitted when structures are identified and evaluated.Evaluation of the success of this work:Successful elucidation of structures.Successful elucidation of BGCs.Entries to publicly available databases such as MIBiG, NPAtlas, and GenBank of sequence data of the BGCs and associated compounds.An estimated four peer-reviewed publications successfully reaching fungal, agricultural, biochemical, and/or chemical journals.Successful training and completion of tasks by undergraduates measured by level of understanding reflected in submitted lab reports, successful transformants, and the development of a measure of independence in experimental work.Graduate students selected to carry on the work.Meetings, conferences, and presentations given. A minimum of two conferences per year as well as presentations outside of these conferences to local scientists at the university.Literature Referenced in MethodsDe Mattos-Shipley, K. M. J.; Lazarus, C. M.; Williams, K. Investigating Fungal Biosynthetic Pathways Using Heterologous Gene Expression: Aspergillus Oryzae as a Heterologous Host. In Engineering Natural Product Biosynthesis; Skellam, E., Ed.; Methods in Molecular Biology; Springer US: New York, NY, 2022; Vol. 2489, pp 23-39. https://doi.org/10.1007/978-1-0716-2273-5_2.Liu, Q.; Johnson, L. J.; Applegate, E. R.; Arfmann, K.; Jauregui, R.; Larking, A.; Mace, W. J.; Maclean, P.; Walker, T.; Johnson, R. D. Identification of Genetic Diversity, Pyrrocidine-Producing Strains and Transmission Modes of Endophytic Sarocladium Zeae Fungi from Zea Crops. Microorganisms 2022, 10 (7), 1415. https://doi.org/10.3390/microorganisms10071415.Johnson, L. J.; Koulman, A.; Christensen, M.; Lane, G. A.; Fraser, K.; Forester, N.; Johnson, R. D.; Bryan, G. T.; Rasmussen, S. An Extracellular Siderophore Is Required to Maintain the Mutualistic Interaction of Epichloë Festucae with Lolium Perenne. PLOS Pathog. 2013, 9 (5), e1003332. https://doi.org/10.1371/journal.ppat.1003332.Thornewell, S. J.; Peery, R. B.; Skatrud, P. L. Cloning and Characterization of the Gene Encoding Translation Elongation Factor 1α from Aureobasidium Pullulans. Gene 1995, 162 (1), 105-110. https://doi.org/10.1016/0378-1119(95)00312-T.Jiang, L.; Lv, K.; Zhu, G.; Lin, Z.; Zhang, X.; Xing, C.; Yang, H.; Zhang, W.; Wang, Z.; Liu, C.; Qu, X.; Hsiang, T.; Zhang, L.; Liu, X. Norditerpenoids Biosynthesized by Variediene Synthase-Associated P450 Machinery along with Modifications by the Host Cell Aspergillus Oryzae. Synth. Syst. Biotechnol. 2022, 7 (4), 1142-1147. https://doi.org/10.1016/j.synbio.2022.08.002.Turner, N. W.; Subrahmanyam, S.; Piletsky, S. A. Analytical Methods for Determination of Mycotoxins: A Review. Anal. Chim. Acta 2009, 632 (2), 168-180. https://doi.org/10.1016/j.aca.2008.11.010.