Safe and Efficient Plant Systems for Antimicrobial PEptide production – SEPSAPE

The objective of the project is to develop economically, sustainable, high yield and safe plant expression systems (plant biofactories) to bring antimicrobial peptides (AMPs) to the market with an advantageous position of research groups and private companies from Europe. Sustainable production of AMPs will benefit the plant protection, food, veterinary and human pharma sectors. The project starts from well established and characterized synthetic AMPs as lead compounds that were developed previously with antibacterial (peptide BP100 as representative of the BP series) and antifungal selectivity (PAF26 from PAF peptides). The project will also consider other AMPs that have been successfully tested against bacterial and/or fungal pathogens. Identification and rational design of novel AMPs will be oriented not only to increase antimicrobial activity and decrease toxicity to non-target organisms, but most importantly to optimize properties for plant expression at high yield and accumulation in different plant tissues. The amount of possible combinations will require a high throughput screening (HTS) platform for peptides that have to be developed on purpose. Synthetic genes and constructions in expression vectors will be made based on a targeting strategy to decrease toxicity, escape proteolysis and increase yield, directing secretion to subcellular compartments (endoplasmic reticulum retention, oil bodies) and/or tissue specific storage (seed endosperm, tuber, green tissue). Efficiency of expression will be also evaluated with different types of AMP transgenes(cecropin A, BP100, PAF26, LfcB) or constructions (tandem, enlarged, with processing elements). Proteomics of specific tissues and subcellular organelles, extraction, purification and analysis of recovered expression products, and bioassays of activity in vitro of the recovered peptide fractions will be then performed. Proteomic analysis will be performed by using staining procedures with visible stains and fluorescent dyes in combination with MALDI-TOF/TOF Mass Spectrometry and by gel-free based proteomics combining chemical protein tagging and nanoLC-MS/MS. Integration of data from proteomics and metabolomics will be performed by data-dimensionality reduction methods. Specific downstream processing of the plant biomass will be developed for each expression strategy, including homogeneization in antioxidant buffer, removal of fiber and plant debris, protein or oil bodies extraction and expression product recovery, enzyme processing (specific tag-fusion removal), AMP recovery, and further purification or formulation. The analysis of the bioactivity of recovered AMPs against bacterial and fungal pathogens will be performed at laboratory scale on selected bacterial and fungal pathogens, and also using plant (Arabidopsis thaliana) and animal (Caenorhabditis elegans) models. Finally, batches of AMPs produced will be selected to perform acute toxicity testing and pilot trials of efficacy of control of representative pathogens of interest such as in a mice model, postharvest pathogens causing fruit rot (apple and pear, citrus, strawberry), greenhouse fungal and bacterial pathogens, and food-borne bacterial pathogens in minimally processed fresh meat and vegetable products.