Source: UNIVERSITY OF ARIZONA submitted to NRP
BIOACTIVE NATURAL PRODUCTS FOR AGRICULTURE AND MEDICINE FROM ARID LAND PLANTS AND MICROORGANISMS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1020652
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2019
Project End Date
Sep 30, 2024
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF ARIZONA
888 N EUCLID AVE
TUCSON,AZ 85719-4824
Performing Department
Natural Resources & the Environment
Non Technical Summary
Discovery of novel bioactive natural products is necessary for the identification of new pharmaceuticals and agricultural agents (herbicides and pesticides). We will use plants and microorganisms from arid and semiarid environments to identify such natural products; characterize the enzymes that produce them; and isolate the genes that encode their biosynthesis. We will use the purified natural products in various biological tests to elucidate their biological activities, and we will determine the chemical structures of the compounds that show interesting activities. We will use the enzymes and their encoding genes to change the chemical structures of these natural products, or to produce completely new "unnatural natural products" using cellular factories in "domesticated" microorganisms. Discovering such novel bioactive chemical compounds, and developing them to practical, useful agents will improve human health, agricultural productivity and safety, and the economic prospects of our State and Nation.Guaranteeing the safety of our produce is important for public health. For example, leafy green vegetables and melons are usually consumed raw, thus pre- or post-harvest decontamination is very important to prevent foodborne illnesses. Interventions currently used by the fresh produce industry include chemical sanitizers, the most common being chlorine. However, studies show that chlorine is not very effective in reducing bacterial populations on fresh produce. Plant-based antimicrobials are well known for their antimicrobial activity and are natural healthy products. Many bacteria produce antibacterial compounds called bacteriocins, which possess killing activities with much higher specificity than commonly used antibiotics. We propose to use plant-based antimicrobials such as plant extracts, essential oils and their active components to treat leafy greens and melons post-harvest and bacteriocins (tailocins) pre-harvest in order to reduce the population of foodborne pathogens such as E. coli O157:H7, Salmonella and L. monocytogenes on these produce. We expect the pathogen population will be largely reduced after the treatments. The results could provide the produce industry with natural alternatives to chemical based sanitizers such as chlorine.
Animal Health Component
30%
Research Effort Categories
Basic
30%
Applied
30%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2157410104030%
5112420200020%
7121420110015%
7121430110015%
5117410118020%
Knowledge Area
511 - New and Improved Non-Food Products and Processes; 215 - Biological Control of Pests Affecting Plants; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
1430 - Greens and leafy vegetables; 7410 - General technology; 1420 - Melons; 2420 - Noncrop plant research;

Field Of Science
1040 - Molecular biology; 1180 - Pharmacology; 1100 - Bacteriology; 2000 - Chemistry;
Goals / Objectives
This proposed project involves the following research objectives:Screen arid-land plants and microorganisms for the presence of bioactive compounds;Conduct bioassay-directed fractionation of extracts with demonstrated biological activity and elucidate the structures of bioactive NPs;Characterize and manipulate the biosynthesis of selected bioactive NPs;Further develop alternative agricultural systems, especially aeroponics system for plant cultivation, and for the production and structural diversification of plant-derived NPs of economic importance;Produce analogues of major NPs by chemical transformations and microbial biotransformations;Produce novel unnatural natural products (uNPs) by combinatorial synthetic microbiology;Evaluate promising lead NPs and their analogues with collaborators in other University of Arizona departments and colleges, other academic institutions, government agencies, and private industry;Evaluate plant-based antimicrobials including plant extracts, essential oils and their active components against E. coli O157:H7, S. enterica, and L. monocytogenes in vitro and on produce;Test whether application of tailocins to spinach and lettuce prevents colonization by E. coli O157:H7;Characterize Pseudomonas strains for the ability to produce tailocins that are effective against E. coli O157:H7, S. enterica and L. monocytogenes.
Project Methods
Natural product extracts will be prepared from plant collection materials and microbial cultures using standard natural product chemistry techniques as described under Procedures. Bioactivities will be determined using standard assays at the Southwest Center for Natural Product Research or at collaborators' laboratories. Bioactive natural products will be isolated by various chromatography procedures for bioassay-guided fractionation, and pure compounds will be isolated by preparative HPLC. Compound structures will be determined by various spectroscopic techniques. Natural product derivatives will be prepared by standard medicinal chemistry procedures. Aeroponic cultivation of plants will be performed as we described earlier. Biosynthetic gene clusters will be isolated by PCR of diagnostic regions, or by bioinformatic searches in genome sequence databases. Biosynthetic genes will be prepared by PCR, or by custom gene synthesis. Traditional genetic recombinant DNA cloning techniques as well as modern in vivo recombination techniques will be used for cloning genes or gene clusters into appropriate vectors, and these will be introduced into appropriate expression hosts such as E. coli or S. cerevisiae. Combinations of biosynthetic gene clusters, and hybrid biosynthetic genes will be prepared as we described earlier. Unnatural natural products will be detected and isolated similar to that of natural products.Natural antimicrobials including plant extracts, essential oils and their active components, and bacteriocins (tailocins) and other NPs and uNPs will be evaluated against E. coli O157:H7, S. enterica Newport and L. monocytogenes on leafy greens and melons. Some of the novel compounds will initially be tested against three foodborne pathogens including E. coli O157:H7, S. enterica and L. monocytogenes in vitro. The most promising compounds will then be used for the washing of produce post-harvest, while the tailocins will be applied to the produce pre-harvest. The artificially inoculated pathogen population will be analyzed before and after treatment to determine the efficacy of the natural antimicrobials and bacteriocins in inactivating pathogens. Appropriate controls washed in deionized water or chlorine will be included. Data analysis will be performed on all experiments using a statistical software package. The analysis of variance (ANOVA) will be performed on mean surviving bacteria from each treatment and control.Significant differences between controls and treatments will be analyzed.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The target audiences for this project include stakeholders in anticancer, agricultural, food production and microbiological research, such as: biotechnology and drug discovery professionals, natural product chemists and biologists, produce growers, packers, processors, and consumers; manufacturers of natural product or unnatural product antimicrobials; students and other scientists involved in the project. Produce growers and manufacturers of natural antimicrobials have been involved in this project by providing in-kind support to the project. The various target audiences will be informed periodically on the results of this project through various forums such as webinars, seminars, workshops, trainings, talks, social media, and demonstrations. Students and other scientific professionals will be receiving training on laboratory skills, experimental design, data analysis and statistical analysis and other activities that will enhance their professional development such as oral and poster presentations and manuscript preparation for publication of their findings in peer reviewed scientific journals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided training and professional development opportunities to students, postdoctoral research associates, research specialists, and visiting scientists. Dr. Molnar's lab provided training and professional development for one graduate and two postdoctoral associates and a visiting postdoctoral scientist in synthetic microbiology, genomics, genetics, combinatorial biosynthesis, and enzymology. Dr. Gunatilaka's lab provided training and professional development for 4 postdoctoral associates and one graduate student in natural product isolation, structure elucidation, bioassays, and medicinal chemistry. Dr Ravishankar' lab has provided the opportunity for one research specialist, four research technicians, one graduate student, and nine undergraduate students to learn numerous techniques and skills related to food safety research, including but not limited to sampling of melons and environmental samples for foodborne pathogens and indicator microorganisms, natural antimicrobial interventions for melons post-harvest treatment, antimicrobial treatments, biophotonic imaging and sample preparation for scanning electron microscopy. All personnel were also trained in oral and writing communication skills by participating in seminars and various outreach activities, and by contributing to the preparation of scientific manuscripts for publication in peer-reviewed scientific journals and posters in scientific meetings. How have the results been disseminated to communities of interest?Scientific results were published in the primary scientific literature. Nucleotide and protein sequences were deposited in public databases. Structure data were made available as supplemental information available at publishers' websites. All PIs and many research personnel presented lectures and posters at regional meetings and at various seminars and meetings at the University of Arizona. These included the 2019 Annual Food Safety Conference hosted by the University of Arizona Food Safety Consortium in Tucson, Arizona, attended by the local food industry professionals, regulatory agency scientists, and academic scientists. Dr. Ravishankar and her team members had the opportunity to participate in the food safety poster session at the 2020 Southwest Agricultural Summit held in Yuma, Arizona to provide information to the stakeholders on melons and other produce safety issues and interventions. Demonstrations on the use of plant antimicrobials and other aspects of melons food safety research were conducted by Dr. Ravishankar's team for the produce industry professionals, University of Arizona alumni/donors and local high school students throughout the year. Dr. Ravishankar and her team members conducted demonstrations of various food safety projects in her lab for high school students from various areas participating in the USDA-Ag Discovery Camp and other camps. Dr. Ravishankar and her team members hosted the University of Arizona- Food Safety Booth at the SARSEF- Future Innovator's Night in which they explained the importance of food safety and microbiology, prevention of cross-contamination and use of natural sanitizers for washing fresh produce to thousands of children and their parents. What do you plan to do during the next reporting period to accomplish the goals?Work towards the major goals will continue in the next reporting period as envisaged in the accepted proposal. Combinatorial biosynthesis and synthetic biology experiments will focus on the sulfation of NPs to improve their pharmacokinetic and pharmacodynamic profiles. Food fermentations will be studied by microbial community analysis. Biosynthesis of various natural products will be further elucidated. Withanolide analogues will be prepared in large-scale for animal studies vs melanoma, to be carried out at UA College of Medicine (Phoenix) Melon samples will be collected from various growing regions to conduct studies on natural antimicrobial interventions, and results will be collected for publication and dissemination to numerous target audiences. Outreach activities related to food safety will also continue.

Impacts
What was accomplished under these goals? Goal 1: Extracts derived from several endophytic fungi and plants of the genus Physalis were screened for their cytotoxic activity using cancer cell lines and normal cells. Surveys of entomopathogenic fungi for their ability to produce bioactive NPs, and for their genomes to encode the capacity to biosynthesize such NPs were conducted. Novel NP derivatives produced by combinatorial biosynthesis were evaluated for their cytotoxic activity using cancer cell lines and normal cells. Goal 2: Bioassay-guided fractionation of Physalis acutifolia afforded five new and six known withanolides including ten physalins. Of these, several withanolides were shown to have cytotoxic activity and physalin F was found to reverse neuropathic pain. Novel naphthoquinone-type NPs were isolated from the fungus Monascus sp. Novel benzenediol lactones and their derivatives were produced by combinatorial biosynthesis using genes identified from various filamentous fungi. These compounds were isolated and their structures were elucidated. Goal 3: The biosynthesis of Monascus azaphilone pigments (widely used food colorants) were further characterized, and redirected towards the production of naphthoquinones with potential antimicrobial activity. The biosynthesis of indole diterpene mycotoxins from Claviceps paspali were clarified by gene knockout studies. These mycotoxins cause tremors in animals and lead to considerable losses in agriculture. The production of antrodin C, the main bioactive ingredient of the medicinal mushroom Anthrodia cinnamomea were improved by optimizing fermentation conditions. Goal 4: Physalis acutifolia was grown under aeroponic growth conditions for the isolation and structure characterization of withanolide and physalin type NPs. Goal 5: Several analogues of 17β-hydroxywithaloides for anticancer activity studies were obtained by chemical transformations. Goal 6: The biosynthesis of amyrines, a common intermediate of bioactive triterpenoids, was studied in the plant Iris tectorum, revealing a novel active site architecture in the key oxidosqualene cyclase enzyme. Mutagenesis of the active site revealed structural contributions towards product specificity and productivity. A novel benzenediol biosynthetic gene cluster was identified from the fungus Rhytidhysteron rufulum, and used to produce >20 novel benzenediol lactone analogues by combinatorial biosynthesis. Goal 7: Withanlolide analogues belonging several classes were submitted to collaborators from UA College of Pharmacy, UA Department of Pharmacology, UA Department of Medicine (Phoenix), University of Geneva, and Sun Pharma Advanced Research Company in India. Goal 8: Melon samples (5 cantaloupe varieties, 3 honeydew varieties and 12 cantaloupe hybrids) were harvested from farms located in Georgia, Arizona, Texas, North Carolina, Indiana and California, and the rinds were inoculated with Salmonella Newport or Listeria monocytogenes. The samples were treated (by immersion) with 5% olive extract or 0.5% oregano oil as an antimicrobial solution. The plant-based antimicrobials reduced S. Newport and L. monocytogenes population on all the rind samples, in most cases to below the detection limit (1 CFU/g) at day 3, regardless of the melon types, varieties, or growing locations. Goal 9: Essential oil-based antimicrobial microemulsions were supplemented to the wash water to evaluate their efficacy against antibiotic-resistant Salmonella Newport and spoilage bacterium L. casei on Iceberg lettuce. The use of microemulsions resulted in a 2.3-4.37 log CFU/g reduction in the Salmonella population at various time points during days 0-28. They were also effective against Lactobacillus, resulting in 0.11-4.25 log CFU/g reduction during storage at days 0-28.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Wang, C., Wang, X., Zhang, L., Yue, Q., Liu, Q., Xu, Y.-m., Gunatilaka, A.A.L., Wei, X., Xu, Y., Moln�r, I.: Intrinsic and extrinsic programming of product chain length and release mode in fungal collaborating iterative polyketide synthases. J. Am. Chem. Soc. 142: 17093-17104, 2020. DOI: 10.1021/jacs.0c07050, PMID: 32833442, PMCID: PMC7659983
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Wu, S., Zhang, F., Xiong, W., Moln�r, I., Liang, J., Ji, A., Li, Y., Wang, C., Wang, S., Liu, Z., Wu, R., Duan, L.: An unexpected oxidosqualene cyclase active site architecture in the Iris tectorum multifunctional ?-amyrin synthase. ACS Catal. 10: 9515-9520, 2020. DOI: 10.1021/acscatal.0c03231
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhang, L., Yue, Q., Wang, C., Xu, Y., Moln�r, I.: Secondary metabolites from hypocrealean entomopathogenic fungi: genomics as a tool to elucidate the encoded parvome. Nat. Prod. Rep. 37: 1160-1180, 2020. DOI: 10.1039/d0np00007h, PMID: 32211677, PMCID: PMC7529689
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Hu, P.F., Huang, J., Chen, L., Ding, Z., Liu, L., Moln�r, I., Zhang, B.B.: Oxidative stress induction is a rational strategy to enhance the productivity of Antrodia cinnamomea fermentations for the antioxidant secondary metabolite antrodin C. J. Agric. Food Chem. 68: 3995-4004, 2020. DOI: 10.1021/acs.jafc.9b07965, PMID: 32133853, PMCID: PMC7351023
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Koz�k, L., Szil�gyi, Z., T�th, L., P�csi, I., Moln�r, I.: Functional characterization of the idtF and idtP genes in the Claviceps paspali indole diterpene biosynthetic gene cluster. Folia Microb. 65: 605-613, 2020. DOI: 10.1007/s12223-020-00777-6, PMID: 32077051, PMCID: PMC7244603
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Li, M., Kang, L., Ding, X., Liu, J., Shao, Y., Moln�r, I., Chen, F.: Monasone naphthoquinone biosynthesis and resistance in Monascus fungi. mBio 11: e02676-19, 2020. DOI: 10.1128/mBio.02676-19, PMID: 32019788, PMCID: PMC7002339
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhang, L., Fasoyin, O.E., Moln�r, I., Xu, Y.: Secondary metabolites from hypocrealean entomopathogenic fungi: novel bioactive compounds. Nat. Prod. Rep. 37: 1181-1206, 2020. DOI: 10.1039/c9np00065h, PMID: 32211639, PMCID: PMC7529686
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhou Y, Cai S, Gomez K, Wijertne EMK, Ji Y, Bellampalli SS, Luo S, Moutal A, Gunatilaka AAL, Khanna R; 1-O-Acetylgeopyxin, a Derivative of a Fungal Metabolite, Blocks Tetrodotoxin-Sensitive Voltage-Gated Sodium, Calcium Channels and Neuronal Excitability Which Correlates With Inhibition of Neuropathic Pain. Mol Brain 13: 73, 2020
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Lacombe J, Cretignier T, Meli L, Wijeratne EMK, Veuthey JL, Cuendet M, Gunatilaka AAL, Zenhausern F; Withanolide D Enhances Radiosensitivity of Human Cancer Cells by Inhibiting DNA Damage Non-homologous End Joining Repair Pathway. Frontiers in Oncology 9: 1468, 2020
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Xu YM, Arnold AE, Ren JMA, Xuan LH, Wang WQ, Gunatilaka AAL. Teratopyrones A-C, dimeric naphtho-?-pyrones and other metabolites from Teratosphaeria sp. AK1128, a fungal endophyte of Equisetum arvense. Molecules 25: 5058, 2020
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: An epigenetic modifier induces production of 3-(4-oxopyrano)-chromen-2-ones in Aspergillus sp. AST0006, an endophytic fungus of Astragalus lentiginosus. M.R. de Amorim, E.M.K. Wijeratne, S. Zhou, A.E. Arnold, A.N.L. Batista, J.M. Batista, L.C. dos Santos, A.A.L. Gunatilaka, Tetrahedron 76: 131525, 2020. doi.org/10.1016/j.tet.2020.131525
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Reversal of peripheral neuropathic pain by the small-molecule natural product physalin F via block of CaV2.3 (R-type) and Cav2.2 (N-type) voltage-gated calcium channels, Z. Shan, S. Cai, J. Yu, Z. Zhang, T.G.M. Vallecillo, M.J. Serafini, A.M. Thomas, N.Y.N. Pham, S.S. Bellampalli, A. Moutal, Y. Zhou, G.-B. Xu, Y.-M. Xu, S. Luo, M. Patek, J.M. Streicher, A.A.L. Gunatilaka, and R. Khanna, ACS Chem. Neurosci. 10: 2939-2955, 2019
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: S. Arellano and S. Ravishankar. 2019. Green Sanitizers: Improved Safety and Shelf-life of Iceberg Lettuce Washed with Plant-based Antimicrobial Microemulsions. Poster presented at the Annual Food Safety Conference. Tucson, AZ
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: S. Arellano and S. Ravishankar. 2019. Green Sanitizers: Improved Safety and Shelf-life of Iceberg Lettuce Washed with Plant-based Antimicrobial Microemulsions. Poster presented at the Annual GIDP Student Research Showcase. Tucson, AZ
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: S. Arellano and S. Ravishankar. 2020 Green Sanitizers: Improved Safety and Shelf-life of Iceberg Lettuce Washed with Plant-based Antimicrobial Microemulsions. Poster presented at the SW Ag Summit. Yuma, AZ
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: L. Zhu, Q. Wei, P. Brierley, M. Porchas, B. Patil and S. Ravishankar. 2020. Plant-based Antimicrobials Inactivate Salmonella enterica and Listeria monocytogenes on Melon Rinds. Poster presented at International Association for Food Protection 2020 annual meeting
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: S. Arellano, S. Ravishankar and B. Law. 2020. Green Sanitizers: Improved Safety and Shelf Life of Iceberg Lettuce Washed with Plant-based Antimicrobial Microemulsions. Poster presented at the International Association for Food Protection 2020 annual meeting